Sample records for atpase regulates surfactant

  1. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari


    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  2. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... a huge amount of energy in form of ATP, to pump out protons. To avoid complete energy depletion in the cells, tight regulation of the PM H+-ATPase is a necessity. The proteins two terminal domains have been identified as autoinhibitory domains that regulate the pumping activity, but due to lack of a high....... In contrast to fungal PM H+-ATPases the terminal phosphorylation sites in the plant counterpart was found highly conserved even in the earliest land plants. The phosphorylation sites were, however, not found in algae, the sister group of land plants. We therefore hypotheses that the delicate regulation...

  3. The Kdp-ATPase system and its regulation

    Indian Academy of Sciences (India)


    Mar 15, 2007 ... K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E. coli, the KdpATPase (comprising of the KdpFABC complex), ...

  4. New ATPase regulators-p97 goes to the PUB

    DEFF Research Database (Denmark)

    Madsen, Louise; Seeger, Michael; Semple, Colin A


    The conserved eukaryotic AAA-type ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast, is involved in a number of cellular pathways, including fusion of homotypic membranes, protein degradation, and activation of membrane-bound transcription factors. Most likely, p97 is directed....... Recently, a small, conserved family of proteins, containing PUB domains, was found to function as p97 adaptors. Intriguingly, their association with p97 is regulated by tyrosine phosphorylation, suggesting that they act as a relay between signalling pathways and p97 functions. Here we give an overview...

  5. Regulation of Na+/K+-ATPase by Estradiol and IGF-1 in Cardio-Metabolic Diseases. (United States)

    Obradovic, Milan; Stanimirovic, Julijana; Panic, Anastasija; Bogdanovic, Nikola; Sudar-Milovanovic, Emina; Cenic-Milosevic, Desanka; Isenovic, Esma R


    The sodium/potassium- adenosine- triphosphatase (Na+/K+-ATPase) is an important mediator in vasculature tone and contractility, and its abnormal regulation has been implicated in many diseases such as obesity, insulin resistance, diabetes, and hypertension. Decreased Na+/K+-ATPase abundance and its altered isoform expression induce cardiomyocytes death and cardiac dysfunction, possibly leading to the development of myocardial dilation and heart failure. Therefore, the regulation of Na+/K+-ATPase activity/expression could be important in treatment and possible prevention of cardio-metabolic diseases. A number of hormones and environmental factors regulate the function of Na+/K+-ATPase in response to changing cellular requirements. Estradiol and insulin like growth factor-1 (IGF-1) are among potent hormones that positively regulate Na+/K+- ATPase activity or de novo synthesis of α - and β - subunits. Both estradiol and IGF-1 have a huge therapeutic potential in treatment of vasculopathy in cardio-metabolic diseases. We searched the MEDLINE and PUBMED databases for all English and non-English articles with an English abstract from April 1978 to May 2016. The main data search terms were: Na+/K+-ATPase; estradiol and Na+/K+-ATPase; estradiol, Na+/K+-ATPase and CVS; estradiol, Na+/K+-ATPase and CVD; estradiol, Na+/K+- ATPase and obesity; estradiol, Na+/K+-ATPase and diabetes; estradiol, Na+/K+-ATPase and hypertension; IGF-1; IGF-1 and Na+/K+-ATPase; IGF-1, Na+/K+-ATPase and CVS; IGF-1, Na+/K+-ATPase and CVD; IGF-1, Na+/K+- ATPase and obesity; IGF-1, Na+/K+-ATPase and diabetes; IGF-1, Na+/K+-ATPase and hypertension. The present review discusses the latest data from animal and human studies which focus on the effects of estradiol and IGF-1 on Na+/K+-ATPase regulation in physiological and pathophysiological conditions in cardiovascular system. Understanding the molecular mechanisms of estradiol and IGF-1 action on Na+/K+-ATPase in humans, may help resolving outstanding

  6. Lung surfactant levels are regulated by Ig-Hepta/GPR116 by monitoring surfactant protein D.

    Directory of Open Access Journals (Sweden)

    Taku Fukuzawa

    Full Text Available Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta(+/+ and Ig-Hepta(-/- mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i balanced synthesis of surfactant lipids and proteins and (ii surfactant secretion, and (iii a stimulating effect on recycling (uptake in response to elevated levels of Sp-D in alveolar space.

  7. Saccharomyces cerevisiae vacuolar H+-ATPase regulation by disassembly and reassembly: one structure and multiple signals. (United States)

    Parra, Karlett J; Chan, Chun-Yuan; Chen, Jun


    Vacuolar H(+)-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Regulation of the Plasma Membrane H+-ATPase

    DEFF Research Database (Denmark)

    Falhof, Janus

    The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

  9. Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated (United States)

    Barkla; Vera-Estrella; Maldonado-Gama; Pantoja


    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

  10. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1α. (United States)

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette; Wojtaszewski, Jørgen F P; Richter, Erik A; Juel, Carsten


    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1α are underlying factors in long-term regulation of Na,K-ATPase isoform (α,β and PLM) abundance and Na(+) affinity. Repeated treatment of mice with the AMPK activator AICAR decreased total PLM protein content but increased PLM phosphorylation, whereas the number of α- and β-subunits remained unchanged. The K(m) for Na(+) stimulation of Na,K-ATPase was reduced (higher affinity) after AICAR treatment. PLM abundance was increased in AMPK kinase-dead mice compared with control mice, but PLM phosphorylation and Na,K-ATPase Na(+) affinity remained unchanged. Na,K-ATPase activity and subunit distribution were also measured in mice with different degrees of PGC-1α expression. Protein abundances of α1 and α2 were reduced in PGC-1α +/- and -/- mice, and the β(1)/β(2) ratio was increased with PGC-1α overexpression (TG mice). PLM protein abundance was decreased in TG mice, but phosphorylation status was unchanged. Na,K-ATPase V (max) was decreased in PCG-1α TG and KO mice. Experimentally in vitro induced phosphorylation of PLM increased Na,K-ATPase Na(+) affinity, confirming that PLM phosphorylation is important for Na,K-ATPase function. In conclusion, both AMPK and PGC-1α regulate PLM abundance, AMPK regulates PLM phosphorylation and PGC-1α expression influences Na,K-ATPase α(1) and α(2) content and β(1)/β(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism.

  11. Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity. (United States)

    Bełtowski, Jerzy; Jamroz-Wiśniewska, Anna; Nazar, Jarosław; Wójcicka, Grazyna


    Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.

  12. V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells (United States)

    Jansen, Eric J. R.; Hafmans, Theo G. M.


    The vacuolar (H+)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing. PMID:20702583

  13. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1a

    DEFF Research Database (Denmark)

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette


    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1a are underlying factors in long-te...... influences Na,K-ATPase a(1) and a(2) content and ß(1)/ß(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism....

  14. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)


    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  15. Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma. (United States)

    Xie, Jeffrey X; Zhang, Shungang; Cui, Xiaoyu; Zhang, Jue; Yu, Hui; Khalaf, Fatimah K; Malhotra, Deepak; Kennedy, David J; Shapiro, Joseph I; Tian, Jiang; Haller, Steven T


    Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis.

  16. Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1 (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar


    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

  17. LASS2 inhibits growth and invasion of bladder cancer by regulating ATPase activity (United States)

    Wang, Haifeng; Zuo, Yigang; Ding, Mingxia; Ke, Changxing; Yan, Ruping; Zhan, Hui; Liu, Jingyu; Wang, Wei; Li, Ning; Wang, Jiansong


    Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2) is a novel suppressor of human cancer metastasis, and downregulation of LASS2 has been associated with a poor prognosis in patients with bladder cancer (BC). However, the molecular mechanism underlying LASS2-mediated inhibition of tumor invasion and metastasis in BC remains unclear. LASS2 has been reported to directly bind to subunit C of vacuolar H+-ATPase (V-ATPase) in various types of cancer, suggesting that LASS2 may inhibit cancer invasion and metastasis by regulating the function of V-ATPase. The present study investigated the effect of LASS2-specific small interfering (si)RNA on the invasion and metastasis of the RT4 human BC cell line, which has a low metastatic potential, and its functional interaction with V-ATPase. Silencing of LASS2 in RT4 cells was able to increase V-ATPase activity, the extracellular hydrogen ion concentration and, in turn, the activation of secreted matrix metalloproteinase (MMP)-2 and MMP-9, which occurred simultaneously with enhanced cell proliferation, cell survival and cell invasion in vitro, as well as acceleration of BC growth in vivo. In this process, it was found that siRNA-LASS2 treatment was able to suppress cell apoptosis induced by doxorubicin. These findings suggest that silencing of LASS2 may enhance the growth, invasion and metastasis of BC by regulating ATPase activity. PMID:28356943

  18. Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase

    DEFF Research Database (Denmark)

    Jorgensen, P.L.


    Gaps in our understanding of the complex regulated expression of isozymes of Na,K-ATPase and the diverse systems for posttranslational modification and short term regulation of active Na,K-transport in animals and humans are the main problems in comprehensive Na,K-pump physiology. In mammalian...... genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular...... mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites....

  19. Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells. (United States)

    Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J


    Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448-19457). This study capitalized on the mechanisms suppressing vacuolar H(+)-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C. (United States)

    Bełtowski, Jerzy; Marciniak, Andrzej; Jamroz-Wiśniewska, Anna; Borkowska, Ewelina; Wójcicka, Grazyna


    We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

  1. Oryza sativa H+-ATPase (OSA) is Involved in the Regulation of Dumbbell-Shaped Guard Cells of Rice. (United States)

    Toda, Yosuke; Wang, Yin; Takahashi, Akira; Kawai, Yuya; Tada, Yasuomi; Yamaji, Naoki; Feng Ma, Jian; Ashikari, Motoyuki; Kinoshita, Toshinori


    The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  2. Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase. (United States)

    Song, Jianliang; Zhang, Xue-Qian; Wang, JuFang; Cheskis, Ellina; Chan, Tung O; Feldman, Arthur M; Tucker, Amy L; Cheung, Joseph Y


    Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct

  3. RIN4 functions with plasma membrane H+-ATPases to regulate stomatal apertures during pathogen attack

    DEFF Research Database (Denmark)

    Liu, Jun; Elmore, James M.; Fuglsang, Anja Thoe


    Abstract Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4- mediated immune signal transduction, we...... exhibit differential PM H+-ATPase activity. PM H+-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H+-ATPase activity and, importantly, its...... stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H+-ATPase to regulate stomatal...

  4. Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase

    Directory of Open Access Journals (Sweden)

    Forest H. Andrews


    Full Text Available MORC3 is linked to inflammatory myopathies and cancer; however, the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA, but it is negatively regulated by the CW domain, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing MORC3 functions and suggest that its modulation may contribute to human disease.

  5. Analysis of the regulation of surfactant phosphatidylcholine metabolism using stable isotopes. (United States)

    Brandsma, Joost; Postle, Anthony D


    The pathways and mechanisms that regulate pulmonary surfactant synthesis, processing, secretion and catabolism have been extensively characterised using classical biochemical and analytical approaches. These have constructed a model, largely in experimental animals, for surfactant phospholipid metabolism in the alveolar epithelial cell whereby phospholipid synthesised on the endoplasmic reticulum is selectively transported to lamellar body storage vesicles, where it is subsequently processed before secretion into the alveolus. Surfactant phospholipid is a complex mixture of individual molecular species defined by the combination of esterified fatty acid groups and a comprehensive description of surfactant phospholipid metabolism requires consideration of the interactions between such molecular species. However, until recently, lipid analytical techniques have not kept pace with the considerable advances in understanding of the enzymology and molecular biology of surfactant metabolism. Refinements in electrospray ionisation mass spectrometry (ESI-MS) can now provide very sensitive platforms for the rapid characterisation of surfactant phospholipid composition in molecular detail. The combination of ESI-MS and administration of phospholipid substrates labelled with stable isotopes extends this analytical approach to the quantification of synthesis and turnover of individual molecular species of surfactant phospholipid. As this methodology does not involve radioactivity, it is ideally suited to application in clinical studies. This review will provide an overview of the metabolic processes that regulate the molecular specificity of surfactant phosphatidylcholine together with examples of how the application of stable isotope technologies in vivo has, for the first time, begun to explore regulation of the molecular specificity of surfactant synthesis in human subjects. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. Regulation of Na+/K+ ATPase transport velocity by RNA editing.

    Directory of Open Access Journals (Sweden)

    Claudia Colina


    Full Text Available Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+/K(+ pumps in order to maintain ion homeostasis. In this study we show that Na(+/K(+ pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+/K(+ ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+ release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

  7. A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1

    NARCIS (Netherlands)

    Prattes, M.; Loibl, M.; Zisser, G.; Luschnig, D.; Kappel, L.; Rossler, I.; Grassegger, M.; Hromic, A.; Krieger, E.; Gruber, K.; Pertschy, B.; Bergler, H.


    AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP

  8. Coordinated regulation of cardiac Na(+)/Ca (2+) exchanger and Na (+)-K (+)-ATPase by phospholemman (FXYD1). (United States)

    Cheung, Joseph Y; Zhang, Xue-Qian; Song, Jianliang; Gao, Erhe; Chan, Tung O; Rabinowitz, Joseph E; Koch, Walter J; Feldman, Arthur M; Wang, JuFang


    Phospholemman (PLM) is the founding member of the FXYD family of regulators of ion transport. PLM is a 72-amino acid protein consisting of the signature PFXYD motif in the extracellular N terminus, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail containing three phosphorylation sites. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. The TM domain of PLM interacts with TM9 of the α-subunit of Na(+)-K(+)-ATPase, while its cytoplasmic tail interacts with two small regions (spanning residues 248-252 and 300-304) of the proximal intracellular loop of Na(+)/Ca(2+) exchanger. Under stress, catecholamine stimulation phosphorylates PLM at serine(68), resulting in relief of inhibition of Na(+)-K(+)-ATPase by decreasing K(m) for Na(+) and increasing V(max), and simultaneous inhibition of Na(+)/Ca(2+) exchanger. Enhanced Na(+)-K(+)-ATPase activity lowers intracellular Na(+), thereby minimizing Ca(2+) overload and risks of arrhythmias. Inhibition of Na(+)/Ca(2+) exchanger reduces Ca(2+) efflux, thereby preserving contractility. Thus, the coordinated actions of PLM during stress serve to minimize arrhythmogenesis and maintain inotropy. In acute cardiac ischemia and chronic heart failure, either expression or phosphorylation of PLM or both are altered. PLM regulates important ion transporters in the heart and offers a tempting target for development of drugs to treat heart failure.

  9. Down-regulated Na(+)/K(+)-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats. (United States)

    Huang, Hao; Chen, Yang-Mei; Zhu, Fei; Tang, Shi-Ting; Xiao, Ji-Dong; Li, Lv-Li; Lin, Xin-Jing


    This study was aimed to examine whether the Na(+)/K(+) adenosine triphosphatase (Na(+)/K(+)-ATPase) activity in ischemic penumbra is associated with the pathogenesis of ischemia/reperfusion-induced brain injury. An experimental model of cerebral ischemia/reperfusion was made by transient middle cerebral artery occlusion (tMCAO) in rats and the changes of Na(+)/K(+)-ATPase activity in the ischemic penumbra was examined by Enzyme Assay Kit. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 6 h, 24 h, 48 h, 3 d and 7 d after tMCAO. Enzyme Assay analyses revealed the activity of Na(+)/K(+)-ATPase was decreased in the ischemic penumbra of model rats after focal cerebral ischemia/reperfusion compared with sham-operated rats, and reduced to its minimum at 48 h, while the infarct volume was enlarged gradually. In addition, accompanied by increased brain water content, apoptosis-related bcl-2 and Bax proteins, apoptotic index and neurologic deficits Longa scores, but fluctuated the ratio of bcl-2/Bax. Correlation analysis showed that the infarct volume, apoptotic index, neurologic deficits Longa scores and brain water content were negatively related with Na(+)/K(+)-ATPase activity, while the ratio of bcl-2/Bax was positively related with Na(+)/K(+)-ATPase activity. Our results suggest that down-regulated Na(+)/K(+)-ATPase activity in ischemic penumbra might be involved in the pathogenesis of cerebral ischemia/reperfusion injury presumably through the imbalance ratio of bcl-2/Bax and neuronal apoptosis, and identify novel target for neuroprotective therapeutic intervention in cerebral ischemic disease.

  10. Regulation of the synthesis and assembly of the plant vacuolar H sup + -ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Taiz, L.


    During the past three years we have focused on four main areas: the characterization of the 5{prime}-upstream sequence of the gene for the V-ATPase 70 kDa (A) subunit gene, the generation of V-ATPase-deficient mutants using antisense constructs of the A subunit cDNA, analysis of V-ATPase ultrastructure by negative staining and the characterization of organelle-specific isoforms of the A subunit of carrot. In addition we have extended our studies on the cellular distribution of the V-ATPase and we have continued our investigation of the evolution of the V-ATPases by characterizing the A and B subunits of two species of the archaebacterium, Methanococcus.

  11. Regulation of Na+-K+-ATPase effected high glucose-induced myocardial cell injury through c-Src dependent NADPH oxidase/ROS pathway. (United States)

    Yan, Xiaofei; Xun, Meng; Dou, Xiaojuan; Wu, Litao; Han, Yan; Zheng, Jin


    Depressed Na+/K+-ATPase activity has long been reported to be involved in diabetic-related cardiomyocyte death and cardiac dysfunction. However, the nature of directly regulating Na+-K+-ATPase in diabetic-related myocardial diseases remains unknown. Hyperglycemia is believed as one of major factors responsible for diabetic-related myocardial apoptosis and dysfunction. In this study, whether inhibiting Na+-K+-ATPase by ouabain or activating Na+-K+-ATPase by DRm217 has functions on high glucose (HG) -induced myocardial injury was investigated. Here we found that addition of DRm217 or ouabain to HG-treated cells had opposite effects. DRm217 decreased but ouabain increased HG-induced cell injury and apoptosis. This was mediated by changing Na+-K+-ATPase activity and Na+-K+-ATPase cell surface expression. The inhibition of Na+-K+-ATPase endocytosis alleviated HG-induced ROS accumulation. Na+-K+-ATPase·c-Src dependent NADPH oxidase/ROS pathway was also involved in the effects of ouabain and DRm217 on HG-induced cell injury. These novel results may help us to understand the important role of the Na+-K+-ATPase in diabetic cardiovascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria. (United States)

    Frees, Dorte; Savijoki, Kirsi; Varmanen, Pekka; Ingmer, Hanne


    Clp proteolytic complexes consisting of a proteolytic core flanked by Clp ATPases are widely conserved in bacteria, and their biological roles have received considerable interest. In particular, mutants in the clp genes in the low-GC-content Gram-positive phyla Bacillales and Lactobacillales display a diverse range of phenotypic changes including general stress sensitivity, aberrant cell morphology, failure to initiate developmental programs, and for pathogens, severely attenuated virulence. Extensive research dedicated to unravelling the molecular mechanisms underlying these complex phenotypes has led to fascinating new insights that will be covered by this review. First, Clp ATPases and ClpP-containing proteolytic complexes play indispensable roles in cellular protein quality control systems by refolding or degrading damaged proteins in both stressed and non-stressed cells. Secondly, ClpP proteases and the chaperone activity of Clp ATPases are important for controlling stability and activity of central transcriptional regulators, thereby exerting tremendous impact on cell physiology. Targets include major stress regulators like Spx (oxidative stress), the antisigma factor RsiW (alkaline stress) and HdiR (DNA damage) in addition to regulators of developmental programs like ComK (competence development), sigmaH and Sda (sporulation). Thus, Clp proteins are central in co-ordinating developmental decisions and stress response in low GC Gram-positive bacteria.

  13. Thermogenic activity of the Ca2+-ATPase from blue marlin heater organ: regulation by KCl and temperature. (United States)

    da Costa, Danielly Cristiny Ferraz; Landeira-Fernandez, Ana Maria


    This work shows that vesicles derived from the blue marlin heater organ retain a sarcoplasmic reticulum (SR) Ca(2+)-ATPase that can interconvert different forms of energy. During the hydrolysis of ATP part of the energy is always converted into heat, and the other part can be converted into work (Ca(2+) transport) or heat, depending on the temperature and the presence of KCl in the reaction medium. At 15 degrees C, where KCl stimulates the activity approximately threefold, measurements of the amount of heat released per mole of ATP hydrolyzed (DeltaH(cal)) show similar values (approximately -11 kcal/mol) in the presence or absence of a Ca(2+) gradient. At 25 degrees C, KCl activates the enzyme to the same extent as at 15 degrees C, but inhibits the production of extra heat by SR Ca(2+)-ATPase when a Ca(2+) gradient is built up across the membrane. The DeltaH(cal) values found in the presence of a Ca(2+)-gradient were -26.2 +/- 2.9 kcal/mol (n = 7) in control experiments and -16.1 +/- 1.5 (n = 14) in the presence of 100 mM KCl. At 35 degrees C, KCl has a smaller effect ( approximately 1.5-fold) on activating the enzyme. Similar to SR Ca(2+)-ATPase from mammals, at this temperature the enzyme produces almost twice the amount of heat per mole of ATP hydrolyzed in the presence of a Ca(2+) gradient and KCl has no effect at all on this increment. These data suggest that the marlin SR Ca(2+)-ATPase may play an important role in heater organ thermogenesis and that KCl has the potential for regulating the heat production catalyzed by the enzyme.

  14. Electrostatic Stabilization Plays a Central Role in Autoinhibitory Regulation of the Na+,K+-ATPase

    DEFF Research Database (Denmark)

    Jiang, Qiucen; Garcia, Alvaro; Han, Minwoo


    The Na+,K+-ATPase is present in the plasma membrane of all animal cells. It plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane, which are essential in numerous physiological processes, e.g., nerve, muscle, and kidney function. Its cellular a...

  15. C. pneumoniae CdsL regulates CdsN ATPase activity, and disruption with a peptide mimetic prevents bacterial invasion

    Directory of Open Access Journals (Sweden)

    Chris Blair Stone


    Full Text Available Chlamydiae are obligate intracellular pathogens that likely require type III secretion (T3S to invade cells and replicate intracellulary within a cytoplasmic vacuole called an inclusion body. C. pneumoniae possess a YscL ortholog, CdsL, that has been shown to interact with the T3S ATPase (CdsN. In this report we demonstrate that CdsL down-regulates CdsN enzymatic activity in a dose-dependent manner. Using PepScan epitope mapping we identified two separate binding domains to which CdsL binds viz. CdsN 221-229 and CdsN265-270. We confirmed the binding domains using a pull-down assay and showed that GST-CdsN221-270, which encompasses these peptides, co-purified with His-CdsL. Next, we used orthology modeling based on the crystal structure of a T3S ATPase ortholog from E. coli, EscN, to map the binding domains on the predicted three dimensional structure of CdsN. The CdsL binding domains mapped to the catalytic domain of the ATPase, one in the central channel of the ATPase hexamer and one on the outer face. Since peptide mimetics have been used to disrupt essential protein interactions of the chlamydial T3S system and inhibit T3S-mediated invasion of HeLa cells, we hypothesized that if CdsL – CdsN binding is essential for regulating T3S then a CdsN peptide mimetic could be used to potentially block T3S and Chlamydial invasion. Treatment of EBs with a CdsN peptide mimetic inhibited C. pneumoniae invasion into HeLa cells in a dose-dependent fashion. This report represents the first use of Pepscan technology to identify binding domains for specific T3S proteins viz. CdsL on the ATPase, CdsN, and demonstrates that peptide mimetics can be used as anti-virulence factors to block bacterial invasion.

  16. Isoform specificity of the Na/K-ATPase association and regulation by phospholemman. (United States)

    Bossuyt, Julie; Despa, Sanda; Han, Fei; Hou, Zhanjia; Robia, Seth L; Lingrel, Jerry B; Bers, Donald M


    Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha1 or CFP-NKA-alpha2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha1 and 27.5 +/- 2.5% for NKA-alpha2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha2 versus PLM-NKA-alpha1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving >or=3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha1 is ouabain-sensitive and NKA-alpha2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha1 and NKA-alpha2 function. Isoproterenol enhanced internal Na(+) affinity of both isoforms (K((1/2)) decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mm for NKA-alpha1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mm for NKA-alpha2) without altering maximum transport rate (V(max)). Protein kinase C activation also decreased K((1/2)) for both NKA-alpha1 and NKA-alpha2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mm, respectively) but increased V(max) only for NKA-alpha2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA

  17. Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+ -ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

    DEFF Research Database (Denmark)

    Madsen, Steffen; Jensen, Lars Nørholm; Tipsmark, Christian Kølbaek


    Effects of salinity and hormones on cystic fibrosis transmembrane conductance regulator (CFTR) and alpha-subunit Na(+),K(+) -ATPase (alpha-NKA) mRNA (analysed by semi-quantitative PCR) and protein expression (analysed by western blotting and immunocytochemistry) were investigated in gills...... filaments at equal intensity. Cortisol decreased serum [Na(+)] in FW fish, but had no effect on gill Na(+),K(+) -ATPase activity, alpha-NKA and CFTR mRNA levels. Incubation of gill tissue with cortisol (24 h, >0.01 micro g/ml) and epidermal growth factor (EGF 10 micro g/ml) decreased CFTR mRNA levels......-regulated kinase (ERK) 1/2 was stimulated by EGF but not affected by IGF-I. This study is the first to report a branchial EGF response and to demonstrate a functional ERK 1/2 pathway in the teleost gill. In conclusion, CFTR and Na(+),K(+) -ATPase are differentially regulated by salinity and hormones in gills...


    Stam, Nicholas J; Wilkens, Stephan


    Eukaryotic vacuolar H + -ATPase (V-ATPase) is a multisubunit enzyme complex that acidifies subcellular organelles and the extracellular space. V-ATPase consists of soluble V 1 -ATPase and membrane-integral V o proton channel sectors. To investigate the mechanism of V-ATPase regulation by reversible disassembly, we recently determined a cryo-EM reconstruction of yeast V o The structure indicated that, when V 1 is released from V o , the N-terminal cytoplasmic domain of subunit a (a NT ) changes conformation to bind rotor subunit d However, insufficient resolution precluded a precise definition of the a NT -d interface. Here we reconstituted V o into lipid nanodiscs for single-particle EM. 3D reconstructions calculated at ∼15-Å resolution revealed two sites of contact between a NT and d that are mediated by highly conserved charged residues. Alanine mutagenesis of some of these residues disrupted the a NT -d interaction, as shown by isothermal titration calorimetry and gel filtration of recombinant subunits. A recent cryo-EM study of holo V-ATPase revealed three major conformations corresponding to three rotational states of the central rotor of the enzyme. Comparison of the three V-ATPase conformations with the structure of nanodisc-bound V o revealed that V o is halted in rotational state 3. Combined with our prior work that showed autoinhibited V 1 -ATPase to be arrested in state 2, we propose a model in which the conformational mismatch between free V 1 and V o functions to prevent unintended reassembly of holo V-ATPase when activity is not needed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Regulation of hepatic Na+/K+-ATPase in obese female and male rats: involvement of ERK1/2, AMPK, and Rho/ROCK. (United States)

    Stanimirovic, Julijana; Obradovic, Milan; Panic, Anastasija; Petrovic, Voin; Alavantic, Dragan; Melih, Irena; Isenovic, Esma R


    In this study, we assessed whether the disturbed regulation of sodium/potassium-adenosine-triphosphatase (Na+/K+-ATPase) occurs as a consequence of obesity-induced IR in sex-specific manner. We also assessed whether alterations of IRS/PI3K/Akt, ERK1/2, AMPKα, and RhoA/ROCK signaling cascades have an important role in this pathology. Female and male Wistar rats (150-200 g, 8 weeks old) were fed a standard laboratory diet or a high-fat (HF) diet (42% fat) for 10 weeks. The activity of hepatic Na+/K+-ATPase and Rho, and the association of IRS-1/p85 were assessed in liver. Furthermore, the protein level of α1 Na+/K+-ATPase in plasma membrane fractions, and protein levels of IRS-1, PI3K-p85, -p110, RhoA, ROCK1, ROCK2, ERK1/2, AMPKα, ERα, and ERβ in liver lysates were assessed. The expression of hepatic α1 Na+/K+-ATPase mRNA was also analyzed by qRT-PCR. The results show that HF-fed female rats exhibited an increase in hepatic ERK1/2 (p K+-ATPase α1 mRNA, decreased level of Na+/K+-ATPase activity (p K+-ATPase protein expression (p K+-ATPase activity (p K+-ATPase mRNA expression and activation of ERK1/2, AMPKα, and Rho in the liver. Exploring the sex-specific factors and pathways that promote obesity-related diseases may lead to a better understanding of pathogenesis and discovering new therapeutic targets.

  20. Vesicles from pH-regulated reversible gemini amino-acid surfactants as nanocapsules for delivery. (United States)

    Lv, Jing; Qiao, Weihong; Li, Zongshi


    Reversible transition from micelles to vesicles by regulating pH were realized by gemini amino-acid surfactants N,N'-dialkyl-N,N'-diacetate ethylenediamine. Measurement results of ζ-potential at different pH and DLS at varying solvents revealed that the protonation between H(+) and double NCH2COO(-) groups (generating NH(+)CH2COO(-)), expressed as pKa1 and pKa2, is the key driving force to control the aggregation behaviors of gemini surfactant molecule. Effect of pH on the bilayer structure was studied in detail by using steady-state fluorescence spectroscopy of hydrophobic pyrene and Coumarin 153 (C153) respectively and fluorescence resonance energy transfer (FRET) from C153 to Rhodamine 6G (R6G). Various pH-regulated and pH-reversible self-assemblies were obtained in one surfactant system. Vitamin D3 was encapsulated in vesicle bilayers to form nano-VD3-capsules as VD3 supplement agent for health care products. By using the electrostatic attraction between Ca(2+) and double -COO(-) groups, nano-VD3-capsules with Ca(2+) coated outermost layers were prepared as a formulation for VD3 and calcium co-supplement agent. DLS and TEM were performed to check stability and morphology of the nano-capsules. It is concluded that the pH-regulated gemini amino-acid surfactants can be used to construct colloidal systems for delivering hydrophobic drugs or nutritions without lipids at human physiological pH level. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Volume regulation of intestinal cells of echinoderms: Putative role of ion transporters (Na(+)/K(+)-ATPase and NKCC). (United States)

    Castellano, Giovanna C; Souza, Marta M; Freire, Carolina A


    Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na(+)/K(+)-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na(+)/K(+)-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase. (United States)

    Sasaki, Tomoyuki; Lian, Shanshan; Khan, Alam; Llop, Jesse R; Samuelson, Andrew V; Chen, Wenbiao; Klionsky, Daniel J; Kishi, Shuji


    Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H+-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H+-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H+ transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.

  3. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments (United States)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul


    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  4. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. (United States)

    Abu-Remaileh, Monther; Wyant, Gregory A; Kim, Choah; Laqtom, Nouf N; Abbasi, Maria; Chan, Sze Ham; Freinkman, Elizaveta; Sabatini, David M


    The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H+-adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  5. Extracellular Na+ levels regulate formation and activity of the NaX/alpha1-Na+/K+-ATPase complex in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Emmanuelle eBerret


    Full Text Available MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na+]out. The mechanism underlying Na+-sensing involves Na+-flow through the NaX channel, directly regulated by the Na+/K+-ATPase α1-isoform which controls Na+-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na+]in. Here we aim to determine whether environmental changes in Na+ could actively modulate the NaX/Na+/K+-ATPase complex activity.We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na+] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na+] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na+/K+-ATPase remained unaltered. This unbalance between NaX and Na+/K+-ATPase pump proportion would induce a bigger portion of Na+/K+-ATPase-control-free NaX channel. Thus we suggest that hypernatremic environment increases NaX/Na+/K+-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.

  6. The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair (United States)

    Xu, Ye; Sun, Yingli; Jiang, Xiaofeng; Ayrapetov, Marina K.; Moskwa, Patryk; Yang, Shenghong; Weinstock, David M.


    The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage. PMID:20876283

  7. Loop 2 in Saccharomyces cerevisiae Rad51 protein regulates filament formation and ATPase activity. (United States)

    Zhang, Xiao-Ping; Galkin, Vitold E; Yu, Xiong; Egelman, Edward H; Heyer, Wolf-Dietrich


    Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 protein increases the interaction with Rad54 protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 A, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 A pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable protein-dsDNA complexes. The mutant protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.

  8. Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens

    Directory of Open Access Journals (Sweden)

    Seema Mishra


    Full Text Available P1B-ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri. Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd2+ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn2+ and toxic Cd2+, but not at deficient Zn2+ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4. Further, in shoots NcHMA4 was more abundant in 10 μM Zn2+ and AhHMA4 in Zn2+ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn2+ when compared to replete Zn2+ and toxic Cd2+ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn2+ supply, but were up-regulated by 10 μM Cd2+. Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri, both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens, mesophyll in A. halleri.

  9. Salt regulation of transcript levels for the c subunit of a leaf vacuolar H(+)-ATPase in the halophyte Mesembryanthemum crystallinum. (United States)

    Tsiantis, M S; Bartholomew, D M; Smith, J A


    The halophyte Mesembryanthemum crystallinum is an inducible crassulacean acid metabolism (CAM) plant native to seasonally arid coastal environments that has been widely used to study plant responses to environmental stress. On exposure of plants to salt, the activities of both the tonoplast (vacuolar) H(+)-ATPase (V-ATPase) and Na+/H+ antiporter increase in leaf cells, thereby energizing vacuolar salt accumulation. To investigate the molecular basis of this response, a cDNA (Vmac1) encoding the H(+)-conducting c subunit (16.6 kDa) of an M. crystallinum V-ATPase has been cloned. Northern analysis of RNA from leaves of plants treated with NaCl or with isoosmotic mannitol solutions demonstrated (i) that NaCl increased steady-state transcript levels for the V-ATPase c subunit, and (ii) that this effect was caused by the ionic rather than the osmotic component of salt stress. Southern analysis of genomic DNA suggested the probable existence of more than one gene for this subunit of the V-ATPase in M. crystallinum. Expression studies using the 3'-untranslated region of the Vmac1 cDNa as a probe showed that the corresponding salt-inducible transcript was preferentially expressed in leaves. Induction by salt was also observed in juvenile plants in addition to adult ones. These findings, as well as the inability of mannitol to upregulate mRNA levels for this gene, clearly differentiate between the induction of transcript for the V-ATPase c subunit and for genes involved in the CAM pathway in M. crystallinum. Further, the plant growth regulator abscisic acid (ABA) was able to mimic the effect of salt on transcript levels for the V-ATPase c subunit, suggesting the possible involvement of ABA in a distinct signal-transduction pathway linked to vacuolar salt accumulation in this highly salt-tolerant species.

  10. Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide. (United States)

    Silva, Elias C C; Masui, Douglas C; Furriel, Rosa P; McNamara, John C; Barrabin, Hector; Scofano, Helena M; Perales, Jonas; Teixeira-Ferreira, André; Leone, Francisco A; Fontes, Carlos Frederico L


    This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. A Surfactant-Induced Functional Modulation of a Global Virulence Regulator from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Sukhendu Mandal

    Full Text Available Triton X-100 (TX-100, a useful non-ionic surfactant, reduced the methicillin resistance in Staphylococcus aureus significantly. Many S. aureus proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in S. aureus. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant S. aureus SarA (rSarA and its derivative (C9W have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future.

  12. ATPase activity tightly regulates RecA nucleofilaments to promote homologous recombination. (United States)

    Zhao, Bailin; Zhang, Dapeng; Li, Chengmin; Yuan, Zheng; Yu, Fangzhi; Zhong, Shangwei; Jiang, Guibin; Yang, Yun-Gui; Le, X Chris; Weinfeld, Michael; Zhu, Ping; Wang, Hailin


    Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange in vitro and HR in vivo. RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR in vitro and in vivo, but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.

  13. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita


    We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3'-diethylo......We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3......,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol...... in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP...

  14. Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression (United States)

    Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A.; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A.; Barrett, Timothy G.


    Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca2+ imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca2+ concentration ([Ca2+]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis. PMID:25274773

  15. Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

    Directory of Open Access Journals (Sweden)

    Mi Young Yang


    Full Text Available GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

  16. Unaccustomed eccentric contractions impair plasma K+ regulation in the absence of changes in muscle Na+,K+-ATPase content.

    Directory of Open Access Journals (Sweden)

    Craig A Goodman

    Full Text Available The Na+,K+-ATPase (NKA plays a fundamental role in the regulation of skeletal muscle membrane Na+ and K+ gradients, excitability and fatigue during repeated intense contractions. Many studies have investigated the effects of acute concentric exercise on K+ regulation and skeletal muscle NKA, but almost nothing is known about the effects of repeated eccentric contractions. We therefore investigated the effects of unaccustomed maximal eccentric knee extensor contractions on K+ regulation during exercise, peak knee extensor muscle torque, and vastus lateralis muscle NKA content and 3-O-MFPase activity. Torque measurements, muscle biopsies, and venous blood samples were taken before, during and up to 7 days following the contractions in six healthy adults. Eccentric contractions reduced peak isometric muscle torque immediately post-exercise by 26±11% and serum creatine kinase concentration peaked 24 h post-exercise at 339±90 IU/L. During eccentric contractions, plasma [K+] rose during Set 1 and remained elevated at ∼4.9 mM during sets 4-10; this was despite a decline in work output by Set 4, which fell by 18.9% at set 10. The rise in plasma [K+] x work(-1 ratio was elevated over Set 2 from Set 4- Set 10. Eccentric contractions had no effect on muscle NKA content or maximal in-vitro 3-O-MFPase activity immediately post- or up to 7 d post-exercise. The sustained elevation in plasma [K+] despite a decrease in work performed by the knee extensor muscles suggests an impairment in K+ regulation during maximal eccentric contractions, possibly due to increased plasma membrane permeability or to excitation-contraction uncoupling.

  17. Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae). (United States)

    Silva, E C C; Masui, D C; Furriel, R P M; Mantelatto, F L M; McNamara, J C; Barrabin, H; Leone, F A; Scofano, H M; Fontes, C F L


    Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine>spermidine>putrescine. Spermidine affected K(0.5) values for Na(+) with minor alterations in K(0.5) values for K(+) and NH(4)(+), causing a decrease in maximal velocities under saturating Na(+), K(+) and NH(4)(+) concentrations. Phosphorylation measurements in the presence of 20 microM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na(+), both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na(+), the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na(+) at the Na(+)-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.

  18. Citrate Secretion Coupled with the Modulation of Soybean Root Tip under Aluminum Stress. Up-Regulation of Transcription, Translation, and Threonine-Oriented Phosphorylation of Plasma Membrane H+-ATPase1 (United States)

    Shen, Hong; He, Long Fei; Sasaki, Takayuki; Yamamoto, Yoko; Zheng, Shao Jian; Ligaba, Ayalew; Yan, Xiao Long; Ahn, Sung Ju; Yamaguchi, Mineo; Sasakawa, Hideo; Matsumoto, Hideaki


    The aluminum (Al)-induced secretion of citrate has been regarded as an important mechanism for Al resistance in soybean (Glycine max). However, the mechanism of how Al induces citrate secretion remains unclear. In this study, we investigated the regulatory role of plasma membrane H+-ATPase on the Al-induced secretion of citrate from soybean roots. Experiments performed with plants grown in full nutrient solution showed that Al-induced activity of plasma membrane H+-ATPase paralleled secretion of citrate. Vanadate and fusicoccin, an inhibitor and an activator, respectively, of plasma membrane H+-ATPase, exerted inhibitory and stimulatory effects on the Al-induced secretion of citrate. Higher activity of plasma membrane H+-ATPase coincided with more citrate secretion in Al-resistant than Al-sensitive soybean cultivars. These results suggested that the effects of Al stress on citrate secretion were mediated via modulation of the activity of plasma membrane H+-ATPase. The relationship between the Al-induced secretion of citrate and the activity of plasma membrane H+-ATPase was further demonstrated by analysis of plasma membrane H+-ATPase transgenic Arabidopsis (Arabidopsis thaliana). When plants were grown on Murashige and Skoog medium containing 30 μm Al (9.1 μm Al3+ activity), transgenic plants exuded more citrate compared with wild-type Arabidopsis. Results from real-time reverse transcription-PCR and immunodetection analysis indicated that the increase of plasma membrane H+-ATPase activity by Al is caused by transcriptional and translational regulation. Furthermore, plasma membrane H+-ATPase activity and expression were higher in an Al-resistant cultivar than in an Al-sensitive cultivar. Al activated the threonine-oriented phosphorylation of plasma membrane H+-ATPase in a dose- and time-dependent manner. Taken together, our results demonstrated that up-regulation of plasma membrane H+-ATPase activity was associated with the secretion of citrate from soybean roots

  19. β-Adrenergic regulation of the cardiac Na+-K+ ATPase mediated by oxidative signaling

    DEFF Research Database (Denmark)

    Galougahi, Keyvan Karimi; Liu, Chia-Chi; Bundgaard, Henning


    Activation of β-adrenergic receptors (ARs) elicits responses arising from protein kinase A (PKA)-mediated phosphorylation of target proteins that regulate Ca(2+)-dependent excitation-contraction coupling. Some important targets for β-AR- and PKA-dependent pathways, including the sarcolemmal Na...... and provides mechanistic explanation for the efficacy of β-AR blockers in heart failure in which raised intracellular Na(+) levels are detrimental-an explanation not provided by traditionally held views on β-AR-mediated regulation of the pump function....

  20. 21-Benzylidene digoxin: a proapoptotic cardenolide of cancer cells that up-regulates Na,K-ATPase and epithelial tight junctions.

    Directory of Open Access Journals (Sweden)

    Sayonarah C Rocha

    Full Text Available Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD, and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1 up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2 cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3 changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.

  1. Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes. (United States)

    Cardouat, G; Duparc, T; Fried, S; Perret, B; Najib, S; Martinez, L O


    Ecto-F 1 -ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F 1 -ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y 13 -mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F 1 -ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F 1 -ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F 1 -ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis. Copyright © 2017. Published by Elsevier B.V.

  2. Regulation of Na(+)/K(+)-ATPase by nuclear respiratory factor 1: implication in the tight coupling of neuronal activity, energy generation, and energy consumption. (United States)

    Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T


    NRF-1 regulates mediators of neuronal activity and energy generation. NRF-1 transcriptionally regulates Na(+)/K(+)-ATPase subunits α1 and β1. NRF-1 functionally regulates mediators of energy consumption in neurons. NRF-1 mediates the tight coupling of neuronal activity, energy generation, and energy consumption at the molecular level. Energy generation and energy consumption are tightly coupled to neuronal activity at the cellular level. Na(+)/K(+)-ATPase, a major energy-consuming enzyme, is well expressed in neurons rich in cytochrome c oxidase, an important enzyme of the energy-generating machinery, and glutamatergic receptors that are mediators of neuronal activity. The present study sought to test our hypothesis that the coupling extends to the molecular level, whereby Na(+)/K(+)-ATPase subunits are regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), found recently by our laboratory to regulate all cytochrome c oxidase subunit genes and some NMDA and AMPA receptor subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutational analysis, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Atp1a1 and Atp1b1 genes but not of the Atp1a3 gene in neurons. The transcripts of Atp1a1 and Atp1b1 subunit genes were up-regulated by KCl and down-regulated by tetrodotoxin. Atp1b1 is positively regulated by NRF-1, and silencing of NRF-1 with small interference RNA blocked the up-regulation of Atp1b1 induced by KCl, whereas overexpression of NRF-1 rescued these transcripts from being suppressed by tetrodotoxin. On the other hand, Atp1a1 is negatively regulated by NRF-1. The binding sites of NRF-1 on Atp1a1 and Atp1b1 are conserved among mice, rats, and humans. Thus, NRF-1 regulates key Na(+)/K(+)-ATPase subunits and plays an important role in mediating the tight coupling between

  3. Helicobacter pylori-induced posttranscriptional regulation of H-K-ATPase α-subunit gene expression by miRNA. (United States)

    Zhang, Yong-Mei; Noto, Jennifer M; Hammond, Charles E; Barth, Jeremy L; Argraves, W Scott; Backert, Steffen; Peek, Richard M; Smolka, Adam J


    Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.

  4. Rotary ATPases (United States)

    Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela


    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

  5. Regulation of branchial V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias)

    National Research Council Canada - National Science Library

    Tresguerres, Martin; Katoh, Fumi; Fenton, Heather; Jasinska, Edyta; Goss, Greg G


    To study the mechanisms of branchial acid-base regulation, Pacific spiny dogfish were infused intravenously for 24 h with either HCl (495+/- 79 micromol kg(-1) h(-1)) or NaHCO(3) (981+/-235 micromol kg(-1) h(-1...

  6. The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C. (United States)

    Duran, Marie-Josée; Pierre, Sandrine V; Carr, Deborah L; Pressley, Thomas A


    Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).

  7. Function and evolution of a MicroRNA that regulates a Ca2+-ATPase and triggers the formation of phased small interfering RNAs in tomato reproductive growth. (United States)

    Wang, Ying; Itaya, Asuka; Zhong, Xuehua; Wu, Yang; Zhang, Jianfeng; van der Knaap, Esther; Olmstead, Richard; Qi, Yijun; Ding, Biao


    MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca(2+)-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca(2+)-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species.

  8. Sarco(endoplasmic Reticulum Ca2+-ATPase-2 Gene: Structure and Transcriptional Regulation of the Human Gene

    Directory of Open Access Journals (Sweden)

    Angel Zarain-Herzberg


    Full Text Available The sarco(endoplasmic reticulum Ca2+-ATPases (SERCAs belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5’ -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE. There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5’-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes.

  9. Regulation of pulmonary surfactant secretion in the developing lizard, Pogona vitticeps. (United States)

    Sullivan, Lucy C; Orgeig, Sandra; Daniels, Christopher B


    Pulmonary surfactant is a mixture of lipids and proteins that is secreted by alveolar type II cells in the lungs of all air-breathing vertebrates. Pulmonary surfactant functions to reduce the surface tension in the lungs and, therefore, reduce the work of breathing. In mammals, the embryonic maturation of the surfactant system is controlled by a host of factors, including glucocorticoids, thyroid hormones and autonomic neurotransmitters. We have used a co-culture system of embryonic type II cells and lung fibroblasts to investigate the ability of dexamethasone, tri-iodothyronine (T(3)), adrenaline and carbamylcholine (carbachol) to stimulate the cellular secretion of phosphatidylcholine in the bearded dragon (Pogona vitticeps) at day 55 (approx. 92%) of incubation and following hatching. Adrenaline stimulated surfactant secretion both before and after hatching, whereas carbachol stimulated secretion only at day 55. Glucocorticoids and triiodothyronine together stimulated secretion at day 55 but did not after hatching. Therefore, adrenaline, carbachol, dexamethasone and T(3), are all involved in the development of the surfactant system in the bearded dragon. However, the efficacy of the hormones is attenuated during the developmental process. These differences probably relate to the changes in the cellular environment during development and the specific biology of the bearded dragon.

  10. Analysis of Conditional Paralytic Mutants in Drosophila Sarco-Endoplasmic Reticulum Calcium ATPase Reveals Novel Mechanisms for Regulating Membrane Excitability


    Sanyal, S.; Consoulas, C.; Kuromi, H.; Basole, A.; Mukai, L.; Kidokoro, Y.; Krishnan, K. S.; Ramaswami, M.


    Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a gene...

  11. Developmental regulation of chicken surfactant protein A and its localization in lung

    DEFF Research Database (Denmark)

    Zhang, Weidong; Cuperus, Tryntsje; van Dijk, Albert


    Surfactant Protein A (SP-A) is a collagenous C-type lectin (collectin) that plays an important role in the early stage of the host immune response. In chicken, SP-A (cSP-A) is expressed as a 26 kDa glycosylated protein in the lung. Using immunohistochemistry, cSP-A protein was detected mainly...

  12. Leptin and the Regulation of Renal Sodium Handling and Renal Na-Transporting ATPases: Role in the Pathogenesis of Arterial Hypertension. (United States)

    Bełtowski, Jerzy


    Leptin, an adipose tissue hormone which regulates food intake, is also involved in the pathogenesis of arterial hypertension. Plasma leptin concentration is increased in obese individuals. Chronic leptin administration or transgenic overexpression increases blood pressure in experimental animals, and some studies indicate that plasma leptin is elevated in hypertensive subjects independently of body weight. Leptin has a dose- and time-dependent effect on urinary sodium excretion. High doses of leptin increase Na(+) excretion in the short run; partially by decreasing renal Na(+),K(+)-ATPase (sodium pump) activity. This effect is mediated by phosphatidylinositol 3-kinase (PI3K) and is impaired in animals with dietary-induced obesity. In contrast to acute, chronic elevation of plasma leptin to the level observed in patients with the metabolic syndrome impairs renal Na(+) excretion, which is associated with the increase in renal Na(+),K(+)-ATPase activity. This effect results from oxidative stress-induced deficiency of nitric oxide and/or transactivation of epidermal growth factor receptor and subsequent stimulation of extracellular signal-regulated kinases. Ameliorating "renal leptin resistance" or reducing leptin level and/or leptin signaling in states of chronic hyperleptinemia may be a novel strategy for the treatment of arterial hypertension associated with the metabolic syndrome.

  13. FXYD5 (dysadherin) may mediate metastatic progression through regulation of the β-Na+-K+-ATPase subunit in the 4T1 mouse breast cancer model. (United States)

    Lubarski-Gotliv, Irina; Dey, Kuntal; Kuznetsov, Yuri; Kalchenco, Vecheslav; Asher, Carol; Garty, Haim


    FXYD5 is a Na+-K+-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5-specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5-related morphological changes are mediated through its interaction with Na+-K+-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression may downregulate the β1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5-expressing cells may correlate with matrix metalloproteinase 9 secretion and adhesion changes in 4T1 wild-type cells. Copyright © 2017 the American Physiological Society.

  14. The Interplay of Cofactor Interactions and Post-translational Modifications in the Regulation of the AAA+ ATPase p97 (United States)

    Hänzelmann, Petra; Schindelin, Hermann


    The hexameric type II AAA ATPase (ATPase associated with various activities) p97 (also referred to as VCP, Cdc48, and Ter94) is critically involved in a variety of cellular activities including pathways such as DNA replication and repair which both involve chromatin remodeling, and is a key player in various protein quality control pathways mediated by the ubiquitin proteasome system as well as autophagy. Correspondingly, p97 has been linked to various pathophysiological states including cancer, neurodegeneration, and premature aging. p97 encompasses an N-terminal domain, two highly conserved ATPase domains and an unstructured C-terminal tail. This enzyme hydrolyzes ATP and utilizes the resulting energy to extract or disassemble protein targets modified with ubiquitin from stable protein assemblies, chromatin and membranes. p97 participates in highly diverse cellular processes and hence its activity is tightly controlled. This is achieved by multiple regulatory cofactors, which either associate with the N-terminal domain or interact with the extreme C-terminus via distinct binding elements and target p97 to specific cellular pathways, sometimes requiring the simultaneous association with more than one cofactor. Most cofactors are recruited to p97 through conserved binding motifs/domains and assist in substrate recognition or processing by providing additional molecular properties. A tight control of p97 cofactor specificity and diversity as well as the assembly of higher-order p97-cofactor complexes is accomplished by various regulatory mechanisms, which include bipartite binding, binding site competition, changes in oligomeric assemblies, and nucleotide-induced conformational changes. Furthermore, post-translational modifications (PTMs) like acetylation, palmitoylation, phosphorylation, SUMOylation, and ubiquitylation of p97 have been reported which further modulate its diverse molecular activities. In this review, we will describe the molecular basis of p97

  15. Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations

    DEFF Research Database (Denmark)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena


    cellular acidification during KCl-stimulated Ca2+ influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu......+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient......Plasma membrane Ca2+-ATPase (PMCA) by extruding Ca2+ outside the cell, actively participates in the regulation of intracellular Ca2+ concentration. Acting as Ca2+/H+ counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four...

  16. A specific phospholipase C activity regulates phosphatidylinositol levels in lung surfactant of patients with acute respiratory distress syndrome. (United States)

    Spyridakis, Spyros; Leondaritis, George; Nakos, George; Lekka, Marilena E; Galanopoulou, Dia


    Lung surfactant (LS) is a lipid-rich material lining the inside of the lungs. It reduces surface tension at the liquid/air interface and thus, it confers protection of the alveoli from collapsing. The surface-active component of LS is dipalmitoyl-phosphatidylcholine, while anionic phospholipids such as phosphatidylinositol (PtdIns) and primarily phosphatidylglycerol are involved in the stabilization of the LS monolayer. The exact role of PtdIns in this system is not well-understood; however, PtdIns levels change dramatically during the acute respiratory distress syndrome (ARDS) evolution. In this report we present evidence of a phosphoinositide-specific phospholipase C (PI-PLC) activity in bronchoalveolar lavage (BAL) fluid, which may regulate PtdIns levels. Characterization of this extracellular activity showed specificity for PtdIns and phosphatidylinositol 4,5-bisphosphate, sharing the typical substrate concentration-, pH-, and calcium-dependencies with mammalian PI-PLCs. Fractionation of BAL fluid showed that PI-PLC did not co-fractionate with large surfactant aggregates, but it was found mainly in the soluble fraction. Importantly, analysis of BAL samples from control subjects and from patients with ARDS showed that the PI-PLC specific activity was decreased by 4-fold in ARDS samples concurrently with the increase in BAL PtdIns levels. Thus, we have identified for the first time an extracellular PI-PLC enzyme activity that may be acutely involved in the regulation of PtdIns levels in LS.

  17. Iron oxide nanoparticles to an Indian major carp, Labeo rohita: Impacts on hematology, iono regulation and gill Na+/K+ ATPase activity

    Directory of Open Access Journals (Sweden)

    Anand Sadanandan Remya


    Full Text Available In this study, the chronic toxicity effects of iron oxide (Fe2O3 nanoparticles (NPs (500 mg l−l on certain hematological, ionoregulatory and gill Na+/K+ ATPase activity of an Indian major carp, Labeo rohita were estimated for a period of 25 days under static bioassay. A significant increase in hemoglobin (Hb content, red blood cell (RBC count and hematocrit (Ht value was noticed throughout the study period when compared to control groups. In contrast, mean cellular volume (MCV, mean cellular hemoglobin (MCH (except on 5th day and mean cellular hemoglobin concentration (MCHC levels and white blood cell (WBC counts were found to be decreased during the above study period. Fe2O3 NPs also caused alterations in iono regulation resulting in hyponatremia (Na+, hypochloremia (Cl− (except on 5th day and hypokalemia (K+ (except up to 15th day. A biphasic trend in gill Na+/K+-ATPase activity was noticed during the above treatment period. Our results demonstrate that high Fe2O3 NP concentrations in the aquatic environment may have adverse physiological effects on fish. These data may be useful to assess the environmental risk posed by NPs. However the toxicity of various sizes of the nanoparticle could be evaluated using different aquatic organisms.

  18. Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells. (United States)

    Hadri, Lahouaria; Pavoine, Catherine; Lipskaia, Larissa; Yacoubi, Sabrina; Lompré, Anne-Marie


    Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1-3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration.

  19. Diseases of Pulmonary Surfactant Homeostasis (United States)

    Whitsett, Jeffrey A.; Wert, Susan E.; Weaver, Timothy E.


    Advances in physiology and biochemistry have provided fundamental insights into the role of pulmonary surfactant in the pathogenesis and treatment of preterm infants with respiratory distress syndrome. Identification of the surfactant proteins, lipid transporters, and transcriptional networks regulating their expression has provided the tools and insights needed to discern the molecular and cellular processes regulating the production and function of pulmonary surfactant prior to and after birth. Mutations in genes regulating surfactant homeostasis have been associated with severe lung disease in neonates and older infants. Biophysical and transgenic mouse models have provided insight into the mechanisms underlying surfactant protein and alveolar homeostasis. These studies have provided the framework for understanding the structure and function of pulmonary surfactant, which has informed understanding of the pathogenesis of diverse pulmonary disorders previously considered idiopathic. This review considers the pulmonary surfactant system and the genetic causes of acute and chronic lung disease caused by disruption of alveolar homeostasis. PMID:25621661

  20. Lung Surfactant Protein D (SP-D) Response and Regulation During Acute and Chronic Lung Injury

    DEFF Research Database (Denmark)

    Gaunsbaek, Maria Quisgaard; Rasmussen, Karina Juhl; Beers, Michael F.


    BACKGROUND: Surfactant protein D (SP-D) is a collection that plays important roles in modulating host defense functions and maintaining phospholipid homeostasis in the lung. The aim of current study was to characterize comparatively the SP-D response in bronchoalveolar lavage (BAL) and serum...... in three murine models of lung injury, using a validated ELISA technology for estimation of SP-D levels. METHODS: Mice were exposed to lipopolysaccharide, bleomycin, or Pneumocystis carinii (Pc) and sacrificed at different time points. RESULTS: In lipopolysaccharide-challenged mice, the level of SP...

  1. Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY

    NARCIS (Netherlands)

    Ridder, Ivo S.; Dijkstra, Bauke W.


    The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and L-2-haloacid dehalogenases. A comparison of the three-dimensional structure of L-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the

  2. Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that is expressed in shoot hairs and floral organs. (United States)

    Tyagi, Wricha; Rajagopal, Divya; Singla-Pareek, Sneh Lata; Reddy, Malireddy K; Sopory, Sudhir K


    We have cloned and characterized the cDNA, genomic clone and upstream promoter region of a vacuolar ATPase (V-ATPase) c subunit (PgVHA-c1) from Pennisetum glaucum. The deduced amino acid sequence shows 98-71% sequence identity with V-ATPase from rice and Arabidopsis, and is a highly hydrophobic protein with four transmembrane regions. PgVHA-c1-GFP fusion protein is expressed in BY2 cells on the endo-membranes surrounding vacuoles; however, PgVHA-c1 could not functionally complement V-ATPase-c deletion mutants of yeast. The sequence analysis of the genomic clone revealed the presence of two introns in the coding region, and the splice junctions followed the typical canonical GU-AG consensus sequence. The transcript analysis showed that the expression of PgVHA-c1 was stimulated more in response to salinity stress and very marginally in response to drought and low temperature stress. Exogenous application of abscisic acid, salicylic acid and calcium stimulated the transcript level in the absence of stress. We have cloned the 5'-flanking regions of PgVHA-c1 and mapped its transcript start site at 78 bp upstream of ATG. Transgenic tobacco with promoter::GUS constructs showed that the region -288/+78 was sufficient for GUS expression. The expression of the reporter gene even with the full-length promoter was limited to shoot hairs and to male and female reproductive organs. The dehydration-responsive element (DRE) and ABA-responsive element (ABRE) in the promoter did not show consensus flanking regions; however, gel mobility shift assays showed that Pennisetum has specific transacting factors that showed binding to the core DRE, ABRE and TCA elements.

  3. ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1. (United States)

    von Blume, Julia; Alleaume, Anne-Marie; Cantero-Recasens, Gerard; Curwin, Amy; Carreras-Sureda, Amado; Zimmermann, Timo; van Galen, Josse; Wakana, Yuichi; Valverde, Miguel Angel; Malhotra, Vivek


    Actin-severing proteins ADF/cofilin are required for the sorting of secretory cargo at the trans-Golgi network (TGN) in mammalian cells. How do these cytoplasmic proteins interact with the cargoes in the lumen of the TGN? Put simply, how are these two sets of proteins connected across the TGN membrane? Mass spectrometry of cofilin1 immunoprecipitated from HeLa cells revealed the presence of actin and the Ca(2+) ATPase SPCA1. Moreover, cofilin1 was localized to the TGN and bound to SPCA1 via dynamic actin. SPCA1 knockdown, like ADF/cofilin1 knockdown, inhibited Ca(2+) uptake into the TGN and caused missorting of secretory cargo. These defects were rescued by the overexpression of the TGN-localized SPCA1. We propose that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca(2+). Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Structure/function analysis of Na(+)-K(+)-ATPase central isoform-specific region: involvement in PKC regulation. (United States)

    Pierre, Sandrine V; Duran, Marie-Josée; Carr, Deborah L; Pressley, Thomas A


    Specific functions served by the various Na(+)-K(+)-ATPase alpha-isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH(2) terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH(2) terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat alpha(1)- and alpha(2)-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K(+) deocclusion pattern and their response to PKC activation. Comparisons with rat full-length alpha(1)- and alpha(2)-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K(+) deocclusion.

  5. Regulation of lung surfactant secretion by the A sub 2 adenosine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Gilfillan, A.M.; Gobran, L.I.; Rooney, S.A. (Yale Univ. School of Medicine, New Haven, CT (USA))


    The authors previously reported that adenosine (A) stimulates secretion of phosphatidylcholine (PC), the major component of surfactant, in type II pneumocytes. To determine how this effect is mediated we examined the effect of P{sub 1} purinoceptor agonists -N{sup 6}-phenylisoprpyl-A (PIA), 5{prime}-N-ethylcarboxyamido-A (NECA), 2-chloro-A (CA) - and antagonists - theophylline (T) and 8-phenyltheophylline (8PT) - on PC secretion and cAMP levels in type II cells isolated from the adult rat. The cells were preincubated with {sup 3}H-choline for 20 h, transferred to fresh medium and incubated {plus minus} test agents for 1.5 h after which {sup 3}H-PC in the cells and medium was measured. A and its analogs stimulated PC secretion in a dose-dependent manner. At the optimal concentration (A, 1 mM; analogs, 0.01 mM) secretion was stimulated approx. 2-fold from a basal rate of 0.08-1.02% of total PC in the medium after 1.5 h. The potency order was NECA>CA=L-PIA>A>D-PIA. The EC{sub 50} for NECA was 8.9 {times} 10{sup {minus}8}M. The effect of NECA was significantly inhibited by 8PT (0.01 mM) and T (0.05 mM). NECA, A and L-PIA increased cellular cAMP levels 34, 12 and 8 fold, respectively, from a basal level of 0.23-0.28 pmol/10{sup 6} cells. These data suggest that the A{sub 2} subtype of the P{sub 1} receptor mediates the effect of A. In newborn rabbits, lung lavage PC increased form 24.1 {plus minus} 1.6 ug P/g lung dry wt at 0 h to 62.6 {plus minus} 7.7 after breathing for 3 h (n=13). 8PT (15 mg/kg, i.m.) at 0 h decreased the PC content at 3 h by 29% to 44.4 {plus minus} 5.1 ug/g. This suggests a functional role for the P{sub 1} receptor in lung surfactant secretion.

  6. Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase. (United States)

    Han, Fei; Bossuyt, Julie; Martin, Jody L; Despa, Sanda; Bers, Donald M


    Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na(+)-K(+)-ATPase (NKA), mainly by reducing its affinity for internal Na(+). The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-α(1) and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na(+) concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na(+) and had no significant effect on the maximum pump rate (V(max)). PKA activation with forskolin (20 μM) restored NKA Na(+) affinity in cells expressing WT but not AA PLM and did not affect V(max) in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na(+) affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.

  7. Regulation of the synthesis and assembly of the plant vacuolar H{sup +}-ATPase. Progress report, [April 1, 1991--March 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Taiz, L.


    During the past three years we have focused on four main areas: the characterization of the 5{prime}-upstream sequence of the gene for the V-ATPase 70 kDa (A) subunit gene, the generation of V-ATPase-deficient mutants using antisense constructs of the A subunit cDNA, analysis of V-ATPase ultrastructure by negative staining and the characterization of organelle-specific isoforms of the A subunit of carrot. In addition we have extended our studies on the cellular distribution of the V-ATPase and we have continued our investigation of the evolution of the V-ATPases by characterizing the A and B subunits of two species of the archaebacterium, Methanococcus.

  8. BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis.

    Directory of Open Access Journals (Sweden)

    Rafal Archacki

    Full Text Available SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.

  9. BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis (United States)

    Archacki, Rafal; Buszewicz, Daniel; Sarnowski, Tomasz J.; Sarnowska, Elzbieta; Rolicka, Anna T.; Tohge, Takayuki; Fernie, Alisdair R.; Jikumaru, Yusuke; Kotlinski, Maciej; Iwanicka-Nowicka, Roksana; Kalisiak, Katarzyna; Patryn, Jacek; Halibart-Puzio, Joanna; Kamiya, Yuji; Davis, Seth J.; Koblowska, Marta K.; Jerzmanowski, Andrzej


    SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways. PMID:23536800

  10. Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations. (United States)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena; Kowalski, Antoni; Stepinski, Dariusz; Wiktorska, Magdalena; Zylinska, Ludmila


    Plasma membrane Ca(2+)-ATPase (PMCA) by extruding Ca(2+) outside the cell, actively participates in the regulation of intracellular Ca(2+) concentration. Acting as Ca(2+)/H(+) counter-transporter, PMCA transports large quantities of protons which may affect organellar pH homeostasis. PMCA exists in four isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca(2+) overload evoked by 59 mM KCl. We observed that manipulation in PMCA expression elevated pHmito and pHcyto but only in PMCA2-downregulated cells higher mitochondrial pH gradient (ΔpH) was found in steady-state conditions. Our data also demonstrated that PMCA2 or PMCA3 knock-down delayed Ca(2+) clearance and partially attenuated cellular acidification during KCl-stimulated Ca(2+) influx. Because SERCA and NCX modulated cellular pH response in neglectable manner, and all conditions used to inhibit PMCA prevented KCl-induced pH drop, we considered PMCA2 and PMCA3 as mainly responsible for transport of protons to intracellular milieu. In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca(2+) overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca(2+)-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient.

  11. The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes. (United States)

    Turner, Andrew G; Ong, Cheryl-Lynn Y; Djoko, Karrera Y; West, Nicholas P; Davies, Mark R; McEwan, Alastair G; Walker, Mark J


    Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen. Copyright © 2017 American Society for Microbiology.

  12. Distinct hormonal regulation of Na+,K+-ATPase genes in the salmonid gill

    DEFF Research Database (Denmark)

    Tipsmark, Christian Kølbæk; Madsen, Steffen


    Igf1 stimulated only a1b. The data elaborate our understanding of Prl and Gh as being antagonists in the control of gill ion regulation, and support a dual role for Gh involving endocrine and  paracrine Igf1 action. Gh and Prl may be the decisive stimuli that direct cortisol-aided mitochondrion...

  13. Differential regulation of the fiber type-specific gene expression of the sarcoplasmic reticulum calcium-ATPase isoforms induced by exercise training. (United States)

    Morissette, Marc P; Susser, Shanel E; Stammers, Andrew N; O'Hara, Kimberley A; Gardiner, Phillip F; Sheppard, Patricia; Moffatt, Teri L; Duhamel, Todd A


    The regulatory role of adenosine monophosphate-activated protein kinase (AMPK)-α2 on sarcoplasmic reticulum calcium-ATPase (SERCA) 1a and SERCA2a in different skeletal muscle fiber types has yet to be elucidated. Sedentary (Sed) or exercise-trained (Ex) wild-type (WT) and AMPKα2-kinase dead (KD) transgenic mice, which overexpress a mutated and inactivated AMPKα2 subunit, were utilized to characterize how genotype or exercise training influenced the regulation of SERCA isoforms in gastrocnemius. As expected, both Sed and Ex KD mice had >40% lower AMPK phosphorylation and 30% lower SERCA1a protein than WT mice (P SERCA2a protein was not different among KD and WT mice. Exercise increased SERCA1a and SERCA2a protein content among WT and KD mice, compared with their Sed counterparts. Maximal SERCA activity was lower in KD mice, compared with WT. Total phospholamban protein was higher in KD mice than in WT and lower in Ex compared with Sed mice. Exercise training increased phospholamban Ser(16) phosphorylation in WT mice. Laser capture microdissection and quantitative PCR indicated that SERCA1a mRNA expression among type I fibers was not altered by genotype or exercise, but SERCA2a mRNA was increased 30-fold in WT+Ex, compared with WT+Sed. In contrast, the exercise-stimulated increase for SERCA2a mRNA was blunted in KD mice. Exercise upregulated SERCA1a and SERCA2a mRNA among type II fibers, but was not altered by genotype. Collectively, these data suggest that exercise differentially influences SERCA isoform expression in type I and type II fibers. Additionally, AMPKα2 influences the regulation of SERCA2a mRNA in type I skeletal muscle fibers following exercise training. Copyright © 2014 the American Physiological Society.

  14. Regulation of ENA1 Na+-ATPase Gene Expression by the Ppz1 Protein Phosphatase Is Mediated by the Calcineurin Pathway (United States)

    Ruiz, Amparo; Yenush, Lynne; Ariño, Joaquín


    Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na+-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at −751 to −667, containing a calcineurin-dependent response element (CDRE), and one downstream region (−573 to −490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants. PMID:14555476

  15. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg


    ) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4......Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases...

  16. Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly* (United States)

    Stransky, Laura A.; Forgac, Michael


    The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

  17. Critical role of the isoform-specific region in alpha1-Na,K-ATPase trafficking and protein Kinase C-dependent regulation. (United States)

    Sottejeau, Yoann; Belliard, Aude; Duran, Marie-Josée; Pressley, Thomas A; Pierre, Sandrine V


    The isoform-specific region (ISR) is a region of structural heterogeneity among the four isoforms of the catalytic alpha-subunit of the Na,K-ATPase and an important structural determinant for isoform-specific functions. In the present study, we examined the role of a potential dileucine clathrin adaptor recognition motif [DE]XXXL[LI] embedded within the alpha1-ISR. To this end, a rat alpha1 construct where leucine 499 was replaced by a valine (as found in the alpha2 isoform sequence) was compared to wild-type rat alpha1 after stable expression in opossum kidney cells. Total Na,K-ATPase expression, activity, or in situ (86)Rb(+) transport was not affected by the L499V mutation. However, surface Na,K-ATPase expression was nearly doubled. This increase was associated with a reduced rate of internalization from the cell surface of about 50% after a 4 h chase and became undetectable if clathrin-coated pit-mediated trafficking was blocked with chlorpromazine. Further, PKC-induced stimulation of Na,K-ATPase-mediated (86)Rb(+) uptake was doubled in mutant-expressing cells, comparable to the chimera containing the intact alpha2-ISR. Similar results were observed when the potential motif was disrupted by means of an E495S mutation. These findings suggest that a dileucine motif embedded within the Na,K-ATPase alpha1-ISR plays a critical role in the surface expression of Na,K-ATPase alpha1 polypeptides at steady state and in the response to PKC activation.

  18. Regulation of the sdsA alkyl sulfatase of Pseudomonas sp. ATCC19151 and its involvement in degradation of anionic surfactants. (United States)

    Jovcic, B; Venturi, V; Davison, J; Topisirovic, L; Kojic, M


    The presented study was aimed to reveal transcriptional regulation of genes involved in SDS degradation (sdsA and sdsB) in Pseudomonas sp. ATCC19151. In addition, the ability of Pseudomonas sp. ATCC19151 to degrade anionic surfactants present in commercial detergent and septic tank drain was analysed. Strain ATCC19151, at 30°C, degrades all SDS present in the liquid medium (up to 4% w/v of SDS) within 48 h. ATCC19151 grows in the presence up to 15% (v/v) 'Fairy' commercial detergent and mineralizes 35% of present anionic surfactants. Analysis of the sdsA (P(sdsA) ) and divergent sdsB (P(sdsB) ) gene promoter activities revealed that SdsB acts as a positive regulator of sdsA and sdsB transcription. P(sdsA) and P(sdsB) activities rose significantly in the presence of the SDS, indicating inducibility of sdsA and sdsB transcription. DNA-binding assay indicated that SdsB directly regulates the transcription of sdsA and sdsB genes. Strain ATCC19151 grew in a sterile septic tank drain and on commercial detergent as sole source of carbon. SdsA enables Pseudomonas sp. ATCC19151 to utilize SDS as a sole carbon source. SdsB is positive transcriptional regulator of sdsA and sdsB genes. Ability of ATCC19151 to degrade anionic surfactants makes Pseudomonas sp. ATCC19151 a good candidate for bioremediation. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  19. Switchable Surfactants

    National Research Council Canada - National Science Library

    Yingxin Liu; Philip G. Jessop; Michael Cunningham; Charles A. Eckert; Charles L. Liotta


    .... We report that long-chain alkyl amidine compounds can be reversibly transformed into charged surfactants by exposure to an atmosphere of carbon dioxide, thereby stabilizing water/alkane emulsions...

  20. Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion. (United States)

    Masui, D C; Furriel, R P M; McNamara, J C; Mantelatto, F L M; Leone, F A


    The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.

  1. Confinement-induced phase behavior and adsorption regulation of ionic surfactants in the aqueous film between charged solids

    NARCIS (Netherlands)

    Lokar, W.J.; Koopal, L.K.; Leermakers, F.A.M.; Ducker, W.A.


    The adsorption of an ionic surfactants in the aqueous film between charged solids was calculated using self-consistent field theory for adsorption and association. It was shown that a change in the separation between the solids leads to a change in interaction energy, which leads to a change in both

  2. The ubiquitous isoform of Na/K-ATPase (ATP1A1) regulates junctional proteins, connexin 43 and claudin 11 via Src-EGFR-ERK1/2-CREB pathway in rat Sertoli cells. (United States)

    Rajamanickam, Gayathri D; Kastelic, John P; Thundathil, Jacob C


    Interaction of Na/K-ATPase with its ligand ouabain has been implicated in the regulation of various biological processes. The objective was to investigate roles of Na/K-ATPase isoforms in formation and function of junctional complexes in Sertoli cells. Primary cultures of Sertoli cells were obtained by enzymatic digestion of 20-day-old rat testes and grown on Matrigel-coated dishes for 7 days. Sertoli cells predominantly expressed the ubiquitous isoform of Na/K-ATPase (ATP1A1), confirmed by immunoblotting, PCR, immunofluorescence, and mass spectrometry. Treatment of Sertoli cells with 50 nM ouabain increased transepithelial electrical resistance (TER) and expression of claudin 11 (tight junctions) and connexin 43 (gap junctions), whereas 1 mM ouabain had opposite effects. Involvement of Src-EGFR-ERK1/2-CREB pathway in ouabain-mediated expression of claudin 11 and connexin 43 was evaluated. Incubation of Sertoli cells with 50 nM ouabain increased content of p-Src, p-EGFR, p-ERK1/2, and p-CREB; in contrast, 1 mM ouabain decreased phosphorylation of these signaling molecules. Preincubation of Sertoli cells with inhibitors of Src and MAPK pathways inhibited ouabain-induced effects on these signaling molecules, TER, and expression of claudin 11 and connexin 43. In conclusion, we inferred that ATP1A1 regulated Sertoli cell tight junctions and gap junctions through the Src-EGFR-ERK1/2-CREB pathway. Ouabain is an endogenous steroid; therefore, its interaction with ATP1A1 may be a critical signaling mechanism for the regulation of Sertoli cell function and male fertility.

  3. Immunomodulatory properties of surfactant preparations. (United States)

    Bersani, Iliana; Kunzmann, Steffen; Speer, Christian P


    Surfactant replacement significantly decreased acute pulmonary morbidity and mortality among preterm neonates with respiratory distress syndrome. Besides improving lung function and oxygenation, surfactant is also a key modulator of pulmonary innate and acquired immunity regulating lung inflammatory processes. In this review, we describe the immunomodulatory features of surfactant preparations. Various surfactant preparations decrease the proinflammatory cytokine and chemokine release, the oxidative burst activity, and the nitric oxide production in lung inflammatory cells such as alveolar neutrophils, monocytes and macrophages; they also affect lymphocyte proliferative response and immunoglobulin production, as well as natural killer and lymphokine-activated killer cell activity. In addition, surfactant preparations are involved in airway remodeling, as they decrease lung fibroblast proliferation capacity and the release of mediators involved in remodeling. Moreover, they increase cell transepithelial resistance and VEGF synthesis in lung epithelial cells. A number of different signaling pathways and molecules are involved in these processes. Because the inhibition of local immune response may decrease lung injury, surfactant therapeutic efficacy may be related not only to its biophysical characteristics but, at least in part, to its anti-inflammatory features and its effects on remodeling processes. However, further studies are required to identify which surfactant preparation ensures the highest anti-inflammatory activity, thereby potentially decreasing the inflammatory process underlying respiratory distress syndrome. In perspective, detailed characterization of these anti-inflammatory effects could help to improve the next generation of surfactant preparations.

  4. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  5. ATPase activity regulation by leader peptide processing of ABC transporter maturation and secretion protein, NukT, for lantibiotic nukacin ISK-1. (United States)

    Zheng, Sen; Nagao, Jun-Ichi; Nishie, Mami; Zendo, Takeshi; Sonomoto, Kenji


    Lantibiotic nukacin ISK-1 is produced by Staphylococcus warneri ISK-1. The dual functional transporter NukT, an ABC transporter maturation and secretion protein, contributes to cleavage of the leader peptide from the prepeptide (modified NukA) and the final transport of nukacin ISK-1. NukT consists of an N-terminal peptidase domain (PEP), a C-terminal nucleotide-binding domain (NBD), and a transmembrane domain (TMD). In this study, NukT and its peptidase-inactive mutant were expressed, purified, and reconstituted into liposomes for analysis of their peptidase and ATPase activities. The ATPase activity of the NBD region was shown to be required for the peptidase activity of the PEP region. Furthermore, we demonstrated for the first time that leader peptide cleavage by the PEP region significantly enhanced the ATPase activity of the NBD region. Taken together, the presented results offer new insights into the processing mechanism of lantibiotic transporters and the necessity of interdomain cooperation.

  6. Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B. (United States)

    Fujita, Hideaki; Umezuki, Yusuke; Imamura, Kanako; Ishikawa, Daisuke; Uchimura, Seiko; Nara, Atsuki; Yoshimori, Tamotsu; Hayashizaki, Yoshihide; Kawai, Jun; Ishidoh, Kazumi; Tanaka, Yoshitaka; Himeno, Masaru


    SKD1 belongs to the AAA-ATPase family and is one of the mammalian class E Vps (vacuolar protein sorting) proteins. Previously we have reported that the overexpression of an ATPase activity-deficient form of SKD1 (suppressor of potassium transport growth defect), SKD1(E235Q), leads the perturbation of membrane transport through endosomes and lysosomes, however, the molecular mechanism behind the action of SKD1 is poorly understood. We have identified two SKD1-binding proteins, SBP1 and mVps2, by yeast two-hybrid screening and we assign them as mammalian class E Vps proteins. The primary sequence of SBP1 indicates 22.5% identity with that of Vta1p from Saccharomyces cerevisiae, which was recently identified as a novel class E Vps protein binding to Vps4p. In fact, SBP1 binds directly to SKD1 through its C-terminal region (198-309). Endogenous SBP1 is exclusively localized to cytosol, however it is redirected to an aberrant endosomal structure, the E235Q compartment, in the cells expressing SKD1(E235Q). The ATPase activity of SKD1 regulates both the membrane association of, and assembly of, a large hetero-oligomer protein complex, containing SBP1, which is potentially involved in membrane transport through endosomes and lysosomes. The N-terminal half (1-157) of human SBP1 is identical to lyst-interacting protein 5 and intriguingly, SKD1 ATPase activity significantly influences the membrane association of lyst protein. The SKD1-SBP1 complex, together with lyst protein, may function in endosomal membrane transport. A primary sequence of mVps2, a mouse homologue of human CHMP2A/BC-2, indicates 44.4% identity with Vps2p/Did4p/Chm2p from Saccharomyces cerevisiae. mVps2 also interacts with SKD1 and is localized to the E235Q compartment. Intriguingly, the N-terminal coiled-coil region of mVps2 is required for the formation of the E235Q compartment but not for binding to SKD1. We propose that both SBP1 and mVps2 regulate SKD1 function in mammalian cells.

  7. Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1). (United States)

    Desmond, Patrick F; Labuza, Amanda; Muriel, Joaquin; Markwardt, Michele L; Mancini, Allison E; Rizzo, Mark A; Bloch, Robert J


    SERCA1, the sarco(endo)plasmic reticulum Ca(2+)-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca(2+) levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca(2+)-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca(2+)-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Rapid regulation of the plasma membrane H+-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa (United States)

    Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey


    Background and Aims The activity of H+-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na+ exclusion via Na+/H+ exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H+-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. Methods The kinetics of salt-induced net H+, Na+ and K+ fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Key Results Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (−144 ± 3·3, −138 ± 5·4 and −128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H+ efflux, Na+ efflux and K+ retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H+ efflux was most pronounced in the root elongation zone. In contrast, H+-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Conclusions Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant’s ability to rapidly upregulate plasma membrane H+-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative

  9. Rapid regulation of the plasma membrane H⁺-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa. (United States)

    Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey


    The activity of H(+)-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na(+) exclusion via Na(+)/H(+) exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H(+)-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. The kinetics of salt-induced net H(+), Na(+) and K(+) fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (-144 ± 3·3, -138 ± 5·4 and -128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H(+) efflux, Na(+) efflux and K(+) retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H(+) efflux was most pronounced in the root elongation zone. In contrast, H(+)-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant's ability to rapidly upregulate plasma membrane H(+)-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative membrane potential values and to

  10. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg


    Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na(+),K(+)- and H(+),K(+)-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two...

  11. Time-dependent transition from H(2)O(2)-extracellular signal-regulated kinase- to O(2)-nitric oxide-dependent mechanisms in the stimulatory effect of leptin on renal Na+/K+/-ATPase in the rat. (United States)

    Marciniak, Andrzej; Borkowska, Ewelina; Kedra, Anna; Rychlik, Monika; Beltowski, Jerzy


    1. Recent studies suggest that leptin, a peptide hormone secreted by white adipose tissue, is involved in the pathogenesis of arterial hypertension, in part by regulating renal sodium handling. Previously, we have demonstrated that in normal rats leptin has a time-dependent effect on renal Na(+)/K(+)-ATPase that drives tubular sodium reabsorption. Short-term leptin infusion results in a transient decrease in Na(+)/K(+)-ATPase activity, whereas prolonged administration stimulates the enzyme. 2. In the present study, we investigated whether these acute effects of leptin are preserved in rats with experimentally induced chronic hyperleptinaemia. 3. Hyperleptinaemia was induced by administration of exogenous leptin (0.25 mg/kg twice daily, s.c., for 7 days). Acute effects of leptin in anaesthetized control (normoleptinaemic) and hyperleptinaemic animals was investigated. Leptin was infused into the abdominal aorta proximally to the renal arteries for 0.5, 1, 2 or 3 h. 4. Leptin (1 microg/min per kg) had a time-dependent effect on renal Na(+)/K(+)-ATPase in both the control and hyperleptinaemic groups. The inhibitory effect observed after 0.5 h infusion was impaired in the hyperleptinaemic group. However, in both groups this effect was abolished by the Janus kinase inhibitor tyrphostin AG490 (100 nmol/min per kg), as well as by the phosphatidylinositol 3-kinase inhibitors wortmannin (10 nmol/min per kg) and LY294002 (1 micromol/min per kg). 5. The stimulatory effect of leptin on Na(+)/K(+)-ATPase activity was observed after 3 h of infusion and was of similar magnitude in control and hyperleptinaemic groups. In the control group, the stimulatory effect of leptin was abolished by the NADPH oxidase inhibitor apocynin (1 micromol/min per kg), the H(2)O(2) scavenger catalase (1 mg/min per kg) and the extracellular signal-regulated kinase (ERK) inhibitor PD98059 (100 nmol/min per kg). In contrast, in the hyperleptinaemic group, the stimulatory effect of leptin was abolished

  12. The role of the plasma membrane H+-ATPase in plant-microbe interactions. (United States)

    Elmore, James Mitch; Coaker, Gitta


    Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant-pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility.

  13. The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens1[C][W][OPEN (United States)

    Langen, Gregor; von Einem, Sabrina; Koch, Aline; Imani, Jafargholi; Pai, Subhash B.; Manohar, Murli; Ehlers, Katrin; Choi, Hyong Woo; Claar, Martina; Schmidt, Rebekka; Mang, Hyung-Gon; Bordiya, Yogendra; Kang, Hong-Gu; Klessig, Daniel F.; Kogel, Karl-Heinz


    MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn2+-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other’s function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species. PMID:24390392

  14. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

    Directory of Open Access Journals (Sweden)

    Summer R. Hayek


    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

  15. Testis-specific isoform of Na/K-ATPase (ATP1A4) regulates sperm function and fertility in dairy bulls through potential mechanisms involving reactive oxygen species, calcium and actin polymerization. (United States)

    Rajamanickam, G D; Kroetsch, T; Kastelic, J P; Thundathil, J C


    Traditional bull breeding soundness evaluation (BBSE) eliminates bulls that are grossly abnormal; however, bulls classified as satisfactory potential breeders still vary in field fertility, implying submicroscopic differences in sperm characteristics. The testis-specific isoform of Na/K-ATPase (ATP1A4) is involved in regulation of sperm motility and capacitation in bulls through well-established enzyme activity and signaling functions. The objective was to determine ATP1A4 content, activity and their relationship to post-thaw sperm function and field fertility, using semen samples from low-fertility (LF) and high-fertility (HF) Holstein bulls (n = 20 each) with known FERTSOL rates (measure of field fertility, based on non-return rate). Frozen-thawed sperm from HF bulls had increased ATP1A4 content and activity compared to LF bulls. Furthermore, post-thaw sperm from HF bulls had increased tyrosine phosphorylation, ROS, F-actin content, and low intracellular calcium compared to LF bulls. Subsequent incubation of HF bull sperm with ouabain (a specific ligand of Na/K-ATPase) further augmented the post-thaw increase in tyrosine phosphorylation, ROS production, and F-actin content, whereas the increase in intracellular calcium was still low compared to LF bull sperm. ATP1A4 content and activity, ROS, F-actin and calcium were significantly correlated with fertility. In conclusion, we inferred that ATP1A4 content and activity differed among dairy bulls with satisfactory semen characteristics and that ATP1A4 may regulate sperm function through mechanisms involving ROS, F-actin and calcium in frozen-thawed sperm of HF and LF dairy bulls. © 2017 American Society of Andrology and European Academy of Andrology.

  16. An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour

    NARCIS (Netherlands)

    Verweij, W.; Spelt, C.; di Sansebastiano, G.-P.; Vermeer, J.; Reale, L.; Ferranti, F.; Koes, R.; Quattrocchio, F.


    The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H+-ATPase1 (V-ATPase), which, in plants, act together with

  17. Vacuolar H+-ATPase: An Essential Multitasking Enzyme in Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    L. Shannon Holliday


    Full Text Available Vacuolar H+-ATPases (V-ATPases are large multisubunit proton pumps that are required for housekeeping acidification of membrane-bound compartments in eukaryotic cells. Mammalian V-ATPases are composed of 13 different subunits. Their housekeeping functions include acidifying endosomes, lysosomes, phagosomes, compartments for uncoupling receptors and ligands, autophagosomes, and elements of the Golgi apparatus. Specialized cells, including osteoclasts, intercalated cells in the kidney and pancreatic beta cells, contain both the housekeeping V-ATPases and an additional subset of V-ATPases, which plays a cell type specific role. The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one or more of the subunits. Three human diseases caused by mutations of isoforms of subunits have been identified. Cancer cells utilize V-ATPases in unusual ways; characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer. Two accessory proteins to the V-ATPase have been identified that regulate the proton pump. One is the (prorenin receptor and data is emerging that indicates that V-ATPase may be intimately linked to renin/angiotensin signaling both systemically and locally. In summary, V-ATPases play vital housekeeping roles in eukaryotic cells. Specialized versions of the pump are required by specific organ systems and are involved in diseases.


    Directory of Open Access Journals (Sweden)

    Pirog T. Р.


    Full Text Available The review is devoted to surface-active glycolipids. The general characteristics, the physiological role of the rhamnolipids, trehalose lipids, sophorolipids, mannosylerythritol lipids and their traditional producers — the representatives of the genera Pseudozyma, Pseudomonas, Rhodococcus and Candida are given. The detailed analysis of the chemical structure, the stages of the biosynthesis and the regulation of some low molecular glycolipids are done. The own experimental data concerning the synthesis intensification, the physiological role and the practical use of Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii IMV B-7405 surfactants, which are a complex of the glyco-, phospho-, amino- and neutral lipids (glycolipids of all strains are presented by trehalose mycolates are summarized. It was found that R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 surfactants have protective, antimicrobial and antiadhesive properties. It was shown that R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii IMV B-7405 surfactants preparation of cultural liquid intensified the degradation of oil in water due to the activation of the natural petroleum-oxidizing microflora.


    Directory of Open Access Journals (Sweden)

    T. P. Pirog


    Full Text Available The classification and the chemical structure of the lipopeptides and their producers (bacteria of the genera Bacillus and Pseudomonas are given. The role of the lipopeptides in cells motility, biofilm formation, metal binding and xenobiotics degradation and their action on the cells of pro- and eukaryotes is summarized. The stages of the nonribosomal lipopeptides synthesis and the role of two-component (GacA/GacS, ComA/ComP and the quorum system regulation of this process are shown. The potential of lactic acid bacteria and marine microorganisms as alternative surfactants producers (glycolipids, lipopeptides, phospholipids and fatty acids, glycolipopeptides are discussed. Their productivity and advantages over traditional producers are given as well. The properties of surfactants synthesized by lactic acid bacteria (the reduction of the surface tension, the critical micelle concentration, the stability in a wide range of pH, the temperature, the biological activity are summarized. Surfactants of nonpathogenic probiotic bacteria could be used as effective antimicrobial agents and antiadhesive and marine producers which able to synthesize unique metabolites that are not produced by other microorganisms.

  20. Expression of gill vacuolar-type H+-ATPase B subunit, and Na+, K+-ATPase alpha- and beta- subunit messenger RNAs in smolting Salmo salar

    DEFF Research Database (Denmark)

    Seidelin, Michel; Madsen, Steffen; Cutler, Christopher P


    Changes in gill vacuolar-type H+-ATPase B subunit, and Na+,K+-ATPase alpha and beta subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H+-ATPase (V-H+-ATPase) B subunit, ii) Na......+,K+-ATPase alpha (1) subunit, and iii) Na+,K+-ATPase beta (1) subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na+,K+-ATPase activity and improved hypo-osmoregulatory ability as judged by their ability to regulate plasma [Cl-] in a 24-hr...... seawater challenge test (35 ppt). Gill Na+,K+-ATPase alpha (1) and beta (1) subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na+,K+-ATPase activity, underlining the importance of increased mRNA levels for increased...

  1. HNF-1B specifically regulates the transcription of the {gamma}a-subunit of the Na{sup +}/K{sup +}-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Ferre, Silvia [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands); Veenstra, Gert Jan C. [Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen (Netherlands); Bouwmeester, Rianne; Hoenderop, Joost G.J. [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands); Bindels, Rene J.M., E-mail: [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands)


    Research highlights: {yields} Defects in HNF-1B transcription factor affect Mg{sup 2+} handling in the distal kidney. {yields} {gamma}a- and {gamma}b- subunits of the Na{sup +}/K{sup +}-ATPase colocalize in the distal convoluted tubule of the nephron. {yields} HNF-1B specifically activates {gamma}a expression. {yields} HNF-1B mutants have a dominant negative effect on wild type HNF-1B activity. {yields} Defective transcription of {gamma}a may promote renal Mg{sup 2+} wasting. -- Abstract: Hepatocyte nuclear factor-1B (HNF-1B) is a transcription factor involved in embryonic development and tissue-specific gene expression in several organs, including the kidney. Recently heterozygous mutations in the HNF1B gene have been identified in patients with hypomagnesemia due to renal Mg{sup 2+} wasting. Interestingly, ChIP-chip data revealed HNF-1B binding sites in the FXYD2 gene, encoding the {gamma}-subunit of the Na{sup +}/K{sup +}-ATPase. The {gamma}-subunit has been described as one of the molecular players in the renal Mg{sup 2+} reabsorption in the distal convoluted tubule (DCT). Of note, the FXYD2 gene can be alternatively transcribed into two main variants, namely {gamma}a and {gamma}b. In the present study, we demonstrated via two different reporter gene assays that HNF-1B specifically acts as an activator of the {gamma}a-subunit, whereas the {gamma}b-subunit expression was not affected. Moreover, the HNF-1B mutations H69fsdelAC, H324S325fsdelCA, Y352finsA and K156E, previously identified in patients with hypomagnesemia, prevented transcription activation of {gamma}a-subunit via a dominant negative effect on wild type HNF1-B. By immunohistochemistry, it was shown that the {gamma}a- and {gamma}b-subunits colocalize at the basolateral membrane of the DCT segment of mouse kidney. On the basis of these data, we suggest that abnormalities involving the HNF-1B gene may impair the relative abundance of {gamma}a and {gamma}b, thus affecting the transcellular Mg{sup 2

  2. ALT1, a Snf2 family chromatin remodeling ATPase, negatively regulates alkaline tolerance through enhanced defense against oxidative stress in rice.

    Directory of Open Access Journals (Sweden)

    Mingxin Guo

    Full Text Available Alkaline salt stress adversely affects rice growth, productivity and grain quality. However, the mechanism underlying this process remains elusive. We characterized here an alkaline tolerant mutant, alt1 in rice. Map-based cloning revealed that alt1 harbors a mutation in a chromatin remodeling ATPase gene. ALT1-RNAi transgenic plants under different genetic background mimicked the alt1 phenotype, exhibiting tolerance to alkaline stress in a transcript dosage-dependent manner. The predicted ALT1 protein belonged to the Ris1 subgroup of the Snf2 family and was localized in the nucleus, and transcription of ALT1 was transiently suppressed after alkaline treatment. Although the absorption of several metal ions maintained well in the mutant under alkaline stress, expression level of the genes involved in metal ions homeostasis was not altered in the alt1 mutant. Classification of differentially expressed abiotic stress related genes, as revealed by microarray analysis, found that the majority (50/78 were involved in ROS production, ROS scavenging, and DNA repair. This finding was further confirmed by that alt1 exhibited lower levels of H2O2 under alkaline stress and tolerance to methyl viologen treatment. Taken together, these results suggest that ALT1 negatively functions in alkaline tolerance mainly through the defense against oxidative damage, and provide a potential two-step strategy for improving the tolerance of rice plants to alkaline stress.

  3. [Exogenous surfactant therapy: new synthetic surfactants]. (United States)

    Lacaze-Masmonteil, Th


    There are numerous pulmonary conditions in which qualitative or quantitative anomalies of the surfactant system have been demonstrated. In premature newborns with immature lungs, a functional deficit in surfactant is the main physiopathologic mechanism of the neonatal respiratory distress syndrome (RDS). Since the landmark pilot study of Fujiwara, published more than 20 years ago, the efficacy of exogenous surfactant for the treatment of neonatal RDS has been established by numerous controlled studies and meta-analyses. Enlightened by a growing insight into both the structure and function of the different surfactant components, a new generation of synthetic surfactants has been developed. Various complementary approaches have confirmed the fundamental role of the two hydrophobic proteins, SP-B and SP-C, in the surfactant system, thus opening the way to the design of analogues, either by chemical synthesis or expression in a prokaryotic system. An example of these peptide-containing synthetic surfactant preparations, lucinactant (Surfaxin), has been recently tested in comparison to a synthetic surfactant that does not contain protein as well as to animal derived surfactant preparations. Major clinical outcomes between lucinactant and animal-derived surfactant preparations were fund similar in two randomized controlled trials, opening the way to a new generation of synthetic surfactants in the near future.

  4. Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells. (United States)

    Dey, Kuntal; Roy, Soumitra; Ghosh, Biswarup; Chakraborti, Sajal


    We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  5. Estrogen, progesterone, and genistein differentially regulate levels of expression of α-, β-, and γ-epithelial sodium channel (ENaC) and α-sodium potassium pump (Na⁺/K⁺-ATPase) in the uteri of sex steroid-deficient rats. (United States)

    Chinigarzadeh, Asma; Muniandy, Sekaran; Salleh, Naguib


    Estrogen, progesterone, and genistein could induce changes in uterine fluid volume and Na(+) concentration. Progesterone upregulates expression of epithelial sodium channel (ENaC) and Na(+)/K(+)-ATPase which contributed toward these changes. However, effects of estrogen and genistein were unknown. This study therefore investigated changes in expression of these proteins in the uterus under estrogen, progesterone, and genistein influences to further understand mechanisms underlying sex steroids and phytoestrogen effects on uterine fluid Na(+) regulation. In this study, uteri of ovariectomized female rats receiving 7-day treatment with genistein (25, 50, and 100 mg/kg/day), estrogen (0.8 × 10(-4) mg/kg/day), or progesterone (4 mg/kg/day) were harvested, and expression levels of α-, β-, and γ-ENaC proteins and messenger RNAs (mRNAs) and α-Na(+)/K(+)-ATPase protein were determined by Western blotting (proteins) and real-time polymerase chain reaction (mRNA). Meanwhile, distribution of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase proteins in the uterus was identified by immunohistochemistry. Our findings indicated that expression of α-, β-, and γ-ENaC proteins and mRNAs and α-Na(+)/K(+)-ATPase protein were enhanced under progesterone influence. Lower expressions were noted under estrogen and genistein influences compared to progesterone. Under estrogen, progesterone, and genistein influences, α- and β-ENaC were distributed at apical membrane and γ-ENaC was distributed at apical and basolateral membranes of uterine luminal epithelia. Under progesterone influence, α-Na(+)/K(+)-ATPase was highly expressed at basolateral membrane. In conclusion, high expression of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase under progesterone influence would contribute toward increased uterine fluid Na(+) reabsorption, whereas lesser expression of these proteins under estrogen and genistein influences would contribute toward lower reabsorption of uterine fluid Na

  6. Molecular characterization and transcriptional regulation of the Na +/K+ ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis). (United States)

    Armesto, Paula; Campinho, Marco A; Rodríguez-Rúa, Ana; Cousin, Xavier; Power, Deborah M; Manchado, Manuel; Infante, Carlos


    In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Surfactant protein D, Club cell protein 16, Pulmonary and activation-regulated chemokine, C-reactive protein, and Fibrinogen biomarker variation in chronic obstructive lung disease

    DEFF Research Database (Denmark)

    Johansson, Sofie Lock; Vestbo, J.; Sorensen, G. L.


    Chronic obstructive pulmonary disease (COPD) is a multifaceted condition that cannot be fully described by the severity of airway obstruction. The limitations of spirometry and clinical history have prompted researchers to investigate a multitude of surrogate biomarkers of disease for the assessm......Chronic obstructive pulmonary disease (COPD) is a multifaceted condition that cannot be fully described by the severity of airway obstruction. The limitations of spirometry and clinical history have prompted researchers to investigate a multitude of surrogate biomarkers of disease...... for the assessment of patients, prediction of risk, and guidance of treatment. The aim of this review is to provide a comprehensive summary of observations for a selection of recently investigated pulmonary inflammatory biomarkers (Surfactant protein D (SP-D), Club cell protein 16 (CC-16), and Pulmonary...... and activation-regulated chemokine (PARC/CCL-18)) and systemic inflammatory biomarkers (C-reactive protein (CRP) and fibrinogen) with COPD. The relevance of these biomarkers for COPD is discussed in terms of their biological plausibility, their independent association to disease and hard clinical outcomes...

  8. Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats. (United States)

    Barati, Michelle T; Ketchem, Corey J; Merchant, Michael L; Kusiak, Walter B; Jose, Pedro A; Weinman, Edward J; LeBlanc, Amanda J; Lederer, Eleanor D; Khundmiri, Syed J


    Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

  9. Thermally cleavable surfactants (United States)

    McElhanon, James R.; Simmons, Blake A.; Zifer, Thomas; Jamison, Gregory M.; Loy, Douglas A.; Rahimian, Kamyar; Long, Timothy M.; Wheeler, David R.; Staiger, Chad L.


    Two new surfactant molecules are reported which contain thermally labile Diels-Alder adducts connecting the polar and non-polar sections of each molecule. The two surfactants possess identical non-polar dodecyl tail segments but exhibit different polar headgroups. The surfactants become soluble in water when anionic salts are formed through the deprotonation of the surfactant headgroups by the addition of potassium hydroxide. When either surfactant is exposed to temperature above about C., the retro Diels-Alder reaction occurs, yielding hydrophilic and hydrophobic fragments and the aqueous solutions of the surfactants subsequently exhibit loss of all surface-active behavior.

  10. Surfactant in airway disease. (United States)

    Enhorning, Goran


    Beta(2)-adrenergic agonists cause a release of pulmonary surfactant into lung airways. The surfactant phospholipids maintain the patency of the conducting airways, but this function is inhibited by plasma proteins entering an inflamed airway. The physical behavior of the surfactant can be studied with a pulsating bubble surfactometer and a capillary surfactometer. Calf lung surfactant extract was found to be inhibited by plasma proteins and by a lowering of temperature. Severe breathing difficulties and malfunctioning surfactant developed in BALB/c mice inhaling ozone or infected with respiratory syncytial virus, mainly as a result of proteins invading the airways. Patients with asthma were challenged with allergens in an area of one lung. BAL fluid (BALF) from such an area contained a surfactant that functioned poorly (ie, an inability to maintain airway openness) compared with BALF from the other lung or from the lungs of healthy volunteers. When proteins in the BALF were removed, surfactant performance clearly improved. Eosinophils, so prominent in asthmatic patients, synthesize the enzyme lysophospholipase, which, together with the enzyme phospholipase A(2), catalyzes the hydrolysis of the main component of the surfactant, phosphatidylcholine. Such hydrolysis incapacitates the ability of the surfactant to maintain airway patency. The treatment of asthma with beta(2)-adrenergic agonists and steroids will have a valuable effect on the surfactant system. It will cause a release of fresh surfactant into terminal airways. Surfactant can also be nebulized and inhaled, which has been shown to be an effective treatment.

  11. Na/K-ATPase Signaling and Salt Sensitivity: The Role of Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Jiang Liu


    Full Text Available Other than genetic regulation of salt sensitivity of blood pressure, many factors have been shown to regulate renal sodium handling which contributes to long-term blood pressure regulation and have been extensively reviewed. Here we present our progress on the Na/K-ATPase signaling mediated sodium reabsorption in renal proximal tubules, from cardiotonic steroids-mediated to reactive oxygen species (ROS-mediated Na/K-ATPase signaling that contributes to experimental salt sensitivity.

  12. Changes in cellular distribution regulate SKD1 ATPase activity in response to a sudden increase in environmental salinity in halophyte ice plant. (United States)

    Jou, Yingtzy; Chiang, Chih-Pin; Yen, Hungchen Emilie


    Halophyte Mesembryanthemum crystallinum L. (ice plant) rapidly responds to sudden increases in salinity in its environment by activating specific salt-tolerant mechanisms. One major strategy is to regulate a series of ion transporters and proton pumps to maintain cellular Na(+)/K(+) homeostasis. Plant SKD1 (suppressor of K(+) transport growth defect 1) proteins accumulate in cells actively engaged in the secretory processes, and play a critical role in intracellular protein trafficking. Ice plant SKD1 redistributes from the cytosol to the plasma membrane hours after salt stressed. In combination with present knowledge of this protein, we suggest that stress facilitates SKD1 movement to the plasma membrane where ADP/ATP exchange occurs, and functions in the regulation of membrane components such as ion transporters to avoid ion toxicity.

  13. Leptin and the Regulation of Renal Sodium Handling and Renal Na+-Transporting ATPases: Role in the Pathogenesis of Arterial Hypertension


    Bełtowski, Jerzy


    Leptin, an adipose tissue hormone which regulates food intake, is also involved in the pathogenesis of arterial hypertension. Plasma leptin concentration is increased in obese individuals. Chronic leptin administration or transgenic overexpression increases blood pressure in experimental animals, and some studies indicate that plasma leptin is elevated in hypertensive subjects independently of body weight. Leptin has a dose- and time-dependent effect on urinary sodium excretion. High doses of...

  14. Silicon mitigates heavy metal stress by regulating P-type heavy metal ATPases, Oryza sativa low silicon genes, and endogenous phytohormones. (United States)

    Kim, Yoon-Ha; Khan, Abdul Latif; Kim, Duk-Hwan; Lee, Seung-Yeol; Kim, Kyung-Min; Waqas, Muhammad; Jung, Hee-Young; Shin, Jae-Ho; Kim, Jong-Guk; Lee, In-Jung


    Silicon (Si) application has been known to enhance the tolerance of plants against abiotic stresses. However, the protective mechanism of Si under heavy metals contamination is poorly understood. The aim of this study was to assess the role of Si in counteracting toxicity due to cadmium (Cd) and copper (Cu) in rice plants (Oryza sativa). Si significantly improved the growth and biomass of rice plants and reduced the toxic effects of Cd/Cu after different stress periods. Si treatment ameliorated root function and structure compared with non-treated rice plants, which suffered severe root damage. In the presence of Si, the Cd/Cu concentration was significantly lower in rice plants, and there was also a reduction in lipid peroxidation and fatty acid desaturation in plant tissues. The reduced uptake of metals in the roots modulated the signaling of phytohormones involved in responses to stress and host defense, such as abscisic acid, jasmonic acid, and salicylic acid. Furthermore, the low concentration of metals significantly down regulated the mRNA expression of enzymes encoding heavy metal transporters (OsHMA2 and OsHMA3) in Si-metal-treated rice plants. Genes responsible for Si transport (OsLSi1 and OsLSi2), showed a significant up-regulation of mRNA expression with Si treatment in rice plants. The present study supports the active role of Si in the regulation of stresses from heavy metal exposure through changes in root morphology.

  15. Retinoschisin is linked to retinal Na/K-ATPase signaling and localization. (United States)

    Plössl, Karolina; Royer, Melanie; Bernklau, Sarah; Tavraz, Neslihan N; Friedrich, Thomas; Wild, Jens; Weber, Bernhard H F; Friedrich, Ulrike


    Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We recently showed that retinoschisin, the protein encoded by RS1 , regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase, whereas the α-subunit is exchangeable. Our investigations revealed no effect of retinoschisin on Na/K-ATPase-mediated ATP hydrolysis and ion transport. However, we identified an influence of retinoschisin on Na/K-ATPase-regulated signaling cascades and Na/K-ATPase localization. In addition to the known ERK deactivation, retinoschisin treatment of retinoschisin-deficient ( Rs1h -/Y ) murine retinal explants decreased activation of Src, an initial transmitter in Na/K-ATPase signal transduction, and of Ca 2+ signaling marker Camk2. Immunohistochemistry on murine retinae revealed an overlap of the retinoschisin-Na/K-ATPase complex with proteins involved in Na/K-ATPase signaling, such as caveolin, phospholipase C, Src, and the IP3 receptor. Finally, retinoschisin treatment altered Na/K-ATPase localization in photoreceptors of Rs1h -/Y retinae. Taken together, our results suggest a regulatory effect of retinoschisin on Na/K-ATPase signaling and localization, whereas Na/K-ATPase-dysregulation caused by retinoschisin deficiency could represent an initial step in XLRS pathogenesis. © 2017 Plössl et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (

  16. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.


    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-AT...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  17. The effect of exercise and beta2-adrenergic stimulation on glutathionylation and function of the Na,K-ATPase in human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten; Hostrup, Morten; Bangsbo, Jens


    subunits are further glutathionylated by exercise and beta2-adrenergic stimulation. Our data suggest that glutathionylation contributes to the complex regulation of Na,K-ATPase function in human skeletal muscle. Glutathionylation of the Na,K-ATPase may explain reductions in maximal Na,K-ATPase activity......Potassium and sodium displacements across the skeletal muscle membrane during exercise may cause fatigue and are in part controlled by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for muscle functioning. We investigated the effect of oxidative stress (glutathionylation...

  18. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe


    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...... transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na...

  19. Dissecting the role of conformational change and membrane binding by the bacterial cell division regulator MinE in the stimulation of MinD ATPase activity. (United States)

    Ayed, Saud H; Cloutier, Adam D; McLeod, Laura J; Foo, Alexander C Y; Damry, Adam M; Goto, Natalie K


    The bacterial cell division regulators MinD and MinE together with the division inhibitor MinC localize to the membrane in concentrated zones undergoing coordinated pole-to-pole oscillation to help ensure that the cytokinetic division septum forms only at the mid-cell position. This dynamic localization is driven by MinD-catalyzed ATP hydrolysis, stimulated by interactions with MinE's anti-MinCD domain. This domain is buried in the 6-β-stranded MinE "closed" structure, but is liberated for interactions with MinD, giving rise to a 4-β-stranded "open" structure through an unknown mechanism. Here we show that MinE-membrane interactions induce a structural change into a state resembling the open conformation. However, MinE mutants lacking the MinE membrane-targeting sequence stimulated higher ATP hydrolysis rates than the full-length protein, indicating that binding to MinD is sufficient to trigger this conformational transition in MinE. In contrast, conformational change between the open and closed states did not affect stimulation of ATP hydrolysis rates in the absence of membrane binding, although the MinD-binding residue Ile-25 is critical for this conformational transition. We therefore propose an updated model where MinE is brought to the membrane through interactions with MinD. After stimulation of ATP hydrolysis, MinE remains bound to the membrane in a state that does not catalyze additional rounds of ATP hydrolysis. Although the molecular basis for this inhibited state is unknown, previous observations of higher-order MinE self-association may explain this inhibition. Overall, our findings have general implications for Min protein oscillation cycles, including those that regulate cell division in bacterial pathogens. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis. (United States)

    Takahashi, Koji; Hayashi, Ken-ichiro; Kinoshita, Toshinori


    The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

  1. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. (United States)

    Zhao, Jianhua; Benlekbir, Samir; Rubinstein, John L


    Eukaryotic vacuolar H(+)-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H(+) ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.

  2. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA. (United States)

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


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

  3. Pulmonary surfactant therapy.


    Poulain, F R; Clements, J. A.


    Surfactant replacement therapy is now an integral part of the care of neonates since several clinical trials of natural surfactant extracts and synthetic preparations have shown efficacy in the treatment of infants with hyaline membrane disease. In these studies, early treatment with exogenous surfactant substantially reduced mortality and the incidence of air leak, although it did not appear to reduce the incidence of other complications, in particular bronchopulmonary dysplasia. Early repor...

  4. Surfactants in tribology

    CERN Document Server

    Biresaw, Girma


    Surface science and tribology play very critical roles in many industries. Manufacture and use of almost all consumer and industrial products rely on the application of advanced surface and tribological knowledge. The fourth in a series, Surfactants in Tribology, Volume 4 provides an update on research and development activities connecting surfactants and tribological phenomena. Written by renowned subject matter experts, the book demonstrates how improved design of surfactants can be harnessed to control tribological phenomena. Profusely illustrated and copiously referenced, the chapters also

  5. Towards unravelling surfactant transport (United States)

    Sellier, Mathieu; Panda, Satyananda


    Surfactant transport arises in many natural or industrial settings. Examples include lipid tear layers in the eye, pulmonary surfactant replacement therapy, or industrial coating flows. Flows driven by the surface tension gradient which arises as a consequence of surfactant concentration inhomogeneity, also known as Marangoni-driven flows, have attracted the attention of fluid dynamists for several decades and has led to the development of sophisticated models and the undeniable advancement of the understanding of such flows. Yet, experimental confirmation of these models has been hampered by the difficulty in reliably and accurately measuring the surfactant concentration and its temporal evolution. In this contribution, we propose a methodology which may help shed some light on surfactant transport at the surface of thin liquid films. The surface stress induced by surfactant concentration induces a flow at the free surface which is visible and measurable. In the context of thin film flows for which the lubrication approximation hold, we demonstrate how the knowledge of this free surface flow field provides sufficient information to reconstruct the surfactant tension field. From the surface tension and an assumed equation of state, the local surfactant concentration can also be calculated and other transport parameters such as the surfactant surface diffusivity indirectly inferred. In this contribution, the proposed methodology is tested with synthetic data generated by the forward solution of the governing partial differential equations in order to illustrate the feasibility of the algorithm and highlight numerical challenges.

  6. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation in the se......Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation...... in the secretory and endocytic pathways. The inward-oriented translocation of phospholipids is catalyzed by proteins called P4-ATPases or flippases, which belong to a subfamily of P-type ATPases. P4-ATPases, together with their CDC50 β-subunits, are essential for eukaryotic life but the key features...... of their activity and regulation remain to be elucidated. Therefore, these studies focus on the role of the catalytic and CDC50 β-subunit in the phospholipid translocation and the regulation processes behind it. Recent studies suggested that P4-ATPase complex functionality is highly dependent on the conformation...

  7. Evolution of plant P-type ATPases

    Directory of Open Access Journals (Sweden)

    Christian N.S. Pedersen


    Full Text Available Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauria and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a moss, Selaginella moellendorffii (a primitive vascular plant, and Arabidopsis thaliana (a model flowering plant. Each organism contained sequences for all five subfamilies of P-type ATPases. Our analysis demonstrates when specific subgroups of P-type ATPases disappeared in the evolution of Angiosperms. Na/K-pump related P2C ATPases were lost with the evolution of streptophytes whereas Na+ or K+ pumping P2D ATPases and secretory pathway Ca2+-ATPases remained until mosses. An N-terminally located calmodulin binding domain in P2B ATPases can only be detected in pumps from Streptophytae, whereas, like in animals, a C-terminally localized calmodulin binding domain might be present in chlorophyte P2B Ca2+-ATPases. Chlorophyte genomes encode P3A ATPases resembling protist plasma membrane H+-ATPases and a C-terminal regulatory domain is missing. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps.

  8. Mechanisms to explain surfactant responses. (United States)

    Jobe, Alan H


    Surfactant is now standard of care for infants with respiratory distress syndrome. Surfactant treatments are effective because of complex metabolic interactions between surfactant and the preterm lung. The large treatment dose functions as substrate; it is taken up by the preterm lung and is reprocessed and secreted with improved function. The components of the treatment surfactant remain in the preterm lung for days. If lung injury is avoided, then surfactant inhibition is minimized. Prenatal corticosteroids complement surfactant to further enhance lung function. The magic of surfactant therapy results from the multiple interactions between surfactant and the preterm lung. Copyright (c) 2006 S. Karger AG, Basel.

  9. Pulmonary surfactant surface tension influences alveolar capillary shape and oxygenation. (United States)

    Ikegami, Machiko; Weaver, Timothy E; Grant, Shawn N; Whitsett, Jeffrey A


    Alveolar capillaries are located in close proximity to the alveolar epithelium and beneath the surfactant film. We hypothesized that the shape of alveolar capillaries and accompanying oxygenation are influenced by surfactant surface tension in the alveolus. To prove our hypothesis, surfactant surface tension was regulated by conditional expression of surfactant protein (SP)-B in Sftpb(-/-) mice, thereby inhibiting surface tension-lowering properties of surfactant in vivo within 24 hours after depletion of Sftpb. Minimum surface tension of isolated surfactant was increased and oxygen saturation was significantly reduced after 2 days of SP-B deficiency in association with deformation of alveolar capillaries. Intravascularly injected 3.2-mum-diameter microbeads through jugular vein were retained within narrowed pulmonary capillaries after reduction of SP-B. Ultrastructure studies demonstrated that the capillary protrusion typical of the normal alveolar-capillary unit was reduced in size, consistent with altered pulmonary blood flow. Pulmonary hypertension and intrapulmonary shunting are commonly associated with surfactant deficiency and dysfunction in neonates and adults with respiratory distress syndromes. Increased surfactant surface tension caused by reduction in SP-B induced narrowing of alveolar capillaries and oxygen desaturation, demonstrating an important role of surface tension-lowering properties of surfactant in the regulation of pulmonary vascular perfusion.

  10. Dynamic covalent surfactants

    NARCIS (Netherlands)

    Minkenberg, C.B.


    In this thesis the development of surfactant aggregates with fast exchange dynamics between the aggregated and non-aggregated state is described. Dynamic surfactant exchange plays an important role in natural systems, for instance in cell signaling, cell division, and uptake and release of cargo.

  11. Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-ATPase

    NARCIS (Netherlands)

    Duarte, A.M.; Wolfs, C.J.A.M.; Nuland, van N.A.J.; Harrison, M.A.; Findlay, J.B.C.; Mierlo, van C.P.M.; Hemminga, M.A.


    Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific

  12. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)


    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  13. Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis). (United States)

    Komisarczuk, Anna Z; Kongshaug, Heidi; Nilsen, Frank


    Na+/K+-ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na+/K+-ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na+/K+-ATPase (LsalNa+/K+-ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa+/K+-ATPase revealed all typical features of Na+/K+-ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa+/K+-ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa+/K+-ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa+/K+-ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na+/K+-ATPase is directly or indirectly involved. Copyright © 2018. Published by Elsevier Inc.

  14. Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E2P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed; Christensen, Søren Brøgger


    anthroylouabain, a fluorescent ouabain analog, revealed that the increased ouabain affinity is unique to oleic and linoleic acids, as compared with ¿-linolenic acid, which decreased pump-mediated ATP hydrolysis but did not equally increase ouabain interaction with the pump. Thus, the dynamic changes in plasma...... levels of oleic and linoleic acids are important in the modulation of the sensitivity of the sodium pump to cardiac glycosides. Given the possible involvement of the cardiac glycoside binding site on Na,K-ATPase in the regulation of hypertension, we suggest oleic acid to be a specific chaperon...... that modulates interaction of cardiac glycosides with the sodium pump....

  15. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast. (United States)

    Banerjee, Subhrajit; Kane, Patricia M


    Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H+-ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of Vo a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (

  16. Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle

    DEFF Research Database (Denmark)

    Galuska, Dana; Kotova, Olga; Barres, Romain


    . Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na(+)-K(+)-ATPase alpha(1)-subunit protein expression was increased 1.6-fold (P alpha(2)- and beta(1)-subunits and protein expression were decreased twofold (P ...(+)-K(+)-ATPase activity after 4 wk of HFD. Exercise training restored alpha(1)-, alpha(2)-, and beta(1)-subunit expression and Na(+)-K(+)-ATPase activity to control levels and reduced beta(2)-subunit expression 2.2-fold (P alpha(1)-subunit-regulating transcription factor ZEB (AREB6......) and alpha(1) mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of alpha(2)- and beta(1)-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na(+)-K(+)-ATPase regulatory protein...

  17. Characterization of Bovine Brain ATPase (United States)


    with a complex structure (presence of galactose, vannose and sialic acid ). This structure raises the possibility of immobilizing via immobilized...SYNAPIIC The (Ca2+ + M9*2)- dependent ATPase-Calcitu Channel Complex is found at the synaptic termini of neurons ( 1). With the exception of Mojave toxin...for 60% inhibition. We examined the effects of various phospholipids on catalytic activity (Table IX). With the exception of phosphatidic acid , all

  18. Pulmonary surfactant and lung transplantation

    NARCIS (Netherlands)

    Erasmus, Michiel Elardus


    Pulmonary surfactant lowers the surface tension at the air-water interface inside the alveolus. This is achieved by adsorption of surfactant phospholipids at the air-water interface, a process controlled by surfactant-associated proteins, such as SP-A. In this way, surfactant prevents collapse of

  19. Metathesis depolymerizable surfactants (United States)

    Jamison, Gregory M.; Wheeler, David R.; Loy, Douglas A.; Simmons, Blake A.; Long, Timothy M.; McElhanon, James R.; Rahimian, Kamyar; Staiger, Chad L.


    A class of surfactant molecules whose structure includes regularly spaced unsaturation in the tail group and thus, can be readily decomposed by ring-closing metathesis, and particularly by the action of a transition metal catalyst, to form small molecule products. These small molecules are designed to have increased volatility and/or enhanced solubility as compared to the original surfactant molecule and are thus easily removed by solvent extraction or vacuum extraction at low temperature. By producing easily removable decomposition products, the surfactant molecules become particularly desirable as template structures for preparing meso- and microstructural materials with tailored properties.

  20. Phosphine oxide surfactants revisited. (United States)

    Stubenrauch, Cosima; Preisig, Natalie; Laughlin, Robert G


    This review summarizes everything we currently know about the nonionic surfactants alkyl dimethyl (C(n)DMPO) and alkyl diethyl (C(n)DEPO) phosphine oxide (PO surfactants). The review starts with the synthesis and the general properties (Section 2) of these compounds and continues with their interfacial properties (Section 3) such as surface tension, surface rheology, interfacial tension and adsorption at solid surfaces. We discuss studies on thin liquid films and foams stabilized by PO surfactants (Section 4) as well as studies on their self-assembly into lyotropic liquid crystals and microemulsions, respectively (Section 5). We aim at encouraging colleagues from both academia and industry to take on board PO surfactants whenever possible and feasible because of their broad variety of excellent properties. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase. (United States)

    Madan, Namrata; Xu, Yunhui; Duan, Qiming; Banerjee, Moumita; Larre, Isabel; Pierre, Sandrine V; Xie, Zijian


    The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na+ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na+ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1. Copyright © 2017 the American Physiological Society.

  2. Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

    DEFF Research Database (Denmark)

    Cornelius, Flemming; Mahmmoud, Yasser Ahmed


    acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K...... with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino......+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither...

  3. Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy

    Directory of Open Access Journals (Sweden)

    Bondar Alexander


    Full Text Available Abstract Background The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform in the postsynaptic region of the spine. Conclusions A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

  4. Immunocytolocalization of Plasma Membrane H-ATPase. (United States)

    Parets-Soler, A; Pardo, J M; Serrano, R


    The localization of plasma membrane H(+)-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific anti-ATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a ;two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H(+) currents, which leave nongrowing parts and enter growing tips.

  5. Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET (United States)

    Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael


    We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

  6. Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

    DEFF Research Database (Denmark)

    Hartmann-Petersen, R; Tanaka, K; Hendil, K B


    and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking......, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7...

  7. The Role of the Plasma Membrane H(+)-ATPase in Plant Responses to Aluminum Toxicity. (United States)

    Zhang, Jiarong; Wei, Jian; Li, Dongxu; Kong, Xiangying; Rengel, Zed; Chen, Limei; Yang, Ye; Cui, Xiuming; Chen, Qi


    Aluminum (Al) toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H(+)-ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H(+)-ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H(+)-ATPase mediated H(+) influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H(+)-ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H(+)-ATPase through application of its activators (e.g., magnesium or IAA) or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H(+)-ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

  8. Structural determinants for the ouabain-stimulated increase in Na-K ATPase activity. (United States)

    Khundmiri, Syed J; Salyer, Sarah A; Farmer, Brandon; Qipshidze-Kelm, Natia; Murray, Rebecca D; Clark, Barbara J; Xie, Zijian; Pressley, Thomas A; Lederer, Eleanor D


    Recent studies suggest that at low concentrations, ouabain increases Na-K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na-K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1μgkg body wt(-1)day(-1)) for 9days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na-K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of (86)Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na-K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na-K ATPase α1 is required for translocation of both Na-K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na-K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na-K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity

    DEFF Research Database (Denmark)

    Martuseviciene, Giedre; Hofman-Bang, Jacob; Clausen, Torben


    The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested......-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect...

  10. Sodium, potassium-atpases in algae and oomycetes. (United States)

    Barrero-Gil, Javier; Garciadeblás, Blanca; Benito, Begoña


    We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.

  11. Gemini (dimeric) Surfactants

    Indian Academy of Sciences (India)

    carboxylate, sulphonate), or nonionic (polyether, sugar). (iv) gemini surfactants. Symmetrical and nonsymmetrical GS are known (see[2]), that is, these mayor may not have two identical polar groups and two identical chains. (v) GS with three or more polar groups or chains are also known. Some representative examples [2] ...

  12. Gemini (dimeric) Surfactants

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 9; Issue 3. Gemini (dimeric) Surfactants - The Two-Faced Molecules. B S Sekhon. General Article Volume 9 Issue 3 March 2004 pp 42-49. Fulltext. Click here to view fulltext PDF. Permanent link: ...

  13. Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. (United States)

    Chan, Chun-Yuan; Parra, Karlett J


    V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V1 domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. (United States)

    Efendiev, R; Bertorello, A M; Pressley, T A; Rousselot, M; Féraille, E; Pedemonte, C H


    Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

  15. The basidiomycete Ustilago maydis has two plasma membrane H⁺-ATPases related to fungi and plants. (United States)

    Robles-Martínez, Leobarda; Pardo, Juan Pablo; Miranda, Manuel; Mendez, Tavis L; Matus-Ortega, Macario Genaro; Mendoza-Hernández, Guillermo; Guerra-Sánchez, Guadalupe


    The fungal and plant plasma membrane H⁺-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H⁺-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H⁺-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H⁺-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H⁺-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H⁺-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H⁺-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H⁺-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.

  16. Proton Pumping and Slippage Dynamics of a Eukaryotic P-Type ATPase Studied at the Single-Molecule Level

    DEFF Research Database (Denmark)

    Veshaguri, Salome

    In all eukaryotes the plasma membrane potential and secondary transport systems are energized by P-type ATPases whose regulation however remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform...... pumping rates remained constant. Titration of ATP down to ~1% of apparent Km for ATPase activity exclusively affected the distributions of the durations the pump spends in active and inactive states. The dramatic consequence of our findings is that ATP reduction decreased ATP/H+ stoichiometry of the pump...

  17. [ATPase and phosphatase activity of drone brood]. (United States)

    Bodnarchuk, L I; Stakhman, O S


    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  18. Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis (United States)

    Jansen, Eric J. R.; van Bakel, Nick. H. M.; Loohuis, Nikkie F. M. Olde; Hafmans, Theo G. M.; Arentsen, Tim; Coenen, Anthon J. M.; Scheenen, Wim J. J. M.; Martens, Gerard J. M.


    The vacuolar (H+)-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V0-sector is involved in Ca2+-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V0-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca2+-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca2+-dependent-regulated exocytosis. PMID:22736765

  19. ε subunit of Bacillus subtilis F1-ATPase relieves MgADP inhibition.

    Directory of Open Access Journals (Sweden)

    Junya Mizumoto

    Full Text Available MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1, which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition.

  20. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.


    that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel...... site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM Hþ-ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger...... an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM Hþ-ATPase regulation....

  1. The F-ATPase operon from the oral streptococci S. mutans and S. sanguis: How structure relates to function (United States)

    Kuhnert, Wendi Lee


    The oral microbe, Streptococcus mutans is known to be a primary contributor to the most common infection in humans, dental caries. In the plaque environment, resident bacteria metabolize dietary sucrose which results in the production of organic acids and a decrease in plaque pH. The proton-translocating ATPase (F-ATPase) protects the bacteria from acidification by extruding protons, at the expense of ATP, to maintain an internal pH which is more neutral than the external environment. Examination of this enzyme will help us to gain insight regarding its contribution to the aciduricity characteristics of oral bacteria. In this work, our goal was to begin the molecular dissection of the mechanism by which streptococcal ATPases are regulated and function enzymatically. Sequence analysis of the F-ATPase from the non-pathogenic S. sanguis revealed that the structural genes are homologous to S. mutans as well as other sequenced F-ATPases. Cloned subunits were functionally similar as shown by complementing E. coli ATPase mutants. S. sanguis/E. coli hybrid enzymes hydrolyzed ATP, but proton conduction was uncoupled as demonstrated with inhibition studies. Transcriptional regulation of the F-ATPase operon from S. mutans was examined using chloramphenicol acetyltransferase gene fusions. Fusions containing 136 bp of DNA upstream of the promoter showed higher levels of expression as compared to those with only 16 bp. Similar to ATPase enzymatic activity, CAT expression also increased during growth at low pH. Analysis of RNA demonstrated that ATPase mRNA levels were higher at low pH, which supported the CAT activity data. Therefore, the F-ATPase from S. mutans was regulated, at least partially, by both the DNA located upstream of the promoter as well as by pH. Examination of structural models of the F-ATPase from the pathogenic oral organisms S. mutans and Lactobacillus casei and the non- pathogenic S. sanguis showed that the differences noted in the sequence of the catalytic

  2. Mixed surfactant systems for enhanced oil recovery

    Energy Technology Data Exchange (ETDEWEB)

    Llave, F.M.; Gall, B.L.; Noll, L.A.


    The results of an evaluation of mixed surfactant systems for enhanced oil recovery are described. Several surfactant combinations have been studied. These include alkyl aryl sulfonates as primary surfactants and carboxymethylated ethoxylated (CME) surfactants and ethoxylated sulfonates (ES) as secondary surfactants. The ethoxylated surfactants increase the salinity tolerance of the primary surfactants and, in theory, allow tailoring of the surfactant system to match selected reservoir conditions. The experiments conducted included interfacial tension (IFT) measurements, phase behavior measurements, adsorption and/or chromatographic separation of mixed surfactant systems, measurements of solution properties such as the critical micelle concentration (CMC) of surfactant mixtures, and crude oil displacement experiments. The effects of temperature, surfactant concentration, salinity, presence of divalent ions, hydrocarbon type, and component proportions in the mixed surfactant combinations, and injection strategies on the performance potential of the targeted surfactant/hydrocarbon systems were studied. 40 refs., 37 figs., 8 tabs.

  3. Effect of preparations Methyure and Ivine on Са(2+)-ATPases activity in plasma and vacuolar membrane of corn seedling roots under salt stress conditions. (United States)

    Rudnytska, M V; Palladina, T A


    Ca2+-ATPases regulate the functioning of Ca2+-dependent signaling pathway SOS which provides removal of Na+ from the cytoplasm of cells via Na+/H+-antiporters in saline conditions. The influence of synthetic preparations Methyure and Ivine on the Ca2+-ATPase activity was investigated. It was shown that exposition of corn seedlings in the presence of 0.1 M NaCl rather enhanced hydrolytic than transport activity of Ca2+-ATPases in plasma and vacuolar membrane of root cells. It was found that seed treatment with such preparations, especially Methyure, caused intensification of the both activities of Ca2+-ATPases, mainly in vacuolar membrane. The results indicate than salt protective activity of preparations, especially Methyure, is associated with increased Ca2+-ATPase activity, which regulates the functioning of Na+/H+-antiporters.

  4. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: [Department of Physiology, University of Tübingen (Germany)


    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  5. Surfactants at the Design Limit. (United States)

    Czajka, Adam; Hazell, Gavin; Eastoe, Julian


    This article analyzes how the individual structural elements of surfactant molecules affect surface properties, in particular, the point of reference defined by the limiting surface tension at the aqueous cmc, γcmc. Particular emphasis is given to how the chemical nature and structure of the hydrophobic tails influence γcmc. By comparing the three different classes of surfactants, fluorocarbon, silicone, and hydrocarbon, a generalized surface packing index is introduced which is independent of the chemical nature of the surfactants. This parameter ϕcmc represents the volume fraction of surfactant chain fragments in a surface film at the aqueous cmc. It is shown that ϕcmc is a useful index for understanding the limiting surface tension of surfactants and can be useful for designing new superefficient surfactants.

  6. Obg-like ATPase 1 regulates global protein serine/threonine phosphorylation in cancer cells by suppressing the GSK3β-inhibitor 2-PP1 positive feedback loop. (United States)

    Xu, Dong; Song, Renduo; Wang, Guohui; Jeyabal, Prince V S; Weiskoff, Amanda M; Ding, Kefeng; Shi, Zheng-Zheng


    OLA1 is an Obg family P-loop NTPase that possesses both GTP- and ATP-hydrolyzing activities. Here we report that OLA1 is a GSK3β interacting protein, and through its ATPase activity, inhibits the GSK3β-mediated activation of protein serine/threonine phosphatase 1 (PP1). It is hypothesized that GSK3β phosphorylates inhibitor 2 (I-2) of PP1 at Thr-72 and activates the PP1 · I-2 complex, which in turn dephosphorylates and stimulates GSK3β, thus forming a positive feedback loop. We revealed that the positive feedback loop is normally suppressed by OLA1, and becomes over-activated under OLA1 deficiency, resulting in increased cellular PP1 activity and dephosphorylation of multiple Ser/Thr phosphoproteins, and more strikingly, decreased global protein threonine phosphorylation. Furthermore, using xenograft models of colon cancer (H116) and ovarian cancer (SKOV3), we established a correlation among downregulation of OLA1, over-activation of the positive feedback loop as indicated by under-phosphorylation of I-2, and more aggressive tumor growth. This study provides the first evidence for the existence of a GSK3β-I-2-PP1 positive feedback loop in human cancer cells, and identifies OLA1 as an endogenous suppressor of this signaling motif.

  7. [Na+, K(+)-ATPase, endogenous cardiotonic steroids and their transducing role]. (United States)

    Tsymbaliuk, O V; Kosterin, S O


    Na+, K(+)-ATPase--a protein complex of plasmatic membrane, which performs the dual function: firstly, it supports the Na+ and K+ homeostasis, and also transmembrane potential gradient, secondly, it serves as the transducer of signals and as the regulator of the expression of many key genes. Endogenous cardiotonic steroids, which are synthesized in the adrenal glands and hypothalamus, serve as the signal molecules. New concepts about the mechanisms of the realization of the Na+, K(+)-ATPase signal function and their connection with cellular functions, apoptosis, and with pathologies of cardiovascular system and water-salt homeostasis are described in the survey.

  8. Retinoschisin is linked to retinal Na/K-ATPase signaling and localization


    Plössl, Karolina; Royer, Melanie; Bernklau, Sarah; Tavraz, Neslihan N.; Friedrich, Thomas; Wild, Jens; Weber, Bernhard H. F.; Friedrich, Ulrike


    Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We recently showed that retinoschisin, the protein encoded by RS1, regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase, whereas the α-subunit is exchangeable. O...

  9. Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

    DEFF Research Database (Denmark)

    Grønberg, Christina; Sitsel, Oleg; Lindahl, Erik


    Cu(+)-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu(+)-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu(+) entry using molecular-dynamics...... simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu(+) delivery. Mutational analyses...... and provide a molecular understanding of ion entry in Cu(+)-transporting P-type ATPases....

  10. The dynamic stator stalk of rotary ATPases (United States)

    Stewart, Alastair G.; Lee, Lawrence K.; Donohoe, Mhairi; Chaston, Jessica J.; Stock, Daniela


    Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases. PMID:22353718

  11. Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904. (United States)

    Kuhnert, Wendi L; Quivey Jr, Robert G


    Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.

  12. Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

    Directory of Open Access Journals (Sweden)

    Bartlett Marilyn S


    Full Text Available Abstract Background Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level. Results The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B to the same cells by two-color fluorescent in situ hybridization. Conclusions The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

  13. Maintained inspiratory activity during proportional assist ventilation in surfactant-depleted cats early after surfactant instillation: phrenic nerve and pulmonary stretch receptor activity

    Directory of Open Access Journals (Sweden)

    Schaller Peter


    Full Text Available Abstract Background Inspiratory activity is a prerequisite for successful application of patient triggered ventilation such as proportional assist ventilation (PAV. It has recently been reported that surfactant instillation increases the activity of slowly adapting pulmonary stretch receptors (PSRs followed by a shorter inspiratory time (Sindelar et al, J Appl Physiol, 2005 [Epub ahead of print]. Changes in lung mechanics, as observed in preterm infants with respiratory distress syndrome and after surfactant treatment, might therefore influence the inspiratory activity when applying PAV early after surfactant treatment. Objective To investigate the regulation of breathing and ventilatory response in surfactant-depleted young cats during PAV and during continuous positive airway pressure (CPAP early after surfactant instillation in relation to phrenic nerve activity (PNA and the activity of PSRs. Methods Seven anesthetized, endotracheally intubated young cats were exposed to periods of CPAP and PAV with the same end-expiratory pressure (0.2–0.5 kPa before and after lung lavage and after surfactant instillation. PAV was set to compensate for 75% of the lung elastic recoil. Results Tidal volume and respiratory rate were higher with lower PaCO2 and higher PaO2 during PAV than during CPAP both before and after surfactant instillation (p Conclusion PSR activity and the control of breathing are maintained during PAV in surfactant-depleted cats early after surfactant instillation, with a higher ventilatory response and a lower breathing effort than during CPAP.

  14. Studies of Ca2+ ATPase (SERCA) inhibition. (United States)

    Inesi, Giuseppe; Hua, Suming; Xu, Cheng; Ma, Hailun; Seth, Malini; Prasad, Anand M; Sumbilla, Carlota


    The Ca(2+) transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br(2)-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.


    Surfactants used to enhance remediation of soils by soil washing are often lost in the process. Neither the amount nor the cause of this loss is known. It is assumed that clays present in the soil are responsible for the loss of the surfactant. In this papere, adsorption prope...

  16. Novel Approaches to Surfactant Administration


    Gupta, Samir; Donn, Steven M.


    Surfactant replacement therapy has been the mainstay of treatment for preterm infants with respiratory distress syndrome for more than twenty years. For the most part, surfactant is administered intratracheally, followed by mechanical ventilation. In recent years, the growing interest in noninvasive ventilation has led to novel approaches of administration. This paper will review these techniques and the associated clinical evidence.

  17. V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus.

    Directory of Open Access Journals (Sweden)

    Peng Lü

    Full Text Available Silkworms are usually susceptible to the infection of Bombyx mori (B. mori nucleopolyhedrovirus (BmNPV, which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells of BmNPV-resistant strains (NB and BC8, the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi, the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.

  18. Structural studies of Ca2+-ATPase ligand and regulatory complexes

    DEFF Research Database (Denmark)

    Drachmann, Nikolaj Düring


    is regulated by the Sarcolipin homologue Phospholamban. They act upon binding by increasing the apparent Ca2+ affinity of the ATPases, thus regulating the activity in the physiologically relevant Ca2+ concentrations. In the first part of the thesis, a purification protocol of native SERCA2a from pig hearts...... is presented. The purified protein was used for X-ray crystallographic studies aiming at determining the three dimensional structure of the SERCA2a isoform in a Ca2+-free conformation. Crystals of the Ca2+ free state of SERCA2a stabilised by the inhibitor cyclopiazonic acid was obtained and a dataset...... was collected scaling to 3.26 Å resolution, allowing a preliminary structural analysis. The overall crystal structure is very similar to SERCA1a. Additionally, co-crystallisation studies have been initiated of SERCA2a and recombinantly expressed Phospholamban. Besides the above mentioned regulatory peptides...

  19. Disruption of the vacuolar-type H(+)-ATPase complex in liver causes MTORC1-independent accumulation of autophagic vacuoles and lysosomes. (United States)

    Kissing, Sandra; Rudnik, Sönke; Damme, Markus; Lüllmann-Rauch, Renate; Ichihara, Atsuhiro; Kornak, Uwe; Eskelinen, Eeva-Liisa; Jabs, Sabrina; Heeren, Jörg; De Brabander, Jef K; Haas, Albert; Saftig, Paul


    The vacuolar-type H(+)-translocating ATPase (v-H(+)-ATPase) has been implicated in the amino acid-dependent activation of the mechanistic target of rapamycin complex 1 (MTORC1), an important regulator of macroautophagy. To reveal the mechanistic links between the v-H(+)-ATPase and MTORC1, we destablilized v-H(+)-ATPase complexes in mouse liver cells by induced deletion of the essential chaperone ATP6AP2. ATP6AP2-mutants are characterized by massive accumulation of endocytic and autophagic vacuoles in hepatocytes. This cellular phenotype was not caused by a block in endocytic maturation or an impaired acidification. However, the degradation of LC3-II in the knockout hepatocytes appeared to be reduced. When v-H(+)-ATPase levels were decreased, we observed lysosome association of MTOR and normal signaling of MTORC1 despite an increase in autophagic marker proteins. To better understand why MTORC1 can be active when v-H(+)-ATPase is depleted, the activation of MTORC1 was analyzed in ATP6AP2-deficient fibroblasts. In these cells, very little amino acid-elicited activation of MTORC1 was observed. In contrast, insulin did induce MTORC1 activation, which still required intracellular amino acid stores. These results suggest that in vivo the regulation of macroautophagy depends not only on v-H(+)-ATPase-mediated regulation of MTORC1.

  20. Innovation in surfactant therapy II: surfactant administration by aerosolization. (United States)

    Pillow, J Jane; Minocchieri, S


    Instilled bolus surfactant is the only approved surfactant treatment for neonatal respiratory distress syndrome. However, recent trends towards increased utilization of noninvasive respiratory support for preterm infants with surfactant deficiency have created a demand for a similarly noninvasive means of administering exogenous surfactant. Past approaches to surfactant nebulization met with varying success due to inefficient aerosol devices resulting in low intrapulmonary delivery doses of surfactant with variable clinical effectiveness. The recent development of vibrating membrane nebulizers, coupled with appropriate positioning of the interface device, indicates that efficient delivery of aerosolized surfactant is now a realistic goal in infants. Evidence of clinical effect despite low total administered dose in pilot studies, together with suggestions of enhanced homogeneity of pulmonary distribution indicate that this therapy may be applied in a cost-effective manner, with minimal patient handling and disruption. These studies need to be subjected to appropriately designed randomized controlled trials. Further work is also required to determine the optimum delivery route (mask, intranasal prong, nasopharyngeal or laryngeal), dosing amount and redosing interval. Copyright © 2012 S. Karger AG, Basel.

  1. V-ATPase, ScNhx1p and yeast vacuole fusion. (United States)

    Qiu, Quan-Sheng


    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 summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p 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. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion. Copyright © 2012. Published by Elsevier Ltd.


    Directory of Open Access Journals (Sweden)

    T. P. Pirog


    Full Text Available It was shown literature and own experimental data concerning the use of microbial surface active glycolipids (rhamno-, sophoro- and trehalose lipids and lipopeptides for water and soil purification from oil and other hydrocarbons, removing toxic heavy metals (Cu2+, Cd2+, Ni2+, Pb2+, degradation of complex pollution (oil and other hydrocarbons with heavy metals, and the role of microbial surfactants in phytoremediation processes. The factors that limit the use of microbial surfactants in environmental technologies are discussed. Thus, at certain concentrations biosurfactant can exhibit antimicrobial properties and inhibit microorganisms destructing xenobiotics. Microbial biodegradability of surfactants may also reduce the effectiveness of bioremediation. Development of effective technologies using microbial surfactants should include the following steps: monitoring of contaminated sites to determine the nature of pollution and analysis of the autochthonous microbiota; determining the mode of surfactant introduction (exogenous addition of stimulation of surfactant synthesis by autochthonous microbiota; establishing an optimal concentration of surfactant to prevent exhibition of antimicrobial properties and rapid biodegradation; research both in laboratory and field conditions.

  3. Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. (United States)

    Lenoir, Guillaume; Menguy, Thierry; Corre, Fabienne; Montigny, Cédric; Pedersen, Per A; Thinès, Denyse; le Maire, Marc; Falson, Pierre


    Large amounts of heterologous C-terminally his-tagged SERCA1a Ca(2+)-ATPase were expressed in yeast using a galactose-regulated promoter and purified by Ni(2+) affinity chromatography followed by Reactive red chromatography. Optimizing the number of galactose inductions and increasing the amount of Gal4p transcription factor improved expression. Lowering the temperature from 28 degrees C to 18 degrees C during expression enhanced the recovery of solubilized and active Ca(2+)-ATPase. In these conditions, a 4 l yeast culture produced 100 mg of Ca(2+)-ATPase, 60 and 22 mg being pelleted with the heavy and light membrane fractions respectively, representing 7 and 1.7% of total proteins. The Ca(2+)-ATPase expressed in light membranes was 100% solubilized with L-alpha-lysophosphatidylcholine (LPC), 50% with n-dodecyl beta-D-maltoside (DM) and 25% with octaethylene glycol mono-n-dodecyl ether (C(12)E(8)). Compared to LPC, DM preserved specific activity of the solubilized Ca(2+)-ATPase during the chromatographic steps. Starting from 1/6 (3.8 mg) of the total amount of Ca(2+)-ATPase expressed in light membranes, 800 microg could be routinely purified to 50% purity by metal affinity chromatography and then 200 microg to 70% with Reactive red chromatography. The purified Ca(2+)-ATPase displayed the same K(m) for calcium and ATP as the native enzyme but a reduced specific activity ranging from 4.5 to 7.3 micromol ATP hydrolyzed/min/mg Ca(2+)-ATPase. It was stable and active for several days at 4 degrees C or after removal of DM with Bio-beads and storage at -80 degrees C.

  4. ABCG2/V-ATPase was associated with the drug resistance and tumor metastasis of esophageal squamous cancer cells

    Directory of Open Access Journals (Sweden)

    Huang Lijun


    Full Text Available Abstract Background ATP-binding cassette sub-family G member 2 (ABCG2 is a protein that in humans is encoded by the ABCG2 gene. ABCG2 participates in efflux of many chemotherapeutic agents. ABCG2 is often expressed in hematopoietic progenitor or stem cells. Vacuolar-H + −ATPase (V-ATPase plays a key role in adjusting and maintaining intracellular pH and in regulating the drug tolerance of cells. The TNM Classification of Malignant Tumours (TNM is a cancer staging system that describes the extent of cancer in a patient’s body. In this study, the expression of ABCG2 and V-ATPase in esophageal squamous cancer cells was detected. Methods Immunohistochemistry staining and Immunofluorescence double staining were used to detect the expression of ABCG2 and V-ATPase in in 66 cases of esophageal squamous cancer cells. Associations and differences in expression of ABCG2 with that of V-ATPase were analyzed. Results Positive staining patterns for both ABCG2 (66.67% and V-ATPase (68.18% were located mainly in the plasma membrane and cytoplasm. Marked differences in expression were also shown (P  Conclusions Both ABCG2 and V-ATPase were over-expressed in esophageal squamous cancer cells. Their expression was associated with pathological grade, TNM stage and tumor metastasis in esophageal squamous cancer cells, suggesting interaction relationship between them. ABCG2 and V-ATPase expression may be strongly associated with drug resistance and tumor metastasis. Virtual slides The virtual slide(s for this article can be found here:


    Directory of Open Access Journals (Sweden)

    Т. P.


    Full Text Available The aim of the work was investigation of antimicrobial effect of Nocardia vaccinii ІMV B-7405 surfactants, synthesized in various culture conditions, against phytopathogenic bacteria of genera Pseudomonas, Xanthomonas, and Pectobacterium. The antimicrobial properties of surfactant were determined in suspension culture by Koch method and also by index of the minimum inhibitory concentration. Surfactants were extracted from supernatant of cultural liquid using mixture of chloroform and methanol (2: 1. It has been established that antimicrobial properties of surfactants depend on the nature of the carbon source in the medium (refined vegetable oil, as well as waste oil after frying potatoes and meat, glycerol, the duration of the cultivation (5 and 7 days, the degree of purification of the surfactants (the supernatant of cultural liquid, purified surfactants solution and the test culture type. The highest antimicrobial activity was exhibited by purified surfactants solutions synthesized by microorganisms on the waste oil after potato frying (decreased survival of pathogenic bacteria by 50–95%, and surfactants formed within 7 days of strain B-7405 ІMV cultivation on all test substrates (minimum inhibitory concentration 7–40 µg/mL, which is several times lower than the surfactant, synthesized for 5 days. These data are promising for the development of ecologically friendly biopreparations for the regulation of the number of phytopathogenic bacteria.

  6. Treatment with exogenous surfactant stimulates endogenous surfactant synthesis in premature infants with respiratory distress syndrome

    NARCIS (Netherlands)

    Bunt, JEH; Carnielli, VP; Janssen, DJ; Wattimena, JLD; Hop, WC; Sauer, PJ; Zimmermann, LJI


    Objective: Treatment of preterm infants with respiratory distress syndrome (RDS) with exogenous surfactant has greatly improved clinical outcome. Some infants require multiple doses, and it has not been studied whether these large amounts of exogenous surfactant disturb endogenous surfactant

  7. Chemistry and technology of surfactants

    National Research Council Canada - National Science Library

    Farn, Richard J


    ... 2.3.1 Micelles and critical micelle concentration 2.3.2 Aggregate structures and shapes 2.4 Adsorption of surfactants at surfaces 2.4.1 Adsorption at liquid- gas and liquid- liquid interfaces 2.4....

  8. Molecular basis for interaction of Na+/K+-ATPase with other transporters in membrane microdomains of vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Hansen, Anne Kirstine; Matchkov, Vladimir; Bouzinova, Elena


    small arteries and in the SMC cell line A7r5. Confocal microscopy and conventional patch clamp were used in functional studies. The Na+/K+-ATPase subunits in SMCs were found to be α1 and α2. As indicated by loss of mechanical synchronization and synchronization of Ca2+ transients between SMCs...... and by direct measurements of electrical coupling between SMCs, ouabain effectively uncouples SMCs in micromolar concentrations (1-10 µM). Since rodent α1 Na+/K+-ATPase subunits are ouabain-resistant, we conclude that α2 Na+/K+-ATPase subunits is involved in regulation of the intercellular communications via...

  9. Cell-specific modulation of surfactant proteins by ambroxol treatment. (United States)

    Seifart, Carola; Clostermann, Ursula; Seifart, Ulf; Müller, Bernd; Vogelmeier, Claus; von Wichert, Peter; Fehrenbach, Heinz


    Ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanole hydrochloride], a mucolytic agent, was postulated to provide surfactant stimulatory properties and was previously used to prevent surfactant deficiency. Currently, the underlying mechanisms are not exactly clear. Because surfactant homeostasis is regulated by surfactant-specific proteins (SP), we analyzed protein amount and mRNA expression in whole lung tissue, isolated type II pneumocytes and bronchoalveolar lavage of Sprague-Dawley rats treated with ambroxol i.p. (75 mg/kg body weight, twice a day [every 12 h]). The methods used included competitive polymerase chain reaction (RT-PCR), Northern blotting, Western immunoblotting, and immunohistochemistry. In isolated type II pneumocytes of ambroxol-treated animals, SP-C protein and mRNA content were increased, whereas SP-A, -B and -D protein, mRNA, and immunoreactivity remained unaffected. However, ambroxol treatment resulted in a significant increase of SP-B and in a decrease of SP-D in whole lung tissue with enhanced immunostaining for SP-B in Clara Cells. SP-A and SP-D were significantly decreased in BAL fluid of ambroxol-treated animals. The data suggest that surfactant protein expression is modulated in a cell-specific manner by ambroxol, as type II pneumocytes exhibited an increase in SP-C, whereas Clara cells exhibited an increase in the immunoreactivity for SP-B accounting for the increased SP-B content of whole lung tissue. The results indicate that ambroxol may exert its positive effects, observed in the treatment of diseases related to surfactant deficiency, via modulation of surfactant protein expression.

  10. Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype. (United States)

    Vali, Shireen; Pallavi, Rani; Kapoor, Shweta; Tatu, Utpal


    Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC(50) value (30-100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs. The online version of this article (doi:10.1007/s11693-009-9046-3) contains supplementary material, which is available to authorized users.

  11. The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea. (United States)

    Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin


    The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na+ sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H+-ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H+-PPase (V-PPase) and vacuolar Na+/H+ antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H+-PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na+ staining showed the reduced vacuolar Na+ sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na+ sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

  12. Partial Purification of a Tonoplast ATPase from Corn Coleoptiles 1 (United States)

    Mandala, Suzanne; Taiz, Lincoln


    The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO3 with an estimated Ki of 10 millimolar. The specific activity of the KNO3-sensitive ATPase was increased 29-fold during purification. N,N′-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl− and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins. Images Fig. 4 PMID:16664239

  13. Overproduction of PIB-Type ATPases

    DEFF Research Database (Denmark)

    Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo


    Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purifi...... and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals....

  14. Mechanism of the asymmetric activation of the MinD ATPase by MinE (United States)

    Park, Kyung-Tae; Wu, Wei; Lovell, Scott; Lutkenhaus, Joe


    Summary MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE, however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE, however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE stimulated ATPase activity and suggest that it is a key residue affected by MinE binding. PMID:22651575

  15. [Peroxidative vulnerability of synaptosomal high affinity Ca++-ATPase and pharmacologic effects]. (United States)

    Blaschke, M; Fischer, H D; Schmidt, J


    The high affinity Ca++-ATPase participates essentially in the regulation of intrasynaptosomal calcium homeostasis. Related to posthypoxically restricted transmitter release, we examined the influence of newly-generated free radicals (ascorbic acid-ferric salt mixture) or sodium dodecyl sulfate in vitro and of a mild hypobaric hypoxia in vivo on the activity of synaptosomal high affinity Ca++-ATPase. Moreover we tested the effectiveness of piracetam, meclofenoxate hydrochloride, pyritinol and verapamil on the changed enzyme activity subsequent to a hypoxic exposure. The activity of synaptosomal high affinity Ca++-ATPase (1.04 +/- 0.03 mumol Pi/mg.h) is reduced by not more than 40% depending on the concentration of the ascorbic acid-ferric salt mixture used but is nearly totally inhibited by sodium dodecyl sulfate (0.2 mg/ml). Hypobaric hypoxia (18 h, 8.7 kPa) decreases the enzyme activity to 0.79 +/- 0.03 mumol Pi/mg.h. Piracetam, meclofenoxate hydrochloride and pyritinol are protectively effective on the decrease of enzyme activity induced by hypoxia. The results emphasize the importance of intact protein-phospholipid interactions for the enzyme activity and support relations between synaptosomal high affinity Ca++-ATPase and transmitter release.

  16. Sodium potassium adenosine triphosphatase (Na/K-ATPase) as a therapeutic target for uremic cardiomyopathy. (United States)

    Wang, Xiaoliang; Liu, Jiang; Drummond, Christopher A; Shapiro, Joseph I


    Clinically, patients with significant reductions in renal function present with cardiovascular dysfunction typically termed, uremic cardiomyopathy. It is a progressive series of cardiac pathophysiological changes, including left ventricular diastolic dysfunction and hypertrophy (LVH) which sometimes progress to left ventricular dilation (LVD) and systolic dysfunction in the setting of chronic kidney disease (CKD). Uremic cardiomyopathy is almost ubiquitous in patients afflicted with end stage renal disease (ESRD). Areas covered: This article reviews recent epidemiology, pathophysiology of uremic cardiomyopathy and provide a board overview of Na/K-ATPase research with detailed discussion on the mechanisms of Na/K-ATPase/Src/ROS amplification loop. We also present clinical and preclinical evidences as well as molecular mechanism of this amplification loop in the development of uremic cardiomyopathy. A potential therapeutic peptide that targets on this loop is discussed. Expert opinion: Current clinical treatment for uremic cardiomyopathy remains disappointing. Targeting the ROS amplification loop mediated by the Na/K-ATPase signaling function may provide a novel therapeutic target for uremic cardiomyopathy and related diseases. Additional studies of Na/K-ATPase and other strategies that regulate this loop will lead to new therapeutics.

  17. Biomimicry of surfactant protein C. (United States)

    Brown, Nathan J; Johansson, Jan; Barron, Annelise E


    Since the widespread use of exogenous lung surfactant to treat neonatal respiratory distress syndrome, premature infant survival and respiratory morbidity have dramatically improved. Despite the effectiveness of the animal-derived surfactant preparations, there still remain some concerns and difficulties associated with their use. This has prompted investigation into the creation of synthetic surfactant preparations. However, to date, no clinically used synthetic formulation is as effective as the natural material. This is largely because the previous synthetic formulations lacked analogues of the hydrophobic proteins of the lung surfactant system, SP-B and SP-C, which are critical functional constituents. As a result, recent investigation has turned toward the development of a new generation of synthetic, biomimetic surfactants that contain synthetic phospholipids along with a mimic of the hydrophobic protein portion of lung surfactant. In this Account, we detail our efforts in creating accurate mimics of SP-C for use in a synthetic surfactant replacement therapy. Despite SP-C's seemingly simple structure, the predominantly helical protein is extraordinarily challenging to work with given its extreme hydrophobicity and structural instability, which greatly complicates the creation of an effective SP-C analogue. Drawing inspiration from Nature, two promising biomimetic approaches have led to the creation of rationally designed biopolymers that recapitulate many of SP-C's molecular features. The first approach utilizes detailed SP-C structure-activity relationships and amino acid folding propensities to create a peptide-based analogue, SP-C33. In SP-C33, the problematic and metastable polyvaline helix is replaced with a structurally stable polyleucine helix and includes a well-placed positive charge to prevent aggregation. SP-C33 is structurally stable and eliminates the association propensity of the native protein. The second approach follows the same design

  18. A model of flow and surfactant transport in an oscillatory alveolus partially filled with liquid (United States)

    Wei, Hsien-Hung; Fujioka, Hideki; Hirschl, Ronald B.; Grotberg, James B.


    The flow and transport in an alveolus are of fundamental importance to partial liquid ventilation, surfactant transport, pulmonary drug administration, cell-cell signaling pathways, and gene therapy. We model the system in which an alveolus is partially filled with liquid in the presence of surfactants. By assuming a circular interface due to sufficiently strong surface tension and small surfactant activity, we combine semianalytical and numerical techniques to solve the Stokes flow and the surfactant transport equations. In the absence of surfactants, there is no steady streaming because of reversibility of Stokes flow. The presence of surfactants, however, induces a nontrivial cycle-averaged surfactant concentration gradient along the interface that generates steady streaming. The steady streaming patterns (e.g., number of vortices) particularly depend on the ratio of inspiration to expiration periods (I :E ratio) and the sorption parameter K. For an insoluble surfactant, a single vortex is formed when the I :E ratio is either smaller or larger than 1:1, but the recirculations have opposite directions in the two cases. A soluble surfactant can lead to more complex flow patterns such as three vortices or saddle-point flow structures. The estimated unsteady velocity is 10-3cm/s, and the corresponding Péclet number for transporting respiratory gas is O(1). For a cell-cell signaling molecule such as surfactant-associated protein-A for regulating surfactant secretion, the Péclet number could be O(10) or higher. Convection is either comparable to or more dominant than diffusion in these processes. The estimated steady velocity ranges from 10-6to10-4cm /s, depending on I :E and K, and the corresponding steady Péclet number is between 10-8/Dm and 10-6/Dm (Dm is the molecular diffusivity with units of cm2/s). Therefore, for Dm⩽10-8cm2/s, the convective transport dominates.

  19. Na,K-ATPase β1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity. (United States)

    Lee, Seung Joon; Litan, Alisa; Li, Zhiqin; Graves, Bruce; Lindsey, Stephan; Barwe, Sonali P; Langhans, Sigrid A


    The Sonic hedgehog (Shh) signaling pathway plays an important role in cerebellar development, and mutations leading to hyperactive Shh signaling have been associated with certain forms of medulloblastoma, a common form of pediatric brain cancer. While the fundamentals of this pathway are known, the molecular targets contributing to Shh-mediated proliferation and transformation are still poorly understood. Na,K-ATPase is a ubiquitous enzyme that maintains intracellular ion homeostasis and functions as a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and subunit expression have been reported in several cancers and loss of the β1-subunit has been associated with a poorly differentiated phenotype in carcinoma but its role in medulloblastoma progression is not known. Human medulloblastoma cell lines and primary cultures of cerebellar granule cell precursors (CGP) were used to determine whether Shh regulates Na,K-ATPase expression. Smo/Smo medulloblastoma were used to assess the Na,K-ATPase levels in vivo. Na,K-ATPase β1-subunit was knocked down in DAOY cells to test its role in medulloblastoma cell proliferation and tumorigenicity. Na,K-ATPase β1-subunit levels increased with differentiation in normal CGP cells. Activation of Shh signaling resulted in reduced β1-subunit mRNA and protein levels and was mimicked by overexpression of Gli1and Bmi1, both members of the Shh signaling cascade; overexpression of Bmi1 reduced β1-subunit promoter activity. In human medulloblastoma cells, low β1-subunit levels were associated with increased cell proliferation and in vivo tumorigenesis. Na,K-ATPase β1-subunit is a target of the Shh signaling pathway and loss of β1-subunit expression may contribute to tumor development and progression not only in carcinoma but also in medulloblastoma, a tumor of neuronal origin.

  20. Pulmonary surfactant and bacterial lipopolysaccharide: the interaction and its functional consequences. (United States)

    Kolomaznik, M; Nova, Z; Calkovska, A


    The respiratory system is constantly exposed to pathogens which enter the lungs by inhalation or via blood stream. Lipopolysaccharide (LPS), also named endotoxin, can reach the airspaces as the major component of the outer membrane of Gram-negative bacteria, and lead to local inflammation and systemic toxicity. LPS affects alveolar type II (ATII) cells and pulmonary surfactant and although surfactant molecule has the effective protective mechanisms, excessive amount of LPS interacts with surfactant film and leads to its inactivation. From immunological point of view, surfactant specific proteins (SPs) SP-A and SP-D are best characterized, however, there is increasing evidence on the involvement of SP-B and SP-C and certain phospholipids in immune reactions. In animal models, the instillation of LPS to the respiratory system induces acute lung injury (ALI). It is of clinical importance that endotoxin-induced lung injury can be favorably influenced by intratracheal instillation of exogenous surfactant. The beneficial effect of this treatment was confirmed for both natural porcine and synthetic surfactants. It is believed that the surfactant preparations have anti-inflammatory properties through regulating cytokine production by inflammatory cells. The mechanism by which LPS interferes with ATII cells and surfactant layer, and its consequences are discussed below.

  1. Toward an understanding of the Cdc48/p97 ATPase [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Nicholas Bodnar


    Full Text Available A conserved AAA+ ATPase, called Cdc48 in yeast and p97 or VCP in metazoans, plays an essential role in many cellular processes by segregating polyubiquitinated proteins from complexes or membranes. For example, in endoplasmic reticulum (ER-associated protein degradation (ERAD, Cdc48/p97 pulls polyubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome. Cdc48/p97 consists of an N-terminal domain and two ATPase domains (D1 and D2. Six Cdc48 monomers form a double-ring structure surrounding a central pore. Cdc48/p97 cooperates with a number of different cofactors, which bind either to the N-terminal domain or to the C-terminal tail. The mechanism of Cdc48/p97 action is poorly understood, despite its critical role in many cellular systems. Recent in vitro experiments using yeast Cdc48 and its heterodimeric cofactor Ufd1/Npl4 (UN have resulted in novel mechanistic insight. After interaction of the substrate-attached polyubiquitin chain with UN, Cdc48 uses ATP hydrolysis in the D2 domain to move the polypeptide through its central pore, thereby unfolding the substrate. ATP hydrolysis in the D1 domain is involved in substrate release from the Cdc48 complex, which requires the cooperation of the ATPase with a deubiquitinase (DUB. Surprisingly, the DUB does not completely remove all ubiquitin molecules; the remaining oligoubiquitin chain is also translocated through the pore. Cdc48 action bears similarities to the translocation mechanisms employed by bacterial AAA ATPases and the eukaryotic 19S subunit of the proteasome, but differs significantly from that of a related type II ATPase, the NEM-sensitive fusion protein (NSF. Many questions about Cdc48/p97 remain unanswered, including how it handles well-folded substrate proteins, how it passes substrates to the proteasome, and how various cofactors modify substrates and regulate its function.

  2. Synthesis of carbohydrate-based surfactants (United States)

    Pemberton, Jeanne E.; Polt, Robin L.; Maier, Raina M.


    The present invention provides carbohydrate-based surfactants and methods for producing the same. Methods for producing carbohydrate-based surfactants include using a glycosylation promoter to link a carbohydrate or its derivative to a hydrophobic compound.

  3. Fungal growth on anion surfactant medium. (United States)

    Hamada, Nobuo; Abe, Niichiro


    Before the present study, no fungi using anion surfactant as a nutrient had been identified, although some fungi were known to use nonion surfactant. Washing water collected from 63 washing machines was inoculated onto 0.1% LAS (Sodium dodecyl benzenesulfonate) anion surfactant media to identify fungi that can feed on anion-surfactant. Small dark colonies of fungi were found on several of the Petri-dishes from 12 days after inoculation. These were identified as Cladophialophora boppii and Exophiala spinifera using morphological features and rDNA data. A number of the isolates of C. boppii specifically were recognized as using anion surfactant as a nutrient. The growth characteristics of the two fungal species were examined on surfactant media of three kinds. Apart from anion surfactant, the fungi were also able to grow on nonion surfactant and on soap. The application of these fungi for environmental cleansing after detergent pollution is also discussed.

  4. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.


    The H translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ approx. = 70,000 and approx. = 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-( UC)ethylmaleimide and 7-chloro-4-nitro( UC)benzo-2-oxa-1,3-diazole, labeled the M/sub r/ approx. = 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-( UC)dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ approx. = 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10V, 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ approx. = 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

  5. Dissolution of anionic surfactant mesophases. (United States)

    Poulos, Andreas S; Jones, Christopher S; Cabral, João T


    Linear and circular solvent penetration experiments are used to study the dissolution of anionic SLE3S surfactant mesophases in water. We show that a lamellar (Lα) phase in contact with water will transit through a series of cubic, hexagonal, and micellar phase bands with sharp interfaces identified from their optical textures. In both linear and circular geometries, the kinetics of front propagation and eventual dissolution are well described by diffusive penetration of water, and a simple model applies to both geometries, with a different effective diffusion coefficient for water Df as the only fitting parameter. Finally, we show a surprising variation of dissolution rates with initial surfactant concentration that can be well explained by assuming that the driving force for solvent penetration is the osmotic pressure difference between neat water and the aqueous fraction of the mesophase that is highly concentrated in surfactant counterions.

  6. Adaptation to low body temperature influences pulmonary surfactant composition thereby increasing fluidity while maintaining appropriately ordered membrane structure and surface activity. (United States)

    Suri, Lakshmi N M; McCaig, Lynda; Picardi, Maria V; Ospina, Olga L; Veldhuizen, Ruud A W; Staples, James F; Possmayer, Fred; Yao, Li-Juan; Perez-Gil, Jesus; Orgeig, Sandra


    The interfacial surface tension of the lung is regulated by phospholipid-rich pulmonary surfactant films. Small changes in temperature affect surfactant structure and function in vitro. We compared the compositional, thermodynamic and functional properties of surfactant from hibernating and summer-active 13-lined ground squirrels (Ictidomys tridecemlineatus) with porcine surfactant to understand structure-function relationships in surfactant membranes and films. Hibernating squirrels had more surfactant large aggregates with more fluid monounsaturated molecular species than summer-active animals. The latter had more unsaturated species than porcine surfactant. Cold-adapted surfactant membranes displayed gel-to-fluid transitions at lower phase transition temperatures with reduced enthalpy. Both hibernating and summer-active squirrel surfactants exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and DPH anisotropy revealed that surfactant bilayers from both groups of squirrels possessed similar ordered phase characteristics at low temperatures. While ground squirrel surfactants functioned well during dynamic cycling at 3, 25, and 37 degrees C, porcine surfactant demonstrated poorer activity at 3 degrees C but was superior at 37 degrees C. Consequently the surfactant composition of ground squirrels confers a greater thermal flexibility relative to homeothermic mammals, while retaining tight lipid packing at low body temperatures. This may represent the most critical feature contributing to sustained stability of the respiratory interface at low lung volumes. Thus, while less effective than porcine surfactant at 37 degrees C, summer-active surfactant functions adequately at both 37 degrees C and 3 degrees C allowing these animals to enter hibernation. Here further compositional alterations occur which improve function at low temperatures by maintaining adequate stability at low lung volumes and when temperature increases during arousal from

  7. Cationic surfactants as the hydrolytic micellar catalysts


    Janošcová, Petra


    Cationic surfactants as the hydrolytic micellar catalysts Petra Janošcová The effectiveness of hydrolytic cleavage of the pesticide fenitrothionin cationic surfactants micellar media has been tested. All used surfactants increased the rate of fenitrothionhydrolysis, which was the evidence of micellar catalysis. For some surfactants decreases has been evident at the highest rate of hydrolysis concentrations. It has been the result of a phenomenon called the effect of empty micelles. High hydro...

  8. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed


    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence, but not in the absence of K+. Studies with purified membranes revealed that CPZ reduced Na+-dependent phosphorylation by interference with Na+ binding from the intracellular side of the memb...

  9. Review: P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas

    DEFF Research Database (Denmark)

    Andersen, Jens P; Vestergaard, Anna L; Mikkelsen, Stine A


    coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases...... on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms....

  10. Surfactant adsorption to soil components and soils

    NARCIS (Netherlands)

    Ishiguro, Munehide; Koopal, Luuk K.


    Soils are complex and widely varying mixtures of organic matter and inorganic materials; adsorption of surfactants to soils is therefore related to the soil composition. We first discuss the properties of surfactants, including the critical micelle concentration (CMC) and surfactant adsorption on


    A demonstration of surfactant-enhanced aquifer remediation was conducted during the spring of 1999 at Marine Corps Base, Camp LeJeune, NC. A PCE-DNAPL zone was identified and delineated by extensive soil sampling in 1997, and was further characteized by a partitioning interwell t...

  12. A plasma membrane H + ATPase gene is germinationinduced in ...

    African Journals Online (AJOL)

    The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat embryos.

  13. Changes in erythrocyte ATPase activity under different pathological ...

    African Journals Online (AJOL)

    Conclusion: Na+- K+ATPase specific activity is a biomarker for the diagnosis of individuals with different physiological diseases. Keywords: Na+-K+ATPase ... aging and that changes in young females were more re- markable than that in young .... activity which might be due to increased / decreased con- centration of ions ...

  14. Evolution, Development, and Function of the Pulmonary Surfactant System in Normal and Perturbed Environments. (United States)

    Orgeig, Sandra; Morrison, Janna L; Daniels, Christopher B


    Surfactant lipids and proteins form a surface active film at the air-liquid interface of internal gas exchange organs, including swim bladders and lungs. The system is uniquely positioned to meet both the physical challenges associated with a dynamically changing internal air-liquid interface, and the environmental challenges associated with the foreign pathogens and particles to which the internal surface is exposed. Lungs range from simple, transparent, bag-like units to complex, multilobed, compartmentalized structures. Despite this anatomical variability, the surfactant system is remarkably conserved. Here, we discuss the evolutionary origin of the surfactant system, which likely predates lungs. We describe the evolution of surfactant structure and function in invertebrates and vertebrates. We focus on changes in lipid and protein composition and surfactant function from its antiadhesive and innate immune to its alveolar stability and structural integrity functions. We discuss the biochemical, hormonal, autonomic, and mechanical factors that regulate normal surfactant secretion in mature animals. We present an analysis of the ontogeny of surfactant development among the vertebrates and the contribution of different regulatory mechanisms that control this development. We also discuss environmental (oxygen), hormonal and biochemical (glucocorticoids and glucose) and pollutant (maternal smoking, alcohol, and common "recreational" drugs) effects that impact surfactant development. On the adult surfactant system, we focus on environmental variables including temperature, pressure, and hypoxia that have shaped its evolution and we discuss the resultant biochemical, biophysical, and cellular adaptations. Finally, we discuss the effect of major modern gaseous and particulate pollutants on the lung and surfactant system. Copyright © 2015 John Wiley & Sons, Inc.

  15. Surfactant maturation is not delayed in human fetuses with diaphragmatic hernia.

    Directory of Open Access Journals (Sweden)

    Olivier Boucherat


    Full Text Available BACKGROUND: Pulmonary hypoplasia and persistent pulmonary hypertension account for significant mortality and morbidity in neonates with congenital diaphragmatic hernia (CDH. Global lung immaturity and studies in animal models suggest the presence of surfactant deficiency that may further complicate the pathophysiology of CDH. However, data about surfactant status in human fetuses with CDH at birth are contradictory. The lack of a chronological study of surfactant content in late pregnancy has been a significant limitation. The appropriateness of administering surfactant supplements to neonates with CDH is therefore a debated question. METHODS AND FINDINGS: We investigated surfactant content in human fetuses with CDH compared to age-matched fetuses with nonpulmonary diseases used as controls. Concentrations of disaturated phosphatidylcholine and surfactant proteins were found to be similar at a given stage of pregnancy, with both components showing a similar pattern of increase with progressing pregnancy in fetuses with CDH and in control fetuses. Thyroid transcription factor 1, a critical regulator of surfactant protein transcription, similarly displayed no difference in abundance. Finally, we examined the expression of three glucocorticoid-regulated diffusible mediators involved in lung epithelial maturation, namely: keratinocyte growth factor (KGF, leptin, and neuregulin 1 beta 1 (NRG1-beta1. KGF expression decreased slightly with time in control fetuses, but remained unchanged in fetuses with CDH. Leptin and NRG1-beta1 similarly increased in late pregnancy in control and CDH lungs. These maturation factors were also determined in the sheep fetus with surgical diaphragmatic hernia, in which surfactant deficiency has been reported previously. In contrast to the findings in humans, surgical diaphragmatic hernia in the sheep fetus was associated with decreased KGF and neuregulin expression. Fetoscopic endoluminal tracheal occlusion performed in

  16. Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation. (United States)

    Martz, Françoise; Sutinen, Marja-Liisa; Kiviniemi, Sari; Palta, Jiwan P


    -acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.

  17. Aerosol delivery of synthetic lung surfactant

    Directory of Open Access Journals (Sweden)

    Frans J. Walther


    Full Text Available Background. Nasal continuous positive airway pressure (nCPAP is a widely accepted technique of non-invasive respiratory support in premature infants with respiratory distress syndrome due to lack of lung surfactant. If this approach fails, the next step is often intubation, mechanical ventilation (MV and intratracheal instillation of clinical lung surfactant.Objective. To investigate whether aerosol delivery of advanced synthetic lung surfactant, consisting of peptide mimics of surfactant proteins B and C (SP-B and SP-C and synthetic lipids, during nCPAP improves lung function in surfactant-deficient rabbits.Methods. Experimental synthetic lung surfactants were produced by formulating 3% Super Mini-B peptide (SMB surfactant, a highly surface active SP-B mimic, and a combination of 1.5% SMB and 1.5% of the SP-C mimic SP-Css ion-lock 1 (BC surfactant, with a synthetic lipid mixture. After testing aerosol generation using a vibrating membrane nebulizer and aerosol conditioning (particle size, surfactant composition and surface activity, we investigated the effects of aerosol delivery of synthetic SMB and BC surfactant preparations on oxygenation and lung compliance in saline-lavaged, surfactant-deficient rabbits, supported with either nCPAP or MV.Results. Particle size distribution of the surfactant aerosols was within the 1–3 µm distribution range and surfactant activity was not affected by aerosolization. At a dose equivalent to clinical surfactant therapy in premature infants (100 mg/kg, aerosol delivery of both synthetic surfactant preparations led to a quick and clinically relevant improvement in oxygenation and lung compliance in the rabbits. Lung function recovered to a greater extent in rabbits supported with MV than with nCPAP. BC surfactant outperformed SMB surfactant in improving lung function and was associated with higher phospholipid values in bronchoalveolar lavage fluid; these findings were irrespective of the type of ventilatory

  18. Surfactant gene polymorphisms and interstitial lung diseases

    Directory of Open Access Journals (Sweden)

    Pantelidis Panagiotis


    Full Text Available Abstract Pulmonary surfactant is a complex mixture of phospholipids and proteins, which is present in the alveolar lining fluid and is essential for normal lung function. Alterations in surfactant composition have been reported in several interstitial lung diseases (ILDs. Furthermore, a mutation in the surfactant protein C gene that results in complete absence of the protein has been shown to be associated with familial ILD. The role of surfactant in lung disease is therefore drawing increasing attention following the elucidation of the genetic basis underlying its surface expression and the proof of surfactant abnormalities in ILD.

  19. The late administration of surfactant

    African Journals Online (AJOL)

    Arterial/alveolar oxygen ratio following administration of surfactant. Hours After Dosing. The outcome of infants receiving delayed SRT was good. The mean duration of ventilation of surviving infants was. 7,9 ± 4,3 days. Oxygen therapy post ventilation was required for a mean of 5,7 ± 4,5 days and infants were hospitalised ...

  20. The loss-of-function disease-mutation G301R in the Na+/K+-ATPase α2 isoform decreases lesion volume and improves functional outcome after acute spinal cord injury in mice

    DEFF Research Database (Denmark)

    Ellman, Ditte; Isaksen, Toke Jost; Lund, Minna


    BACKGROUND: The Na(+)/K(+)-ATPases are transmembrane ion pumps important for maintenance of ion gradients across the plasma membrane that serve to support multiple cellular functions, such as membrane potentials, regulation of cellular volume and pH, and co-transport of signaling transmitters...... to anaerobic metabolism and lactate accumulation. During ischemia, Na(+)/K(+)-ATPase-related functions will naturally increase the energy demand of the Na(+)/K(+)-ATPase ion pump. However, the role of the α2Na(+)/K(+)-ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice...... for the loss-of-function disease-mutation G301R in the Atp1a2 gene (α 2(+/G301R) ) to study the effect of reduced α2Na(+)/K(+)-ATPase expression in a moderate contusion spinal cord injury (SCI) model. RESULTS: We found that α 2(+/G301R) mice display significantly improved functional recovery and decreased...

  1. RNAi-directed downregulation of vacuolar H(+ -ATPase subunit a results in enhanced stomatal aperture and density in rice.

    Directory of Open Access Journals (Sweden)

    Huiying Zhang

    Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

  2. Interactions of surfactants with lipid membranes. (United States)

    Heerklotz, Heiko


    Surfactants are surface-active, amphiphilic compounds that are water-soluble in the micro- to millimolar range, and self-assemble to form micelles or other aggregates above a critical concentration. This definition comprises synthetic detergents as well as amphiphilic peptides and lipopeptides, bile salts and many other compounds. This paper reviews the biophysics of the interactions of surfactants with membranes of insoluble, naturally occurring lipids. It discusses structural, thermodynamic and kinetic aspects of membrane-water partitioning, changes in membrane properties induced by surfactants, membrane solubilisation to micelles and other phases formed by lipid-surfactant systems. Each section defines and derives key parameters, mentions experimental methods for their measurement and compiles and discusses published data. Additionally, a brief overview is given of surfactant-like effects in biological systems, technical applications of surfactants that involve membrane interactions, and surfactant-based protocols to study biological membranes.

  3. Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms (United States)

    Mobasheri, Ali; Trujillo, Elisa; Arteaga, Mari-Francis; Martín-Vasallo, Pablo


    Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons). Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4) using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes. PMID:22606027

  4. Disaturated-phosphatidylcholine and surfactant protein-B turnover in human acute lung injury and in control patients. (United States)

    Simonato, Manuela; Baritussio, Aldo; Ori, Carlo; Vedovelli, Luca; Rossi, Sandra; Dalla Massara, Lorenza; Rizzi, Sabina; Carnielli, Virgilio P; Cogo, Paola E


    Patients with adult respiratory distress syndrome (ARDS) and acute lung injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover. To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls). 2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured. 1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively). 1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

  5. Identification of β-chain of FoF1-ATPase in apoptotic cell population induced by Microplitis bicoloratus bracovirus and its role in the development of Spodoptera litura. (United States)

    Kou, Tian-Chao; Liu, Yue-Tong; Li, Ming; Yang, Yang; Zhang, Wei; Cui, Ji-Hui; Zhang, Xue-Wen; Dong, Shu-Mei; Xu, Sha; You, Shan; Yu, Dong-Shuai; Pang, Zun-Yu; Luo, Kai-Jun


    Two physiological changes of Spodoptera litura parasitized by Microplitis bicoloratus are hemocyte-apoptosis and retarded immature development. β-Chain of F o F 1 -ATPase was found from a S. litura transcriptome. It belongs to a conserved P-loop NTPase superfamily, descending from a common ancestor of Lepidopteran clade. However, the characterization of β-chain of ATPase in apoptotic cells and its involvement in development remain unknown. Here, the ectopic expression and endogenous F o F 1 -ATPase β-chain occurred on S. litura cell membrane: in vivo, at the late stage of apoptotic hemocyte, endogenous F o F 1 -ATPase β-chain was stably expressed during M. bicoloratus larva development from 4 to 7 days post-parasitization; in vitro, at an early stage of pre-apoptotic Spli221 cells by infecting with M. bicoloratus bracovirus particles, the proteins were speedily recover expression. Furthermore, endogenous F o F 1 -ATPase β-chain was localized on the apoptotic cell membrane. RNA interference (RNAi) of F o F 1 -ATPase β-chain led to significantly decreased head capsule width. This suggested that F o F 1 -ATPase β-chain positively regulated the development of S. litura. The RNAi effect on the head capsule width was enhanced with parasitism. Our research found that F o F 1 -ATPase β-chain was expressed and localized on the cell membrane in the apoptotic cells, and involved in the development of S. litura. © 2017 Wiley Periodicals, Inc.

  6. The Kdp-ATPase system and its regulation

    Indian Academy of Sciences (India)

    Madhu Sudhan


    Mar 15, 2007 ... Potassium (K+), the major intracellular cation, is essential for life in both prokaryotic and eukaryotic cells. The high intracellular concentration of K+ in bacteria reflects an ancient strategy of living cells to accumulate K+ and exclude. Na+ ion (Epstein 2003). K+, being the most dominant intracellular cation ...

  7. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

    Directory of Open Access Journals (Sweden)

    Brian Panicucci


    Full Text Available The mitochondrial (mt FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF, but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF, which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm. Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1 binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1, but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

  8. Perfluorinated surfactants in surface and drinking waters. (United States)

    Skutlarek, Dirk; Exner, Martin; Färber, Harald


    the Ruhr river and the Moehne river (tributary of the Ruhr) (Ruhr: up to 446 ng/L, Moehne: up to 4385 ng/L). The maximum concentration of all drinking water samples taken in the Rhine-Ruhr area was determined at 598 ng/L with the major component PFOA (519 ng/L). The surface water contaminations most likely stem from contaminated inorganic and organic waste materials (so-called 'Abfallgemisch'). This waste material was legally applied to several agricultural areas on the upper reaches of the Moehne. Perfluorinated surfactants could be detected in some suchlike soil samples. They contaminated the river and the reservoir belonging to it, likely by superficial run-off over several months or probably years. Downstream, dilution effects are held responsible for decreasing concentrations of PS in surface waters of the Moehne and the Ruhr river. In analogy to the surface water samples, PS (major component PFOA) can be determined in many drinking water samples of the Rhine-Ruhr area where the water supplies are mainly based on bank filtration and artificial recharge. The concentrations found in drinking waters decreased with the concentrations of the corresponding raw water samples along the flow direction of the Ruhr river (from east to west) and were not significantly different from surface water concentrations. This indicates that perfluorinated surfactants are at present not successfully removed by water treatment steps. Because of their different problematic properties (persistence, mobility, toxicity, bioaccumulation), the concentrations of specific perfluorinated surfactants and their precursors in drinking waters and food have to be minimised. Therefore, it is of utmost importance to take the initiative to establish suitable legal regulations (limitations/ban) concerning the production and use of these surfactants and their precursors. Furthermore, it is indispensable to protect water resources from these compounds. A discussion on appropriate limit values in drinking

  9. A bioassay-guided fractionation system to identify endogenous small molecules that activate plasma membrane H+-ATPase activity in Arabidopsis. (United States)

    Han, Xiuli; Yang, Yongqing; Wu, Yujiao; Liu, Xiaohui; Lei, Xiaoguang; Guo, Yan


    Plasma membrane (PM) H+-ATPase is essential for plant growth and development. Various environmental stimuli regulate its activity, a process that involves many protein cofactors. However, whether endogenous small molecules play a role in this regulation remains unknown. Here, we describe a bio-guided isolation method to identify endogenous small molecules that regulate PM H+-ATPase activity. We obtained crude extracts from Arabidopsis seedlings with or without salt treatment and then purified them into fractions based on polarity and molecular mass by repeated column chromatography. By evaluating the effect of each fraction on PM H+-ATPase activity, we found that fractions containing the endogenous, free unsaturated fatty acids oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) extracted from salt-treated seedlings stimulate PM H+-ATPase activity. These results were further confirmed by the addition of exogenous C18:1, C18:2, or C18:3 in the activity assay. The ssi2 mutant, with reduced levels of C18:1, C18:2, and C18:3, displayed reduced PM H+-ATPase activity. Furthermore, C18:1, C18:2, and C18:3 directly bound to the C-terminus of the PM H+-ATPase AHA2. Collectively, our results demonstrate that the binding of free unsaturated fatty acids to the C-terminus of PM H+-ATPase is required for its activation under salt stress. The bio-guided isolation model described in this study could enable the identification of new endogenous small molecules that modulate essential protein functions, as well as signal transduction, in plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  10. 7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells. (United States)

    Duran, M J; Pierre, S V; Lesnik, P; Pieroni, G; Bourdeaux, M; Dignat-Georges, F; Sampol, J; Maixent, J M


    As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

  11. Overexpression of Na(+)/K (+)-ATPase parallels the increase in sodium transport and potassium recycling in an in vitro model of proximal tubule cellular ageing. (United States)

    Silva, E; Gomes, P; Soares-da-Silva, P


    Na(+)/K(+)-ATPase plays a key role in the transport of Na(+) throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na(+) and K(+) ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na(+)/K(+)-ATPase activity and K(+) conductance were evaluated using electrophysiological methods. Na(+)K(+)-ATPase alpha(1)- and beta(1)-subunit expression was quantified by Western blot techniques. Na(+)/H(+) exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na(+) transport, which results from an increase in the number of Na(+)/K(+)-ATPase alpha(1)- and beta(1)-subunits, in the membrane. Increases in Na(+)/K(+)-ATPase activity were accompanied by increases in K(+) conductance as a result of functional coupling between Na(+)/K(+)-ATPase and basolateral K(+) channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na(+)/H(+) exchanger activity were observed. H(2)O(2) production was increased in aged cells, but exposure for 5 days to 1 and 10 microM: of H(2)O(2) had no effect on Na(+)/K(+)-ATPase expression. Ouabain (100 nM: ) increased alpha(1)-subunit, but not beta(1)-subunit, Na(+)/K(+)-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.

  12. Metal Fluoride Complexes of Na,K-ATPase (United States)

    Cornelius, Flemming; Mahmmoud, Yasser A.; Toyoshima, Chikashi


    The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K+·Pi with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na+, after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. PMID:21708939

  13. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities (United States)

    Caldwell, C.


    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  14. Surfactants in tribology, v.3

    CERN Document Server

    Biresaw, Girma


    The manufacture and use of almost every consumer and industrial product rely on application of advanced knowledge in surface science and tribology. These two disciplines are of critical importance in major economic sectors, such as mining, agriculture, manufacturing (including metals, plastics, wood, computers, MEMS, NEMS, appliances), construction, transportation, and medical instruments, transplants, and diagnostic devices. An up-to-date reference with contributions by experts in surface science and tribology, Surfactants in Tribology, Volume 3 discusses some of the underlying tribological a

  15. The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles (United States)

    Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio


    Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

  16. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R. (Brigham and Women' s Hospital, Boston, MA (USA))


    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake.

  17. A Review of Surfactant Role in Soil Clogging Processes at Wastewater Exfiltration Locations in Sewers. (United States)

    Nikpay, Mitra; Krebs, Peter; Ellis, Bryan


      Wastewater contains significant sources of pollutants and contaminants. often the failure of a pipe, inadequate sealing or corrupt pipe-connections cause the loss of raw sewage, which percolates into the nearby soil. As a consequence, a colmation layer in conjunction with soil clogging is developing, which regulates the exfiltration rate. Recently, literature has emerged that offers findings about the effects of wastewater surfactants on the change of physical properties of the soil. A survey of published literature in this field provides information highlighting the influential mechanisms of surfactants in soil clogging through physical, chemical and biological processes. Therefore, to provide a comprehensive approach, this review describes the adsorption mechanisms of surfactants on organic and inorganic particles, at gas-bubbles and at biomass. We also provided our own input to the description of the adsorption of surfactants at fluid/fluid and fluid/solid interfaces in porous media associated with the clogging process.

  18. Surfactant transport on viscous bilayers (United States)

    Matar, Omar; Craster, Richard; Warner, Mark


    We model the external delivery of surfactant to pulmonary airways, an integral part of Surfactant Replacement Therapy (SRT), a method of treatment of Respiratory Distress Syndrome in neonates. We examine the spreading dynamics of insoluble surfactant by Marangoni stresses along the mucus-perciliary liquid bilayers that line the inside of airways. The bilayer is modelled as a thin highly viscous mucus surface film (mucus) overlying a much less viscous perciliary liquid layer (PCL); this is appropriate for small airways. By exploiting this large viscosity constrast, a variant of standard lubrication theory is adopted wherein terms, which would have otherwise been neglected in the lubrication approximation, are promoted in order to model correctly the presence of the mucus. Inclusion of van der Waals forces in the model permit the study of the effect of this mucus 'skin' on the possibility of bilayer rupture, a potential cause of failure of SRT. We find that increasing the viscosity contrast and initial mucus layer thickness delays the onset of rupture, while increasing the relative significance of Marangoni stresses leads to more marked thinning and rapid bilayer rupture [1]. [1] O. K. Matar, R. V. Craster and M. R. Warner, submitted to J. Fluid Mech. (2001).

  19. Hydrodynamic size of DNA/cationic gemini surfactant complex as a function of surfactant structure. (United States)

    Devínsky, Ferdinand; Pisárcik, Martin; Lacko, Ivan


    The present study deals with the determination of hydrodynamic size of DNA/cationic gemini surfactant complex in sodium bromide solution using the dynamic light scattering method. Cationic gemini surfactants with polymethylene spacer of variable length were used for the interaction with DNA. The scattering experiments were performed at constant DNA and sodium bromide concentrations and variable surfactant concentration in the premicellar and micellar regions as a function of surfactant spacer length. It was found that the DNA conformation strongly depends on the polymethylene spacer length as well as on the surfactant concentration relative to the surfactant critical micelle concentration. Gemini surfactant molecules with 4 methylene groups in the spacer were found to be the least efficient DNA compacting agent in the region above the surfactant cmc. Gemini molecules with the shortest spacer length (2 methylene groups) and the longest spacer length (8 methylene groups) investigated showed the most efficient DNA compaction ability.

  20. Surfactant inhibition in acute respiratory failure : consequences for exogenous surfactant therapy

    NARCIS (Netherlands)

    E.P. Eijking (Eric)


    textabstractThe neonatal respiratory distress syndrome (RDS) is characterized by immaturity of the lung, resulting in relative or absolute absence of pulmonary surfactant. Worldwide, neonates suffering from RDS have been treated successfully with exogenous surfactant preparations. Currently,

  1. Acute Pathophysiological Effects of Intratracheal Instillation of Budesonide and Exogenous Surfactant in a Neonatal Surfactant-depleted Piglet Model

    Directory of Open Access Journals (Sweden)

    Chia-Feng Yang


    Conclusions: Intratracheal instillation of surfactant or surfactant plus budesonide can improve oxygenation and pulmonary histologic outcome in neonatal surfactant-depleted lungs. The additional use of budesonide does not disturb the function of the exogenous surfactant. Intratracheal administration of a corticosteroid combined with surfactant may be an effective method for alleviating local pulmonary inflammation in severe RDS.

  2. Surfactant inhibition in acute respiratory failure : consequences for exogenous surfactant therapy


    Eijking, Eric


    textabstractThe neonatal respiratory distress syndrome (RDS) is characterized by immaturity of the lung, resulting in relative or absolute absence of pulmonary surfactant. Worldwide, neonates suffering from RDS have been treated successfully with exogenous surfactant preparations. Currently, exogenous surfactant administration has been accepted as a valuable treatment for this syndrome. Nevertheless, many questions on exogenous surfactant treatment remain unanswered. It has been observed that...

  3. Influence of surfactant on gas bubble stability. (United States)

    Hanwright, Jennifer; Zhou, James; Evans, Geoffrey M; Galvin, Kevin P


    Gas-bubble stability is achieved either by a reduction in the Laplace pressure or by a reduction in the permeability of the gas-liquid interface. Although insoluble surfactants have been shown definitively in many studies to lower the permeability of the gas-liquid interface and hence increase the resistance to interfacial mass transfer, remarkably little work has been done on the effects of soluble surfactants. An experimental system was developed to measure the effect of the soluble surfactant dodecyl trimethylammonium bromide on the desorption and absorption of carbon dioxide gas through a quiescent planar interface. The desorption experiments conformed to the model of non-steady-state molecular diffusion. The absorption experiments, however, produced an unexpected mass transfer mechanism, with surface renewal, probably because of instability in the density gradient formed by the carbon dioxide. In general, the soluble surfactant produced no measurable reduction in the rate of interfacial mass transfer for desorption or absorption. This finding is consistent with the conclusion of Caskey and Barlage that soluble surfactants produce a significantly lower resistance to interfacial mass transfer than do insoluble surfactants. The dynamic adsorption and desorption of the surfactant molecules at the gas-liquid interface creates short-term vacancies, which presumably permit the unrestricted transfer of the gas molecules through the interface. This surfactant exchange does not occur for insoluble surfactants. Gas bubbles formed in the presence of a high concentration of soluble surfactant were observed to dissolve completely, while those formed in the presence of the insoluble surfactant stearic acid did not dissolve easily, and persisted for very long periods. The interfacial concentration of stearic acid rises during bubble dissolution, as it is insoluble, and must eventually achieve full monolayer coverage and a state of compression, lowering the permeability of the

  4. Surfactant Therapy of ALI and ARDS (United States)

    Raghavendran, K; Willson, D; Notter, RH


    This article examines exogenous lung surfactant replacement therapy and its utility in mitigating clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Biophysical research has documented that lung surfactant dysfunction can be reversed or mitigated by increasing surfactant concentration, and multiple studies in animals with ALI/ARDS have shown that respiratory function and pulmonary mechanics in vivo can be improved by exogenous surfactant administration. Exogenous surfactant therapy is a routine intervention in neonatal intensive care, and is life-saving in preventing or treating the neonatal respiratory distress syndrome (NRDS) in premature infants. In applications relevant for lung injury-related respiratory failure and ALI/ARDS, surfactant therapy has been shown to be beneficial in term infants with pneumonia and meconium aspiration lung injury, and in children up to age 21 with direct pulmonary forms of ALI/ARDS. However, extension of exogenous surfactant therapy to adults with respiratory failure and clinical ALI/ARDS remains a challenge. Coverage here reviews clinical studies of surfactant therapy in pediatric and adult patients with ALI/ARDS, particularly focusing on its potential advantages in patients with direct pulmonary forms of these syndromes. Also discussed is the rationale for mechanism-based therapies utilizing exogenous surfactant in combination with agents targeting other aspects of the multifaceted pathophysiology of inflammatory lung injury. Additional factors affecting the efficacy of exogenous surfactant therapy in ALI/ARDS are also described, including the difficulty of effectively delivering surfactants to injured lungs and the existence of activity differences between clinical surfactant drugs. PMID:21742216

  5. Surfactant-enhanced cellulose nanocrystal Pickering emulsions. (United States)

    Hu, Zhen; Ballinger, Sarah; Pelton, Robert; Cranston, Emily D


    The effect of surfactants on the properties of Pickering emulsions stabilized by cellulose nanocrystals (CNCs) was investigated. Electrophoretic mobility, interfacial tension, confocal microscopy and three-phase contact angle measurements were used to elucidate the interactions between anionic CNCs and cationic alkyl ammonium surfactants didecyldimethylammonium bromide (DMAB) and cetyltrimethylammonium bromide (CTAB). Both surfactants were found to adsorb onto CNCs with concentration-dependent morphology. At low concentrations, individual surfactant molecules adsorbed with alkyl tails pointing outward leading to hydrophobic CNCs. At higher concentrations, above the surfactant's apparent critical micelle concentration, surfactant aggregate morphologies on CNCs were inferred and the hydrophobicity of CNCs decreased. DMAB, which has two alkyl tails, rendered the CNCs more hydrophobic than CTAB which has only a single alkyl tail, at all surfactant concentrations. The change in CNC wettability from surfactant adsorption was directly linked to emulsion properties; adding surfactant increased the emulsion stability, decreased the droplet size, and controlled the internal phase of CNC Pickering emulsions. More specifically, a double transitional phase inversion, from oil-in-water to water-in-oil and back to oil-in-water, was observed for emulsions with CNCs and increasing amounts of DMAB (the more hydrophobic surfactant). With CNCs and CTAB, no phase inversion was induced. This work represents the first report of CNC Pickering emulsions with surfactants as well as the first CNC Pickering emulsions that can be phase inverted. The ability to surface modify CNCs in situ and tailor emulsions by adding surfactants may extend the potential of CNCs to new liquid formulations and extruded/spray-dried materials. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus. (United States)

    Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P


    Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. Copyright © 2014 the American Physiological Society.

  7. Elucidating Functional Aspects of P-type ATPases

    DEFF Research Database (Denmark)

    Autzen, Henriette Elisabeth


    and helped enlighten how thapsigargin, a potent inhibitor of SERCA1a, depends on a water mediated hydrogen bond network when bound to SERCA1a. Furthermore, molecular dynamics (MD) simulations of the same P-type ATPase were used to assess a long-standing question whether cholesterol affects SERCA1a through......P-type ATPases are proteins that act to maintain ion homeostasis and electrochemical gradients through the translocation of cations across cell membranes. Underscoring their significance in humans, dysfunction of the ATPases can lead to crucial diseases. Dysfunction of the sarco......; however, encouraging hits were obtained with AgNO3 and E. coli lipids, potentially underscoring the importance of utilizing native lipids. Finally, atomistic MD simulations of a Zn2+-ATPase (ZntA) were set up and computed in order to elucidate the dynamics of its ion exit pathway, and helped reveal how...


    African Journals Online (AJOL)

    + -K+ ATPase activity of the erythrocytes of HIV/AIDS patients. Whole blood was taken from subjects at the Human Virology Laboratory of the Nigerian Institute of Medical Research. Subjects were judged suitable for the various investigations by ...

  9. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening. (United States)

    Elbatsh, Ahmed M O; Haarhuis, Judith H I; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, René H; Nasmyth, Kim; Rowland, Benjamin D


    Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Cholesterol-mediated surfactant dysfunction is mitigated by surfactant protein A. (United States)

    Hiansen, Joshua Qua; Keating, Eleonora; Aspros, Alex; Yao, Li-Juan; Bosma, Karen J; Yamashita, Cory M; Lewis, James F; Veldhuizen, Ruud A W


    The ability of pulmonary surfactant to reduce surface tension at the alveolar surface is impaired in various lung diseases. Recent animal studies indicate that elevated levels of cholesterol within surfactant may contribute to its inhibition. It was hypothesized that elevated cholesterol levels within surfactant inhibit human surfactant biophysical function and that these effects can be reversed by surfactant protein A (SP-A). The initial experiment examined the function of surfactant from mechanically ventilated trauma patients in the presence and absence of a cholesterol sequestering agent, methyl-β-cyclodextrin. The results demonstrated improved surface activity when cholesterol was sequestered in vitro using a captive bubble surfactometer (CBS). These results were explored further by reconstitution of surfactant with various concentrations of cholesterol with and without SP-A, and testing of the functionality of these samples in vitro with the CBS and in vivo using surfactant depleted rats. Overall, the results consistently demonstrated that surfactant function was inhibited by levels of cholesterol of 10% (w/w phospholipid) but this inhibition was mitigated by the presence of SP-A. It is concluded that cholesterol-induced surfactant inhibition can actively contribute to physiological impairment of the lungs in mechanically ventilated patients and that SP-A levels may be important to maintain surfactant function in the presence of high cholesterol within surfactant. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Surfactant nebulisation : lung function, surfactant distribution and pulmonary blood flow distribution in lung lavaged rabbits

    NARCIS (Netherlands)

    Dijk, Peter H.; Heikamp, A; Bambang Oetomo, Sidarto


    Objective: Surfactant nebulisation is a promising alternative to surfactant instillation in newborns with the respiratory distress syndrome. Although less surfactant is deposited in the lung, it improves gas exchange, probably due to a superior distribution. We hypothesize that a more uniform

  12. Solubilization of Hydrophobic Dyes in Surfactant Solutions

    Directory of Open Access Journals (Sweden)

    Ali Reza Tehrani-Bagha


    Full Text Available In this paper, the use of surfactants for solubilization of hydrophobic organic dyes (mainly solvent and disperse dyes has been reviewed. The effect of parameters such as the chemical structures of the surfactant and the dye, addition of salt and of polyelectrolytes, pH, and temperature on dye solubilization has been discussed. Surfactant self-assemble into micelles in aqueous solution and below the concentration where this occurs—the critical micelle concentration (CMC—there is no solubilization. Above the CMC, the amount of solubilized dye increases linearly with the increase in surfactant concentration. It is demonstrated that different surfactants work best for different dyes. In general, nonionic surfactants have higher solubilization power than anionic and cationic surfactants. It is likely that the reason for the good performance of nonionic surfactants is that they allow dyes to be accommodated not only in the inner, hydrocarbon part of the micelle but also in the headgroup shell. It is demonstrated that the location of a dye in a surfactant micelle can be assessed from the absorption spectrum of the dye-containing micellar solution.

  13. Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

    Directory of Open Access Journals (Sweden)

    Brian Roland eLarsen


    Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

  14. Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

    Directory of Open Access Journals (Sweden)

    I. Stefanon


    Full Text Available Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group underwent left coronary artery ligation (infarcted, Inf or sham-operation (Sham. Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05. The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05, while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

  15. Proton Pumping of the Yeast Plasma Membrane H+-ATPase (United States)


    eucaryotic cells would be more resistant to damage by expression of the ATPase. We started with a plasmid from Dr. Fink’s laboratory tnat uontains the...have begun construction of a Baculovirus expression vector that we will ti-ansfect into insect cells . We hope that this 6 system will not suffer from...causes the bacteria to stop growing. We decided to try to produce the ATPase in insect cells using the baculovirus expression system hoping that the

  16. Structural divergence between the two subgroups of P5 ATPases

    DEFF Research Database (Denmark)

    Sørensen, Danny Mollerup; Buch-Pedersen, Morten Jeppe; Palmgren, Michael Broberg


    been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify....... Together these findings indicate that P5A and P5B ATPases are structurally and functionally different....

  17. Photoisomerization of merocyanine 540 in polymer–surfactant ...

    Indian Academy of Sciences (India)


    surfactant aggregate. 1. Introduction. Many biological and technological processes involve polymer-surfactant interaction 1–17. ... model' (figure 1), structure of the polymer-surfactant aggregate is like a necklace with .... group projected into bulk water.

  18. Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study (United States)

    Sims, Robert Edward; Dale, Nicholas


    In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na+ influxes associated with neuronal signalling activate the Na+-K+ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na+. We have simultaneously imaged intracellular Na+ and measured adenosine release. The accumulation of intracellular Na+ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca2+, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na+-K+ ATPase in the release of adenosine. Under conditions which caused a Na+ influx, brief applications of ouabain increased the accumulation of intracellular Na+ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na+-K+ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na+-K+ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep. PMID:24489921

  19. Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

    Directory of Open Access Journals (Sweden)

    Young Ou

    Full Text Available Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.

  20. Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase α3 sodium pump. (United States)

    Kirshenbaum, Greer S; Clapcote, Steven J; Duffy, Steven; Burgess, Christian R; Petersen, Janne; Jarowek, Karolina J; Yücel, Yeni H; Cortez, Miguel A; Snead, O Carter; Vilsen, Bente; Peever, John H; Ralph, Martin R; Roder, John C


    Bipolar disorder is a debilitating psychopathology with unknown etiology. Accumulating evidence suggests the possible involvement of Na(+),K(+)-ATPase dysfunction in the pathophysiology of bipolar disorder. Here we show that Myshkin mice carrying an inactivating mutation in the neuron-specific Na(+),K(+)-ATPase α3 subunit display a behavioral profile remarkably similar to bipolar patients in the manic state. Myshkin mice show increased Ca(2+) signaling in cultured cortical neurons and phospho-activation of extracellular signal regulated kinase (ERK) and Akt in the hippocampus. The mood-stabilizing drugs lithium and valproic acid, specific ERK inhibitor SL327, rostafuroxin, and transgenic expression of a functional Na(+),K(+)-ATPase α3 protein rescue the mania-like phenotype of Myshkin mice. These findings establish Myshkin mice as a unique model of mania, reveal an important role for Na(+),K(+)-ATPase α3 in the control of mania-like behavior, and identify Na(+),K(+)-ATPase α3, its physiological regulators and downstream signal transduction pathways as putative targets for the design of new antimanic therapies.

  1. Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase. (United States)

    Aggeler, R; Mendel-Hartvig, J; Capaldi, R A


    Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.

  2. Disaturated-phosphatidylcholine and Surfactant protein-B turnover in human acute lung injury and in control patients

    Directory of Open Access Journals (Sweden)

    Rizzi Sabina


    Full Text Available Abstract Background Patients with Adult Respiratory Distress Syndrome (ARDS and Acute Lung Injury (ALI have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover. Objectives To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls. Methods 2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured. Results 1 Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2 In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p Conclusions 1 Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2 In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

  3. Hemolysis by surfactants--A review. (United States)

    Manaargadoo-Catin, Magalie; Ali-Cherif, Anaïs; Pougnas, Jean-Luc; Perrin, Catherine


    An overview of the use of surfactants for erythrocyte lysis and their cell membrane action mechanisms is given. Erythrocyte membrane characteristics and its association with the cell cytoskeleton are presented in order to complete understanding of the erythrocyte membrane distortion. Cell homeostasis disturbances caused by surfactants might induce changes starting from shape modification to cell lysis. Two main mechanisms are hypothesized in literature which are osmotic lysis and lysis by solubilization even if the boundary between them is not clearly defined. Another specific mechanism based on the formation of membrane pores is suggested in the particular case of saponins. The lytic potency of a surfactant is related to its affinity for the membrane and the modification of the lipid membrane curvature. This is to be related to the surfactant shape defined by its hydrophobic and hydrophilic moieties but also by experimental conditions. As a consequence, prediction of the hemolytic potency of a given surfactant is challenging. Several studies are focused on the relation between surfactant erythrolytic potency and their physico-chemical parameters such as the critical micellar concentration (CMC), the hydrophile-lipophile balance (HLB), the surfactant membrane/water partition coefficient (K) or the packing parameter (P). The CMC is one of the most important factors considered even if a lytic activity cut-off effect points out that the only consideration of CMC not enough predictive. The relation K.CMC must be considered in addition to the CMC to predict the surfactant lytic capacity within the same family of non ionic surfactant. Those surfactant structure/lytic activity studies demonstrate the requirement to take into account a combination of physico-chemical parameters to understand and foresee surfactant lytic potency. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe


    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary tran......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  5. Structured fluids polymers, colloids, surfactants

    CERN Document Server

    Witten, Thomas A


    Over the last thirty years, the study of liquids containing polymers, surfactants, or colloidal particles has developed from a loose assembly of facts into a coherent discipline with substantial predictive power. These liquids expand our conception of what condensed matter can do. Such structured-fluid phenomena dominate the physical environment within living cells. This book teaches how to think of these fluids from a unified point of view showing the far-reaching effects ofthermal fluctuations in producing forces and motions. Keeping mathematics to a minimum, the book seeks the simplest expl

  6. C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.

    Directory of Open Access Journals (Sweden)

    Dana Galuska

    Full Text Available Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86Rb(+ uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM. DNA binding activity was determined by electrical mobility shift assay (EMSA. Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1-subunit protein expression, accompanied with increase in (86Rb(+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6, concomitant with Na,K-ATPase α(1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C

  7. Pulmonary surfactant in the airway physiology: a direct relaxing effect on the smooth muscle. (United States)

    Calkovska, A; Uhliarova, B; Joskova, M; Franova, S; Kolomaznik, M; Calkovsky, V; Smolarova, S


    Beside alveoli, surface active material plays an important role in the airway physiology. In the upper airways it primarily serves in local defense. Lower airway surfactant stabilizes peripheral airways, provides the transport and defense, has barrier and anti-edematous functions, and possesses direct relaxant effect on the smooth muscle. We tested in vitro the effect of two surfactant preparations Curosurf® and Alveofact® on the precontracted smooth muscle of intra- and extra-pulmonary airways. Relaxation was more pronounced for lung tissue strip containing bronchial smooth muscle as the primary site of surfactant effect. The study does not confirm the participation of ATP-dependent potassium channels and cAMP-regulated epithelial chloride channels known as CFTR chloride channels, or nitric oxide involvement in contractile response of smooth muscle to surfactant.By controlling wall thickness and airway diameter, pulmonary surfactant is an important component of airway physiology. Thus, surfactant dysfunction may be included in pathophysiology of asthma, COPD, or other diseases with bronchial obstruction. Copyright © 2015. Published by Elsevier B.V.

  8. Surfactants and submicron sea spray generation

    NARCIS (Netherlands)

    Sellegri, K.; O'Dowd, C.D.; Yoon, Y.J.; Jennings, S.G.; Leeuw, G. de


    Laboratory experiments have been carried out to elucidate the role of surfactants on the primary marine aerosol production of submicron marine aerosols. A synthetic surfactant SDS was used in conjunction with artificially generated seawater, and the resultant bubble-mediated aerosol produced was

  9. Hyaluronan decreases surfactant inactivation in vitro. (United States)

    Lu, Karen W; Goerke, Jon; Clements, John A; Taeusch, H William


    Hyaluronan (HA) is an anionic polymer and a constituent of alveolar fluid that can bind proteins, phospholipids, and water. Previous studies have established that nonionic polymers improve the surface activity of pulmonary surfactants by decreasing inactivation of surfactant. In this work, we investigate whether HA can also have beneficial effects when added to surfactants. We used a modified pulsating bubble surfactometer to measure mixtures of several commercially available pulmonary surfactants or native calf surfactant with and without serum inactivation. Surface properties such as equilibrium surface tension, minimum and maximum surface tensions on compression and expansion of a surface film, and degree of surface area reduction required to reach a surface tension of 10 mN/m were measured. In the presence of serum, addition of HA dramatically improved the surface activities of all four surfactants and in some cases in the absence of serum as well. These results indicate that HA reduces inactivation of surfactants caused by serum and add evidence that endogenous HAs may interact with alveolar surfactant under normal and abnormal conditions.

  10. Surfactant Adsorption: A Revised Physical Chemistry Lab (United States)

    Bresler, Marc R.; Hagen, John P.


    Many physical chemistry lab courses include an experiment in which students measure surface tension as a function of surfactant concentration. In the traditional experiment, the data are fit to the Gibbs isotherm to determine the molar area for the surfactant, and the critical micelle concentration is used to calculate the Gibbs energy of micelle…

  11. Surfactant effects on soil aggregate tensile strength (United States)

    Little is known regarding a soil aggregate's tensile strength response to surfactants that may be applied to alleviate soil water repellency. Two laboratory investigations were performed to determine surfactant effects on the tensile strength of 1) Ap horizons of nine wettable, agricultural soils co...

  12. Human leptin administered intraperitoneally stimulates natriuresis and decreases renal medullary Na+, K+-ATPase activity in the rat -- impaired effect in dietary-induced obesity. (United States)

    Bełtowski, Jerzy; W jcicka, Grazyna; Górny, Dionizy; Marciniak, Andrzej


    Leptin, produced by adipose tissue, apart from regulating food intake and energy expenditure, also has natriuretic activity. In this study we examined the effect of leptin on renal Na+, K+-ATPase responsible for active tubular sodium reabsorption, and compared the renal effects of leptin in lean and obese rats. Male Wistar rats were either kept on normal laboratory chow or made obese by a high-calorie diet. The animals were placed in metabolic cages and urine was collected in 2-hour periods. In lean animals, leptin (1 mg/kg i.p.) caused a 139.5% increase in urine output, a 112.4% increase in natriuresis, and a 57.2% increase in the fractional excretion of sodium, but had no effect on the glomerular filtration rate. Leptin at this dose decreased renal medullary Na+, K+-ATPase activity at 30 minutes, 1 hour and 2 hours by 31%, 34.3% and 21.2%, respectively. The effect of leptin on Na+, K+-ATPase at 1 hour was dose-dependent; the lowest dose inducing significant inhibition was 0.25 mg/kg. By contrast, leptin had no effect on either cortical Na+, K+-ATPase or the ouabain-resistant fraction of ATPase. In obese rats, leptin increased urine output by only 29.1% and natriuresis by 28.9%, and had no significant effect on medullary Na+, K+-ATPase. Leptin stimulates natriuresis primarily by inhibiting tubular sodium reabsorption. This effect is mediated, at least partially, by decreased Na+, K+-ATPase activity in the renal medulla, and is impaired in obese rats.

  13. The future of exogenous surfactant therapy. (United States)

    Willson, Douglas F; Notter, Robert H


    Since the identification of surfactant deficiency as the putative cause of the infant respiratory distress syndrome (RDS) by Avery and Mead in 1959, our understanding of the role of pulmonary surfactant in respiratory physiology and the pathophysiology of acute lung injury (ALI) has advanced substantially. Surfactant replacement has become routine for the prevention and treatment of infant RDS and other causes of neonatal lung injury. The role of surfactant in lung injury beyond the neonatal period, however, has proven more complex. Relative surfactant deficiency, dysfunction, and inhibition all contribute to the disturbed physiology seen in ALI and acute respiratory distress syndrome (ARDS). Consequently, exogenous surfactant, while a plausible therapy, has proven to be less effective in ALI/ARDS than in RDS, where simple deficiency is causative. This failure may relate to a number of factors, among them inadequacy of pharmaceutical surfactants, insufficient dosing or drug delivery, poor drug distribution, or simply an inability of the drug to substantially impact the underlying pathophysiology of ALI/ARDS. Both animal and human studies suggest that direct types of ALI (eg, aspiration, pneumonia) may be more responsive to surfactant therapy than indirect lung injury (eg, sepsis, pancreatitis). Animal studies are needed, however, to further clarify aspects of drug composition, timing, delivery, and dosing before additional human trials are pursued, as the results of human trials to date have been inconsistent and largely disappointing. Further study and perhaps the development of more robust pharmaceutical surfactants offer promise that exogenous surfactant will find a place in our armamentarium of treatment of ALI/ARDS in the future. 2011 Daedalus Enterprises

  14. Formation of transfer cells and H(+)-ATPase expression in tomato roots under P and Fe deficiency. (United States)

    Schikora, Adam; Schmidt, Wolfgang


    In roots of tomato ( Lycopersicon esculentum Mill.), extranumerary root hairs and transfer cell-like wall ingrowth depositions in the rhizodermis were developed in response to P and Fe deficiency. Immunocytolocalization of the plasma membrane H(+)-ATPase in roots of P-deficient plants revealed no appreciable increase in H(+)-ATPase density relative to control plants. In transfer cells, immunogold labeling was considerably higher than in ordinary rhizodermal cells. H(+)-ATPase sites were asymmetrically distributed in cells with and without wall ingrowths under P-deficient conditions. A split-root study revealed that the frequency of transfer cells was higher in the low-P half of the root system, but the density of H(+)-ATPase molecules was enhanced only in the high-P half of the split roots, suggesting that formation of transfer cells was controlled directly by the external Pi concentration, whereas ATPase expression was regulated indirectly by the internal nutrient status of the plant. The role of hormones in the induction of transfer cells was investigated by treating plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or various ethylene antagonists. Transfer cells were induced by ACC to an extent similar to that observed after P or Fe starvation, but inhibitors of either ethylene synthesis or action did not decrease their frequency. These results suggest that ethylene was not required for the induction of transfer cells but changes in ethylene levels appeared to modulate the number of cells forming wall ingrowths. In roots of ethylene-insensitive Never-ripe tomato plants the frequency of transfer cells was rather increased than decreased under most growth conditions relative to the wild type, indicating that ethylene responsiveness played no critical role in the differentiation of transfer cells and that the transduction of signals ultimately leading to their formation was independent of the ethylene signaling cascade.

  15. [Na+, K+-ATPase activity of the kidneys and 3,5,3'-triiodothyronine levels in rat plasma during adaptation to cold]. (United States)

    Zamaĭ, T N; Medvedev, L N


    No correlation between Na, K-ATPase activity increase in rat kidney and the level of T3 in blood plasma was revealed in course of the rats adaptation to cold. Spironolactone prevented the enzyme activation caused by cold. Actinomycin D, administered to the acclimated rats two days before the experiment, decreased the enzyme activity. Addition of actinomycin D to membrane preparation did not change the enzyme activity. The increase in Na, K-ATPase activity in kidney during adaptation of rats to cold appears to be related to DNA-dependent synthesis of RNA. The enzyme activation was apparently regulated by aldosterone but it was not the T3-dependent process.

  16. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

    Directory of Open Access Journals (Sweden)

    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  17. Exogenous Pulmonary Surfactant as a Vehicle for Antimicrobials: Assessment of Surfactant-Antibacterial Interactions In Vitro

    Directory of Open Access Journals (Sweden)

    Alexei Birkun


    Full Text Available Owing to its unique surface-active properties, an exogenous pulmonary surfactant may become a promising drug delivery agent, in particular, acting as a vehicle for antibiotics in topical treatment of pneumonia. The purpose of this study was to assess a mutual influence of natural surfactant preparation and three antibiotics (amikacin, cefepime, and colistimethate sodium in vitro and to identify appropriate combination(s for subsequent in vivo investigations of experimental surfactant/antibiotic mixtures. Influence of antibiotics on surface-active properties of exogenous surfactant was assessed using the modified Pattle method. Effects of exogenous surfactant on antibacterial activity of antimicrobials against Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa were evaluated using conventional microbiologic procedures. Addition of amikacin or cefepime to surfactant had no significant influence on surface-active properties of the latter. Obvious reduction of surface-active properties was confirmed for surfactant/colistimethate composition. When suspended with antibiotics, surfactant either had no impact on their antimicrobial activity (amikacin or exerted mild to moderate influence (reduction of cefepime bactericidal activity and increase of colistimethate bacteriostatic activity against S. aureus and P. aeruginosa. Considering favorable compatibility profile, the surfactant/amikacin combination is advisable for subsequent investigation of joint surfactant/antibacterial therapy in animals with bacterial pneumonia.

  18. Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus. (United States)

    Laursen, Ulla Helt; Pihakaski-Maunsbach, Kaarina; Kwon, Tae-Hwan; Østergaard Jensen, Erik; Nielsen, Søren; Maunsbach, Arvid B


    In a rat model, lithium treatment is associated with polyuria and severe downregulation of aquaporin-2 (AQP2) protein in the inner medulla (IM) or in the whole kidney. However, it is not known (1) to what extent this downregulation occurs at the mRNA level; (2) whether the main sodium transporter of the nephron, Na,K-ATPase, is regulated in parallel at the mRNA level, and (3) whether lithium treatment induces zonal or segmental differences in AQP2 and Na,K-ATPase mRNA levels. We examined the changes in mRNA expression levels for AQP2 and Na,K-ATPase in kidney cortex, inner stripe of the outer medulla (ISOM), and IM of rats treated with lithium orally using semiquantitative Northern blot analyses and in situ hybridization at the light and electron microscopic levels. The AQP2 mRNA levels decreased significantly (p dabetes insipidus and moderately decreased Na,K-ATPase mRNA levels in the ISOM and in the IM. The results suggest that decreased mRNA expressions of AQP2 and Na,K-ATPase contribute to the development of lithium-induced nephrogenic diabetes insipidus. Copyright 2004 S. Karger AG, Basel

  19. Modulation by K+ Plus NH4+ of microsomal (Na+, K+-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae.

    Directory of Open Access Journals (Sweden)

    Francisco A Leone

    Full Text Available We investigate the synergistic stimulation by K(+ plus NH4 (+ of (Na(+, K(+-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+, K(+-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+ and NH4 (+ binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+, K(+-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+, K(+-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+ and NH4 (+ of gill (Na(+, K(+-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+ during ontogenetic development in M. amazonicum.

  20. Modulation by K+ Plus NH4+ of microsomal (Na+, K+)-ATPase activity in selected ontogenetic stages of the diadromous river shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae). (United States)

    Leone, Francisco A; Bezerra, Thais M S; Garçon, Daniela P; Lucena, Malson N; Pinto, Marcelo R; Fontes, Carlos F L; McNamara, John C


    We investigate the synergistic stimulation by K(+) plus NH4 (+) of (Na(+), K(+))-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na(+), K(+))-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K(+) and NH4 (+) binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na(+), K(+))-ATPase activity is stimulated synergistically by ≈ 50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na(+), K(+))-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K(+) and NH4 (+) of gill (Na(+), K(+))-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4 (+) during ontogenetic development in M. amazonicum.

  1. A novel role for the yeast protein kinase Dbf2p in vacuolar H+-ATPase function and sorbic acid stress tolerance. (United States)

    Makrantoni, Vasso; Dennison, Paul; Stark, Michael J R; Coote, Peter J


    In Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H(+)-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Delta). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Delta cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.

  2. Tunable, antibacterial activity of silicone polyether surfactants. (United States)

    Khan, Madiha F; Zepeda-Velazquez, Laura; Brook, Michael A


    Silicone surfactants are used in a variety of applications, however, limited data is available on the relationship between surfactant structure and biological activity. A series of seven nonionic, silicone polyether surfactants with known structures was tested for in vitro antibacterial activity against Escherichia coli BL21. The compounds varied in their hydrophobic head, comprised of branched silicone structures with 3-10 siloxane linkages and, in two cases, phenyl substitution, and hydrophilic tail of 8-44 poly(ethylene glycol) units. The surfactants were tested at three concentrations: below, at, and above their Critical Micelle Concentrations (CMC) against 5 concentrations of E. coli BL21 in a three-step assay comprised of a 14-24h turbidometric screen, a live-dead stain and viable colony counts. The bacterial concentration had little effect on antibacterial activity. For most of the surfactants, antibacterial activity was higher at concentrations above the CMC. Surfactants with smaller silicone head groups had as much as 4 times the bioactivity of surfactants with larger groups, with the smallest hydrophobe exhibiting potency equivalent to sodium dodecyl sulfate (SDS). Smaller PEG chains were similarly associated with higher potency. These data link lower micelle stability and enhanced permeability of smaller silicone head groups to antibacterial activity. The results demonstrate that simple manipulation of nonionic silicone polyether structure leads to significant changes in antibacterial activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. A study of surfactant-assisted waterflooding

    Energy Technology Data Exchange (ETDEWEB)

    Scamehorn, J F; Harwell, J H


    In surfactant-assisted waterflooding, a surfactant slug is injected into a reservoir, followed by a brine spacer, followed by second surfactant slug. The charge on the surfactant in the first slug has opposite sign to that in the second slug. When the two slugs mix in the reservoir, a precipitate or coacervate is formed which plugs the permeable region of the reservoir. Subsequently injected water or brine is forced through the low permeability region of the reservoir, increasing sweep efficiency of the waterflood, compared to a waterflood not using surfactants. In this part of the work, two major tasks are performed. First, core floods are performed with oil present to demonstrate the improvement in incremental oil production, as well as permeability modification. Second, a reservoir simulation model will be proposed to further delineate the optimum strategy for implementation of the surfactant-assisted waterflooding, as well as indicate the reservoir types for which it would be most effective. Surfactants utilized were sodium dodecyl sulfate and dodecyl pyridinium chloride. 44 refs., 17 figs., 3 tabs.

  4. Surfactants tailored by the class Actinobacteria

    Directory of Open Access Journals (Sweden)

    Johannes H Kügler


    Full Text Available Gloablly, the drive towards the establishment of a bio-based economy has resulted in an increased need for bio-based applications. This, in turn, has served as a driving force for the discovery and application of novel biosurfactants. The class Actinobacteria represents a vast group of microorganisms with the ability to produce a diverse range of secondary metabolites, including surfactants. Understanding the extensive nature of the biosurfactants produced by actinobacterial strains can assist in finding novel biosurfactants with new potential applications. This review therefore presents a comprehensive overview of the knowledge available on actinobacterial surfactants, the chemical structures that have been completely or partly elucidated, as well as the identity of the biosurfactant-producing strains. Producer strains of not yet elucidated compounds are discussed, as well as the original habitats of all the producer strains, which seems to indicate that biosurfactant production is environmentally driven. Methodology applied in the isolation, purification and structural elucidation of the different types of surface active compounds, as well as surfactant activity tests, are also discussed. Overall, actinobacterial surfactants can be summarized to include the dominantly occurring trehalose-comprising surfactants, other non-trehalose containing glycolipids, lipopeptides and the more rare actinobacterial surfactants. The lack of structural information on a large proportion of actinobacterial surfactants should be considered as a driving force to further explore the abundance and diversity of these compounds. This would allow for a better understanding of actinobacterial surface active compounds and their potential for biotechnological application.

  5. The coupled chemomechanics of the F(1)-ATPase molecular motor. (United States)

    Xu, Lizhong


    The enzyme F(1)-ATPase is a rotary nanomotor in which the central gamma subunit rotates inside the cavity made of alpha(3)beta(3) subunits. The experiments showed that the rotation proceeds in steps of 120 degrees and each 120 degrees step consists of 80 degrees and 40 degrees substeps. Here the Author proposes a stochastic wave mechanics of the F(1)-ATPase motor and combines it with the structure-based kinetics of the F(1)-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F(1) motor. Using the model, several rate-limited situations about gamma subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.

  6. Structural and functional studies of heavy metal ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg


    of SsZntA using a broad range of techniques reveals an array of unique Zn2+-ATPase features relating to ion uptake, binding, discharge and countertransport. These results are then used to comprehensively compare Zn2+-ATPases with their Cu+-transporting counterparts, showing how the two have managed...... to handle heavy metal ions. LpCopA is then compared to its two human homologues ATP7A and ATP7B, which cause the severe Menkes and Wilson diseases when malfunctioning. The differences between the three proteins are described and disease-causing mutations in the human proteins are analyzed. The crystal...... to adapt to transport of distinct ion types while still maintaining all the major features of the P1B-ATPase subclass....

  7. Gemini surfactants from natural amino acids. (United States)

    Pérez, Lourdes; Pinazo, Aurora; Pons, Ramon; Infante, Mrosa


    In this review, we report the most important contributions in the structure, synthesis, physicochemical (surface adsorption, aggregation and phase behaviour) and biological properties (toxicity, antimicrobial activity and biodegradation) of Gemini natural amino acid-based surfactants, and some potential applications, with an emphasis on the use of these surfactants as non-viral delivery system agents. Gemini surfactants derived from basic (Arg, Lys), neutral (Ser, Ala, Sar), acid (Asp) and sulphur containing amino acids (Cys) as polar head groups, and Geminis with amino acids/peptides in the spacer chain are reviewed. © 2013.

  8. Mechanism of bacterial inactivation by cationic surfactants

    Energy Technology Data Exchange (ETDEWEB)

    Pavlova, I.B.; Samoylenko, I.I.


    The mechanism of bacteriocidal action of the cationic surfactant dimethylbenzylammonium chloride was studied on exposure of Staphylococcus aureus, Streptococcus faecium, Bacillus subtilis and Escherichia coli to different concentrations of the agent and determinations of survival plots. The data showed that the surfactant was bacteriocidal for all the bacteria tested at a concentration of 0.0001%, but more efficient in the case of the gram positives. Electron microscopy showed considerable damage and dissarrangement of the cytoplasmic membrane, indicating that the killing mechanism involved this organelle. It appears that cationic surfactants may constitute effective disinfectant preparations. 9 references, 2 figures.

  9. Transport ATPases: structure, motors, mechanism and medicine: a brief overview. (United States)

    Pedersen, Peter L


    Today we know there are four different types of ATPases that operate within biological membranes with the purpose of moving many different types of ions or molecules across these membranes. Some of these ions or molecules are transported into cells, some out of cells, and some in or out of organelles within cells. These ATPases span the biological world from bacteria to eukaryotic cells and have become most simply and commonly known as "transport ATPases." The price that each cell type pays for transport work is counted in molecules of hydrolyzed ATP, a metabolic currency that is itself regenerated by a transport ATPase working in reverse, i.e., the ATP synthase. Four major classes of transport ATPases, the P, V, F, and ABC types are now known. In addition to being involved in many different types of biological/physiological processes, mutations in these proteins also account for a large number of diseases. The purpose of this introductory article to a mini-review series on transport ATPases is to provide the reader with a very brief and focused look at this important area of research that has an interesting history and bears significance to cell physiology, biochemistry, immunology, nanotechnology, and medicine, including drug discovery. The latter involves potential applications to a whole host of diseases ranging from cancer to those that affect bones (osteoporosis), ears (hearing), eyes (macromolecular degeneration), the heart (hypercholesterolemia/cardiac arrest,), immune system (immune deficiency disease), kidney (nephrotoxicity), lungs (cystic fibrosis), pancreas (diabetes and cystic fibrosis), skin (Darier disease), and stomach (ulcers).

  10. Mercuric chloride-induced inhibition of different ATPases in the intestine of mudskipper, Boleophthalmus dentatus. (United States)

    Lakshmi, R; Kundu, R; Thomas, E; Mansuri, A P


    This paper deals with the toxicity of mercuric chloride to different ATPases in the intestine of mudskipper (Boleophthalmus dentatus). Mudskippers were exposed to four sublethal concentrations of mercuric chloride for three durations. The specific activities of Na+, K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase, Ca2+, HCO3(-)-ATPase, and Mg2+, HCO3(-)-ATPase were estimated. There was linear inhibition of all the enzymes with increasing mercuric chloride concentration as well as exposure duration. The Na+,K(+)-ATPase was found to be the enzyme most affected, followed by other ion-dependent ATPases. Inhibition of all the enzymes indicates severe damage to the intestinal cells, resulting in a blockage of the transport of substances across the membrane.

  11. Purification and characterization of ATPase from Nitrobacter winogradskyi. (United States)

    Hara, T; Villalobos, A P; Fukumori, Y; Yamanaka, T


    An ATPase was purified from Nitrobacter winogradskyi, and some of its molecular and enzymatic properties were determined. The enzyme was composed of two subunits of 64 and 59 kDa, respectively. The enzyme had its pH optimum at 9.5 and showed a specific activity of 7 units per mg protein. This activity was about 14% and 18% of that of F1-ATPases obtained from Escherichia coli and Sulfolobus acidocaldarius, respectively. The enzyme was 29% and 6% inhibited by 100 microM dicyclohexylcarbodiimide (DCCD) and 100 microM NaN3, respectively. It was not inhibited by 20 mM NaNO3.

  12. Beneficial renal and pancreatic phenotypes in a mouse deficient in FXYD2 regulatory subunit of Na,K-ATPase

    Directory of Open Access Journals (Sweden)

    Elena eArystarkhova


    Full Text Available The fundamental role of Na,K-ATPase in eukaryotic cells calls for complex and efficient regulation of its activity. Besides alterations in gene expression and trafficking, kinetic properties of the pump are modulated by reversible association with single span membrane proteins, the FXYDs. Seven members of the family are expressed in a tissue-specific manner, affecting pump kinetics in all possible permutations. This mini-review focuses on functional properties of FXYD2 studied in transfected cells, and on noteworthy and unexpected phenotypes discovered in a Fxyd2-/- mouse. FXYD2, the gamma subunit, reduces activity of Na,K-ATPase either by decreasing affinity for Na+, or reducing Vmax. FXYD2 mRNA splicing and editing provide another layer for regulation of Na,K-ATPase. In kidney of knockouts, there was elevated activity for Na,K-ATPase and for NCC and NKCC2 apical sodium transporters. That should lead to sodium retention and hypertension, however, the mice were in sodium balance and normotensive. Adult Fxyd2-/- mice also exhibited a mild pancreatic phenotype with enhanced glucose tolerance, elevation of circulating insulin, but no insulin resistance. There was an increase in beta cell proliferation and beta cell mass that correlated with activation of the PI3K-Akt pathway. The Fxyd2-/- mice are thus in a highly desirable state: the animals are resistant to Na+ retention, and showed improved glucose control, i.e. they display favorable metabolic adaptations to protect against development of salt-sensitive hypertension and diabetes. Investigation of the mechanisms of these adaptations in the mouse has the potential to unveil a novel therapeutic FXYD2-dependent strategy.

  13. Cold stress causes rapid but differential changes in properties of plasma membrane H(+)-ATPase of camelina and rapeseed. (United States)

    Kim, Hyun-Sung; Oh, Jung-Min; Luan, Sheng; Carlson, John E; Ahn, Sung-Ju


    Camelina (Camelina sativa) and rapeseed (Brassica napus) are well-established oil-seed crops with great promise also for biofuels. Both are cold-tolerant, and camelina is regarded to be especially appropriate for production on marginal lands. We examined physiological and biochemical alterations in both species during cold stress treatment for 3 days and subsequent recovery at the temperature of 25°C for 0, 0.25, 0.5, 1, 2, 6, and 24h, with particular emphasis on the post-translational regulation of the plasma membrane (PM) H(+)-ATPase (EC3.6.3.14). The activity and translation of the PM H(+)-ATPase, as well as 14-3-3 proteins, increased after 3 days of cold stress in both species but recovery under normal conditions proceeded differently. The increase in H(+)-ATPase activity was the most dramatic in camelina roots after recovery for 2h at 25°C, followed by decay to background levels within 24h. In rapeseed, the change in H(+)-ATPase activity during the recovery period was less pronounced. Furthermore, H(+)-pumping increased in both species after 15min recovery, but to twice the level in camelina roots compared to rapeseed. Protein gel blot analysis with phospho-threonine anti-bodies showed that an increase in phosphorylation levels paralleled the increase in H(+)-transport rate. Thus our results suggest that cold stress and recovery in camelina and rapeseed are associated with PM H(+)-fluxes that may be regulated by specific translational and post-translational modifications. Copyright © 2013 Elsevier GmbH. All rights reserved.

  14. Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification

    NARCIS (Netherlands)

    Li, Y.; Provenzano, S.; Bliek, M.; Spelt, C.; Appelhagen, I.; Machado de Faria, L.; Verweij, W.; Schubert, A.; Sagasser, M.; Seidel, T.; Weisshaar, B.; Koes, R.; Quattrocchio, F.


    Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H+ P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As

  15. Structural and functional studies of a Cu+-ATPase from Legionella pneumophila

    DEFF Research Database (Denmark)

    Mattle, Daniel

    During his studies, Daniel Mattle explored the copper(I) export mechanism of a P-type Cu+ ATPase from Legionella pneumophila – a homologue to the human Cu+ ATPases. Cu+ ATPases are responsible for the homeostatic control of the physiological relevant – but toxic – copper(I) cations. To assess...

  16. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten


    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase...

  17. Aggregation of sulfosuccinate surfactants in water

    Energy Technology Data Exchange (ETDEWEB)

    Magid, L.J.; Daus, K.A.; Butler, P.D.; Quincy, R.B.


    The aggregation of sodium di-n-alkyl sulfosuccinates in water (H/sub 2/O and D/sub 2/O at 45/sup 0/C) has been investigated. A self-consistent picture of the dependence of sodium ion binding on surfactant concentration is obtained from emf measurements, conductimetry, and small-angle neutron scattering (SANS) measurements. The concentration dependence of the micellar agregation number for the sulfosuccinates and related double-tailed surfactants depends markedly on surfactant solubility. A sphere-to-disk transition in micellar shape, which might have been expected as a precursor to formation of a lamellar mesophase, was not observed as the surfactant concentration was increased. 8 figures, 2 tables.

  18. Enhanced Oil Recovery with Surfactant Flooding

    DEFF Research Database (Denmark)

    Sandersen, Sara Bülow

    behavior inside the reservoir can be manipulated by the injection of surfactants and co-surfactants, creating advantageous conditions in order to mobilize trapped oil. Correctly designed surfactant systems together with the crude oil can create microemulsions at the interface between crude oil and water...... at constant salinity (6.56 %), constant surfactant-alcohol ratio (SAR) but with varying water-oil ratios (WOR). At all temperatures it was very clear that the effect of pressure was significant. The system changed from the two phase region, Winsor II, to the three phase region, Winsor III, as pressure...... characterization of the two crude oils using gas chromatography and SARA analysis confirmed that the heavier components in the crude oils, (in the case of the Latin American crude oil), are correlated to the observed decrease of viscosity, where the viscosity decrease may be explained from change of the shape...

  19. Sphingosine inhibits the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA) activity

    Energy Technology Data Exchange (ETDEWEB)

    Benaim, Gustavo, E-mail: [Instituto de Estudios Avanzados (IDEA), Caracas (Venezuela, Bolivarian Republic of); Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Pimentel, Adriana A., E-mail: [Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Felibertt, Pimali [Facultad de Ciencias, Universidad de Carabobo, Valencia (Venezuela, Bolivarian Republic of); Mayora, Adriana [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Colman, Laura [Instituto Pasteur de Montevideo, Montevideo (Uruguay); Sojo, Felipe [Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Rojas, Héctor [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of); Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas (Venezuela, Bolivarian Republic of); De Sanctis, Juan B. [Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas (Venezuela, Bolivarian Republic of)


    The increase in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca{sup 2+}]{sub i} by inhibiting the sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca{sup 2+} pump. The results showed that addition of sphingosine produced a release of Ca{sup 2+} from the endoplasmic reticulum followed by a Ca{sup 2+} entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca{sup 2+}]{sub I} in mammalian cells.

  20. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy (United States)

    Mohamed, Tamer M. A.; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J.; Oceandy, Delvac


    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition. PMID:27020607

  1. Evaluation of Surfactant Effects on Newborns


    N. Khalessi; K. Kamerani


    Introduction & Objective: One of the standard therapies in neonates with severe respiratory distress syndrome (RDS) is surfactant administration in early course of therapy that cause reduction in mortality, pneumothorax and need to mechanical ventilation. In this study that was carried out in Aliasghar Hospital NICU in 1994-1995 & 2001-2002, the goal was to compare two groups of neonates with severe RDS that had been ventilated in the first 24 hours but one group had received surfactant and ...

  2. Evaluation of Surfactant Effects on Newborns

    Directory of Open Access Journals (Sweden)

    N. Khalessi


    Full Text Available Introduction & Objective: One of the standard therapies in neonates with severe respiratory distress syndrome (RDS is surfactant administration in early course of therapy that cause reduction in mortality, pneumothorax and need to mechanical ventilation. In this study that was carried out in Aliasghar Hospital NICU in 1994-1995 & 2001-2002, the goal was to compare two groups of neonates with severe RDS that had been ventilated in the first 24 hours but one group had received surfactant and the other group (7 years ago was deprived of this substance. Materials & Methods: In our study, 36 neonates that received surfactant and 52 neonates with only mechanical ventilation therapy were compared. Data collected and analyzed using SPSS.Results: We found that mortality in patients with surfactant administration was significantly lower compared to the second group who did not receive surfactant. There were not any significant differences in incidences of HIV, pneumothorax, sepsis, and PDA and also course of hospitalization and need to ventilation between two groups. Conclusion: As a result, all of these findings reflect obligatory surfactant administration in sever RDS in NICU under observation of an educated expert.

  3. [Deficiency of surfactant protein: Case report]. (United States)

    Milet, María Beatriz; Mena N, Patricia; Pérez, Héctor I; Espinoza, Tatiana

    Congenital surfactant deficiency is a condition infrequently diagnosed in newborns. A clinical case is presented of surfactant protein B deficiency. A review is performed on the study, treatment and differential diagnosis of surfactant protein deficiencies and infant chronic interstitial lung disease. The case is presented of a term newborn that developed respiratory distress, recurrent pulmonary opacification, and a transient response to the administration of surfactant. Immunohistochemical and genetic studies confirmed the diagnosis of surfactant protein B deficiency. Pulmonary congenital anomalies require a high index of suspicion. Surfactant protein B deficiency is clinically progressive and fatal in the majority of the cases, similar to that of ATP binding cassette subfamily A member 3 (ABCA3) deficiency. Protein C deficiency is insidious and may present with a radiological pulmonary interstitial pattern. Due to the similarity in the histological pattern, genetic studies help to achieve greater certainty in the prognosis and the possibility of providing adequate genetic counselling. Copyright © 2016 Sociedad Chilena de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. Effects of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in Wistar rats. (United States)

    Olatunji, Lawrence A; Usman, Taofeek O; Adebayo, Joseph O; Olatunji, Victoria A


    To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in rats. The 25 and 50 mg/(kg·d) of aqueous extracts of H. sabdariffa were respectively given to rats in the experimental groups for 28 d, and rats in the control group received an appropriate volume of distilled water as vehicle. Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in the kidney were assayed by spectrophotometric method. Administrations of 25 and 50 mg/(kg·d) of aqueous extract of H. sabdariffa significantly decreased the Ca(2+)-Mg(2+)-ATPase activity in the kidney of rats (Psabdariffa may preserve the renal function despite a decreased renal Ca(2+)-Mg(2+)-ATPase activity.

  5. On Allosteric Modulation of P-Type Cu+-ATPases

    DEFF Research Database (Denmark)

    Mattle, Daniel; Sitsel, Oleg; Autzen, Henriette E.


    of intramembranous Cu+ binding, and we suggest an alternative role for the proposed second site in copper translocation and proton exchange. The class-specific features demonstrate that topological diversity in P-type ATPases may tune a general energy coupling scheme to the translocation of compounds with remarkably...

  6. Changes in erythrocyte ATPase activity under different pathological ...

    African Journals Online (AJOL)

    Background: Studies have shown that Na+-K+ ATPase activity was altered in disrupted red blood cell membranes and this enzyme is believed to be the site of active transport of Na+ and K+ in intact red blood cells. The enzyme is often referred to as Na+- K+ pump because it pumps Na+ out and K+ into the cell against ...

  7. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)



    Jan 18, 2010 ... Cereal embryo development results in the formation of a fully differentiated, immature embryo within 10-12 ... quent root, coleoptile and leaf development. Although the transition from the dormant embryo ... cally stressed cultured Arabidopsis thaliana cells respon- ded by activating H+-ATPase-mediated H+ ...

  8. Towards the structure of yeast and mammalian P4-ATPases

    DEFF Research Database (Denmark)

    Lyons, Joseph; Laban, Milena; Mikkelsen, Stine


    a basis for the analysis of reported mutagenesis data, we aim to solve the first molecular structures of the PS transporting P4-ATPases using electron microscopy. To date, negative stain EM analysis, on detergent, amphipol and saposin-lipoprotein nanoparticle (Salipro) reconstituted of both Drs2p/CDC50p...

  9. Calcium-ATPases: Gene disorders and dysregulation in cancer. (United States)

    Dang, Donna; Rao, Rajini


    Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. RNAi-based silencing of genes encoding the vacuolar- ATPase ...

    African Journals Online (AJOL)


    Nov 9, 2016 ... Searching the PROSITE database revealed no common motifs for both subunits sequences. The phylogenetic tree analysis shows that the V-ATPase subunit a of both. P. gossypiella and Tribolium castaneum are located on the same branch as Bactrocera dorsalis with 94% bootstrap support (Figure 1a).

  11. Surfactant Protein D Levels in Umbilical Cord Blood and Capillary Blood of Premature Infants

    DEFF Research Database (Denmark)

    Dahl, Marianne; Holmskov, Uffe; Husby, Steffen


    Surfactant protein D (SP-D) is a collectin that plays an important role in the innate immune system and takes part in the surfactant homeostasis by regulating the surfactant pool size. The aims of this study were to investigate the values of SP-D in umbilical cord blood and capillary blood...... of premature infants and to relate the levels to perinatal conditions. A total of 254 premature infants were enrolled in the present study. Umbilical cord blood was drawn at the time of birth and capillary blood at regular intervals throughout the admission. The concentration of SP-D in umbilical cord blood...... and capillary blood was measured using ELISA technique. The median concentration of SP-D in umbilical cord blood was twice as high as in mature infants, 769 ng/mL (range 140-2,551), with lowest values in infants with intrauterine growth retardation (IUGR) and rupture of membranes (ROM). The median concentration...

  12. The vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation

    Directory of Open Access Journals (Sweden)

    Babatunde A. Ayodele


    Full Text Available Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2 were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase V0 subunit d2 (ATP6V0D2, was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium. In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.

  13. Murine ameloblasts are immunonegative for Tcirg1, the v-H-ATPase subunit essential for the osteoclast plasma proton pump. (United States)

    Bronckers, Antonius L J J; Lyaruu, Donacian M; Bervoets, Theodore J; Medina, Juan F; DenBesten, Pamela; Richter, Johan; Everts, Vincent


    Maturation stage ameloblasts of rodents express vacuolar type-H-ATPase in the ruffled border of their plasma membrane in contact with forming dental enamel, similar to osteoclasts that resorb bone. It has been proposed that in ameloblasts this v-H-ATPase acts as proton pump to acidify the enamel space, required to complete enamel mineralization. To examine whether this v-H-ATPase in mouse ameloblasts is a proton pump, we determined whether these cells express the lysosomal, T-cell, immune regulator 1 (Tcirg1, v-H-Atp6v(0)a(3)), which is an essential part of the plasma membrane proton pump that is present in osteoclasts. Mutation of this subunit in Tcirg1 null (or oc/oc) mice leads to severe osteopetrosis. No immunohistochemically detectable Tcirg1 was seen in mouse maturation stage ameloblasts. Strong positive staining in secretory and maturation stage ameloblasts however was found for another subunit of v-H-ATPase, subunit b, brain isoform (v-H-Atp6v(1)b(2)). Mouse osteoclasts and renal tubular epithelium stained strongly for both Tcirg1 and v-H-Atp6v(1)b(2). In Tcirg1 null mice osteoclasts and renal epithelium were negative for Tcirg1 but remained positive for v-H-Atp6v(1)b(2). The bone in these mutant mice was osteopetrotic, tooth eruption was inhibited or delayed, and teeth were often morphologically disfigured. However, enamel formation in these mutant mice was normal, ameloblasts structurally unaffected and the mineral content of enamel similar to that of wild type mice. We concluded that Tcirg1, which is essential for osteoclasts to pump protons into the bone, is not appreciably expressed in maturation stage mouse ameloblasts. Our data suggest that the reported v-H-ATPase in maturation stage ameloblasts is not the typical osteoclast-type plasma membrane associated proton pump which acidifies the extracellular space, but rather a v-H-ATPase potentially involved in intracellular acidification. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

    Directory of Open Access Journals (Sweden)

    Karolína Šubrtová


    Full Text Available In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt membrane potential (Δψm is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC. Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells.

  15. Interactions of organic contaminants with mineral-adsorbed surfactants (United States)

    Zhu, L.; Chen, B.; Tao, S.; Chiou, C.T.


    Sorption of organic contaminants (phenol, p-nitrophenol, and naphthalene) to natural solids (soils and bentonite) with and without myristylpyridinium bromide (MPB) cationic surfactant was studied to provide novel insight to interactions of contaminants with the mineral-adsorbed surfactant. Contaminant sorption coefficients with mineral-adsorbed surfactants, Kss, show a strong dependence on surfactant loading in the solid. At low surfactant levels, the Kss values increased with increasing sorbed surfactant mass, reached a maximum, and then decreased with increasing surfactant loading. The Kss values for contaminants were always higher than respective partition coefficients with surfactant micelles (Kmc) and natural organic matter (Koc). At examined MPB concentrations in water the three organic contaminants showed little solubility enhancement by MPB. At low sorbed-surfactant levels, the resulting mineral-adsorbed surfactant via the cation-exchange process appears to form a thin organic film, which effectively "adsorbs" the contaminants, resulting in very high Kss values. At high surfactant levels, the sorbed surfactant on minerals appears to form a bulklike medium that behaves essentially as a partition phase (rather than an adsorptive surface), with the resulting Kss being significantly decreased and less dependent on the MPB loading. The results provide a reference to the use of surfactants for remediation of contaminated soils/sediments or groundwater in engineered surfactant-enhanced washing.

  16. Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge displacement. (United States)

    Salvador, J M; Inesi, G; Rigaud, J L; Mata, A M


    The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.

  17. An Arabidopsis ATPase gene involved in nematode-induced syncytium development and abiotic stress responses (United States)

    Ali, Muhammad Amjad; Plattner, Stephan; Radakovic, Zoran; Wieczorek, Krzysztof; Elashry, Abdelnaser; Grundler, Florian MW; Ammelburg, Moritz; Siddique, Shahid; Bohlmann, Holger


    The beet cyst nematode Heterodera schachtii induces syncytia in the roots of Arabidopsis thaliana, which are its only nutrient source. One gene, At1g64110, that is strongly up-regulated in syncytia as shown by RT-PCR, quantitative RT-PCR, in situ RT-PCR and promoter::GUS lines, encodes an AAA+-type ATPase. Expression of two related genes in syncytia, At4g28000 and At5g52882, was not detected or not different from control root segments. Using amiRNA lines and T-DNA mutants, we show that At1g64110 is important for syncytium and nematode development. At1g64110 was also inducible by wounding, jasmonic acid, salicylic acid, heat and cold, as well as drought, sodium chloride, abscisic acid and mannitol, indicating involvement of this gene in abiotic stress responses. We confirmed this using two T-DNA mutants that were more sensitive to abscisic acid and sodium chloride during seed germination and root growth. These mutants also developed significantly smaller roots in response to abscisic acid and sodium chloride. An in silico analysis showed that ATPase At1g64110 (and also At4g28000 and At5g52882) belong to the ‘meiotic clade’ of AAA proteins that includes proteins such as Vps4, katanin, spastin and MSP1. PMID:23480402

  18. Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis. (United States)

    Leng, Xiangpeng; Mu, Qian; Wang, Xiaomin; Li, Xiaopeng; Zhu, Xudong; Shangguan, Lingfei; Fang, Jinggui


    With more copper and copper-containing compounds used as bactericides and fungicides in viticulture, copper homeostasis in grapevine (Vitis) has become one of the serious environmental crises with great risk. To better understand the regulation of Cu homeostasis in grapevine, grapevine seedlings cultured in vitro with different levels of Cu were utilized to investigate the tolerance mechanisms of grapevine responding to copper availability at physiological and molecular levels. The results indicated that Cu contents in roots and leaves arose with increasing levels of Cu application. With copper concentration increasing, malondialdehyde (MDA) content increased in roots and leaves and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased to protect the plant itself from damage. The expression patterns of 19 genes, encoding transporters, chaperones, and P-type ATPases involved in copper homeostasis in root and leaf of grapevine seedling under various levels of Cu(2+) were further analyzed. The expression patterns indicated that CTr1, CTr2, and CTr8 transporters were significantly upregulated in response both to Cu excess and deficiency. ZIP2 was downregulated in response to Cu excess and upregulated under Cu-deficient conditions, while ZIP4 had an opposite expression pattern under similar conditions. The expression of chaperones and P-type ATPases in response to Cu availability in grapevine were also briefly studied.

  19. Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump? (United States)

    Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc


    We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

  20. Gastrin decreases Na+,K+-ATPase activity via a PI 3-kinase- and PKC-dependent pathway in human renal proximal tubule cells. (United States)

    Liu, Tianbing; Konkalmatt, Prasad R; Yang, Yu; Jose, Pedro A


    The natriuretic effect of gastrin suggests a role in the coordinated regulation of sodium balance by the gastrointestinal tract and the kidney. The renal molecular targets and signal transduction pathways for such an effect of gastrin are largely unknown. Recently, we reported that gastrin induces NHE3 phosphorylation and internalization via phosphatidylinositol (PI) 3-kinase and PKCα. In this study, we show that gastrin induced the phosphorylation of human Na(+),K(+)-ATPase at serine 16, resulting in its endocytosis via Rab5 and Rab7 endosomes. The gastrin-stimulated phosphorylation of Na(+),K(+)-ATPase was dependent on PI 3-kinase because the phosphorylation was blocked by the PI 3-kinase inhibitor wortmannin. The phosphorylation of Na(+),K(+)-ATPase was also blocked by chelerythrine, a pan-PKC inhibitor, Gö-6976, a conventional PKC (cPKC) inhibitor, and BAPTA-AM, an intracellular calcium chelator, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The gastrin-mediated phosphorylation of Na(+),K(+)-ATPase was also inhibited by U-73122, a phospholipase C (PLC) inhibitor. These results suggest that gastrin regulates sodium hydrogen exchanger and pump in renal proximal tubule cells at the apical and basolateral membranes. Copyright © 2016 the American Physiological Society.

  1. Inhibition of Na(+),K(+)-ATPase in the hypothalamus, pons and cerebellum of the offspring rat due to experimentally-induced maternal hypothyroidism. (United States)

    Koromilas, Christos; Liapi, Charis; Zarros, Apostolos; Tsela, Smaragda; Zissis, Konstantinos M; Kalafatakis, Konstantinos; Skandali, Nikolina; Voumvourakis, Konstantinos; Carageorgiou, Haris; Tsakiris, Stylianos


    Neurodevelopment is known to be particularly susceptible to thyroid hormone insufficiency and can result in extensive structural and functional deficits within the central nervous system (CNS), subsequently leading to the establishment of cognitive impairment and neuropsychiatric symptomatology. The current study evaluated the effects of gestational and/or lactational maternal exposure to propylthiouracil (PTU)-induced hypothyroidism (as a suggestive multilevel experimental approach to the study of hypothyroidism-induced changes that has been developed and characterized by the authors) on crucial brain enzyme activities of 21-day-old Wistar rat offspring in a CNS region-specific manner. The activities of acetylcholinesterase (AChE), Na(+),K(+)-ATPase and Mg(2+)-ATPase in the offspring hypothalamus, cerebellum and pons were assessed. The study demonstrated that maternal exposure to PTU (0.05% w/v in the drinking water) during the critical periods of neurodevelopment can result in an inhibition of hypothalamic, pontine and cerebellar Na(+),K(+)-ATPase; a major marker of neuronal excitability and metabolic energy production as well as an important regulator of important systems of neurotransmission. On the other hand, no significant changes in the activities of the herein offspring CNS regions' AChE and Mg(2+)-ATPase were recorded. The observed Na(+),K(+)-ATPase inhibition: (i) is region-specific (and non-detectable in whole brain homogenetes), (ii) could constitute a central event in the pathophysiology of clinically-relevant hypothyroidism-associated developmental neurotoxicity, (iii) occurs under all examined experimental schemes, and (iv) certainly deserves further clarification at a molecular and histopathological level. As these findings are analyzed and compared to the available literature, they also underline the need for the adoption and further study of Na(+),K(+)-ATPase activity as a consistent neurochemical marker within the context of a systematic

  2. Improved Model of Proton Pump Crystal Structure Obtained by Interactive Molecular Dynamics Flexible Fitting Expands the Mechanistic Model for Proton Translocation in P-Type ATPases. (United States)

    Focht, Dorota; Croll, Tristan I; Pedersen, Bjorn P; Nissen, Poul


    The plasma membrane H(+)-ATPase is a proton pump of the P-type ATPase family and essential in plants and fungi. It extrudes protons to regulate pH and maintains a strong proton-motive force that energizes e.g., secondary uptake of nutrients. The only crystal structure of a H(+)-ATPase (AHA2 from Arabidopsis thaliana) was reported in 2007. Here, we present an improved atomic model of AHA2, obtained by a combination of model rebuilding through interactive molecular dynamics flexible fitting (iMDFF) and structural refinement based on the original data, but using up-to-date refinement methods. More detailed map features prompted local corrections of the transmembrane domain, in particular rearrangement of transmembrane helices 7 and 8, and the cytoplasmic N- and P-domains, and the new model shows improved overall quality and reliability scores. The AHA2 structure shows similarity to the Ca(2+)-ATPase E1 state, and provides a valuable starting point model for structural and functional analysis of proton transport mechanism of P-type H(+)-ATPases. Specifically, Asp684 protonation associated with phosphorylation and occlusion of the E1P state may result from hydrogen bond interaction with Asn106. A subsequent deprotonation associated with extracellular release in the E2P state may result from an internal salt bridge formation to an Arg655 residue, which in the present E1 state is stabilized in a solvated pocket. A release mechanism based on an in-built counter-cation was also later proposed for Zn(2+)-ATPase, for which structures have been determined in Zn(2+) released E2P-like states with the salt bridge interaction formed.

  3. Decreased ATPase activity in adriamycin nephrosis is independent of proteinuria

    Energy Technology Data Exchange (ETDEWEB)

    Bakker, W.W.; Kalicharan, D.; Donga, J.; Hulstaert, C.E.; Hardonk, M.J.


    In previous studies from this laboratory it has been shown that ATP-ase activity in situ in the glomerular basement membrane (GBM) is clearly reduced in rats rendered nephrotic after treatment with adriamycin (ADR). The question was raised whether this reduction of ATP-ase activity in the GBM is due to toxic activity of ADR or rather a result of the nephrotic condition per se. Therefore, we studied ATP-ase activity using the cerium-based method in kidneys from ADR-treated rats without proteinuria (48 hr after ADR injection), or with proteinuria (approximately 150 mg/24 hr) several weeks after ADR injection. Also kidneys from rats rendered nephrotic by surgical ablation and from non-nephrotic rats treated with local X-irradiation (2000 rads) as well as from normal control rats were studied. The results show that in the GBM of ADR-treated or irradiated rats, clear reduction of ATP-ase activity is observed irrespective of their proteinuria, whereas in the GBM of rats rendered nephrotic by renal ablation (approximately 156 mg/24 hr mean protein excretion) no reduction of enzyme activity is found. It is concluded that decreased ATP-ase activity of the glomerular filtration barrier in ADR-treated rats is due to an early toxic activity of this drug and not a result of the nephrotic state per se. In view of the identical results in X-irradiated rats, it is likely that ADR may act through production of toxic radicals leading to damage of this membrane-associated enzyme system.

  4. Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

    Directory of Open Access Journals (Sweden)

    Ali Mobasheri


    Full Text Available Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons. Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4 using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes.

  5. Surfactant nebulization versus instillation during high frequency ventilation in surfactant-deficient rabbits

    NARCIS (Netherlands)

    Dijk, Peter H.; Heikamp, A; Bambang Oetomo, Sidarto


    Surfactant nebulization improves lung function at low alveolar doses of surfactant. However, efficiency of nebulization is low, and lung deposition seems to depend on lung aeration. High frequency ventilation (HFV) has been shown to improve lung aeration. We hypothesize that the combination of HFV

  6. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Directory of Open Access Journals (Sweden)

    Prabhanshu Kumar


    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  7. Dilute Surfactant Methods for Carbonate Formations

    Energy Technology Data Exchange (ETDEWEB)

    Kishore K. Mohanty


    There are many fractured carbonate reservoirs in US (and the world) with light oil. Waterflooding is effective in fractured reservoirs, if the formation is water-wet. Many fractured carbonate reservoirs, however, are mixed-wet and recoveries with conventional methods are low (less than 10%). The process of using dilute anionic surfactants in alkaline solutions has been investigated in this work for oil recovery from fractured oil-wet carbonate reservoirs both experimentally and numerically. This process is a surfactant-aided gravity drainage where surfactant diffuses into the matrix, lowers IFT and contact angle, which decrease capillary pressure and increase oil relative permeability enabling gravity to drain the oil up. Anionic surfactants have been identified which at dilute concentration of 0.05 wt% and optimal salinity can lower the interfacial tension and change the wettability of the calcite surface to intermediate/water-wet condition as well or better than the cationic surfactant DTAB with a West Texas crude oil. The force of adhesion in AFM of oil-wet regions changes after anionic surfactant treatment to values similar to those of water-wet regions. The AFM topography images showed that the oil-wetting material was removed from the surface by the anionic surfactant treatment. Adsorption studies indicate that the extent of adsorption for anionic surfactants on calcite minerals decreases with increase in pH and with decrease in salinity. Surfactant adsorption can be minimized in the presence of Na{sub 2}CO{sub 3}. Laboratory-scale surfactant brine imbibition experiments give high oil recovery (20-42% OOIP in 50 days; up to 60% in 200 days) for initially oil-wet cores through wettability alteration and IFT reduction. Small (<10%) initial gas saturation does not affect significantly the rate of oil recovery in the imbibition process, but larger gas saturation decreases the oil recovery rate. As the core permeability decreases, the rate of oil recovery reduces

  8. A Review on Progress in QSPR Studies for Surfactants (United States)

    Hu, Jiwei; Zhang, Xiaoyi; Wang, Zhengwu


    This paper presents a review on recent progress in quantitative structure-property relationship (QSPR) studies of surfactants and applications of various molecular descriptors. QSPR studies on critical micelle concentration (cmc) and surface tension (γ) of surfactants are introduced. Studies on charge distribution in ionic surfactants by quantum chemical calculations and its effects on the structures and properties of the colloids of surfactants are also reviewed. The trends of QSPR studies on cloud point (for nonionic surfactants), biodegradation potential and some other properties of surfactants are evaluated. PMID:20479997

  9. Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation. (United States)

    Huang, Chun-Yen; Chao, Pei-Lin; Lin, Hui-Chen


    The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model. Copyright 2009 Elsevier B.V. All rights reserved.

  10. AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74

    Energy Technology Data Exchange (ETDEWEB)

    Hiraishi, Nobuhiro; Ishida, Yo-ichi; Nagahama, Masami, E-mail:


    Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis. - Highlights: • WDR74 accumulates in MTR4-exosome complex upon expression of dominant-negative NVL2. • WDR74 is co-localized with NVL2 in the nucleolus. • WDR74, along with NVL2, is involved in the synthesis of 60S ribosomal subunits.

  11. Thiourea modulates the expression and activity profile of mtATPase under salinity stress in seeds of Brassica juncea (United States)

    Srivastava, A. K.; Ramaswamy, N. K.; Mukopadhyaya, R.; Jincy, M. G. Chiramal; D'Souza, S. F.


    Background and Aims Large areas of the globe are becoming saline due to evapotranspiration and poor irrigation practices, and sustainability of agriculture is being seriously affected. Thiourea (TU) has been identified as an effective bioregulator imparting stress tolerance to crops. The molecular mechanisms involved in the TU-mediated response are considered in this study. Methods Differential display was performed in order to identify TU-modulated transcripts in Brassica juncea seeds exposed to various treatments (distilled water; 1 m NaCl; 1 m NaCl + 500 p.p.m. TU). The differential regulation of these transcripts was validated by quantitative real-time PCR. Key Results Thiourea treatment maintained the viability of seeds exposed to NaCl for 6 h. Expression analysis showed that the transcript level of alpha, beta, gamma, delta and epsilon subunits of mitochondrial ATPase (mtATPase) varied in seeds subjected to the different treatments for 1 h: expression level was significantly altered by 1 m NaCl relative to controls; however, in the NaCl + TU treatment it reverted back in an integrated manner. Similar results were obtained from time-kinetics studies of beta and delta subunits in roots of 8-d-old seedlings. These observations were also confirmed by the mtATPase activity from isolated mitochondria. The reversal in the expression and activity profile of mtATPase through the application of a bioregulator such as TU is a novel finding for any plant system. Conclusions The results suggest that TU treatment maintains the integrity and functioning of mitochondria in seeds as well as seedlings exposed to salinity. Thus, TU has the potential to be used as an effective bioregulator to impart salinity tolerance under field conditions, and might prove to be of high economic importance by opening new avenues for both basic and applied research. PMID:19033283

  12. Syntheses of surfactants from oleochemical epoxides

    Directory of Open Access Journals (Sweden)

    Warwel Siegfried


    Full Text Available Sugar-based surfactants were obtained in good yields (up to 100% under mild conditions (70°C, methanol or mixtures of methanol and water by ring-opening of terminal epoxides with aminopolyols, derived from glucose. Reaction of N-methyl glucamine with epoxides from even-numbered C4-C18 alpha-olefins or from terminal unsaturated fatty acid methyl esters leads to linear products, while corresponding reactions with N-dodecyl glucamine or glucamine yield surfactants with different Y-structures. Products obtained by conversion of omega-epoxy fatty acid methyl esters were saponificated with NaOH or hydrolyzed enzymatically to sodium salts or free acids respectively, which are amphoteric surfactants. Studies of the surfactants at different pH-values demonstrate different surface active properties in aqueous solutions. Critical micelle concentrations (c.m.c. in a range between 2 and 500mg/l and surface tensions of 25-40mN/m were measured for several of the synthesized sugar-based surfactants. The ring-opening products are rather poor foamers, whereas some of the corresponding hydrobromides show good foaming properties.

  13. Recovering hydrocarbons with surfactants from lignin

    Energy Technology Data Exchange (ETDEWEB)

    Naae, D.G.; Whittington, L.E.; Ledoux, W.A.; Debons, F.E.


    This patent describes a method of recovering hydrocarbons from an underground hydrocarbon formation penetrated by at least one injection well and at least one production well, which comprises: injecting into the formation through an injection well a surfactant slug comprising about 0.1% to about 10% by weight of surfactants produced from lignin, the surfactants produced by placing lignin in contact with water, converting the lignin into low molecular weight lignin phenols by reducing the lignin in the presence of a reducing agent of carbon monoxide or hydrogen creating a reduction reaction mixture comprising oil soluble lignin phenols, the reduction occurring at a temperature greater than about 200/sup 0/C and a pressure greater than about 100 psi, recovering the oil soluble lignin phenols from the reduction mixture, and converting the lignin phenols into lignin surfactants by a reaction selected from the group consisting of alkoxylation, sulfonation, sulfation, aklylation, sulfomethylation, and alkoxysulfation; injecting into the formation through the injection well a drive fluid to push the surfactant slug towards a production well; and recovering hydrocarbons at the production well.

  14. Surfactant effects on SF6 hydrate formation. (United States)

    Lee, Bo Ram; Lee, Ju Dong; Lee, Hyun Ju; Ryu, Young Bok; Lee, Man Sig; Kim, Young Seok; Englezos, Peter; Kim, Myung Hyun; Kim, Yang Do


    Sulfur hexafluoride (SF(6)) has been widely used in a variety of industrial processes, but it is one of the most potent greenhouse gases. For this reason, it is necessary to separate or collect it from waste gas streams. One separation method is through hydrate crystal formation. In this study, SF(6) hydrate was formed in aqueous surfactant solutions of 0.00, 0.01, 0.05, 0.15 and 0.20 wt% to investigate the effects of surfactants on the hydrate formation rates. Three surfactants, Tween 20 (Tween), sodium dodecyl sulfate (SDS) and linear alkyl benzene sulfonate (LABS), were tested in a semi-batch stirred vessel at the constant temperature and pressures of 276.2 K and 0.78 MPa, respectively. All surfactants showed kinetic promoter behavior for SF(6) hydrate formation. It was also found that SF(6) hydrate formation proceeded in two stages with the second stage being the most rapid. In situ Raman spectroscopy analysis revealed that the increased gas consumption rate with the addition of surfactant was possibly due to the increased gas filling rate in the hydrate cavity.

  15. How nanobubbles lose stability: Effects of surfactants (United States)

    Xiao, Qianxiang; Liu, Yawei; Guo, Zhenjiang; Liu, Zhiping; Zhang, Xianren


    In contrast to stability theories of nanobubbles, the molecular mechanism of how nanobubbles lose stability is far from being understood. In this work, we try to interpret recent experimental observations that the addition of surfactants destabilizes nanobubbles with an unclear mechanism. Using molecular dynamics simulations, we identify two surfactant-induced molecular mechanisms for nanobubbles losing stability, either through depinning of a contact line or reducing vapor-liquid surface tension. One corresponds to the case with significant adsorption of surfactants on the substrates, which causes depinning of the nanobubble contact line and thus leads to nanobubble instability. The other stresses surfactant adsorption on the vapor-liquid interface of nanobubbles, especially for insoluble surfactants, which reduces the surface tension of the interface and leads to an irreversible liquid-to-vapor phase transition. Our finding can help improve our understanding in nanobubble stability, and the insight presented here has implications for surface nanobubbles involving with other amphiphilic molecules, such as proteins and contaminations.

  16. Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

    Directory of Open Access Journals (Sweden)

    Kazi Md Kamrul Huda

    Full Text Available Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible

  17. Fullerene surfactants and their use in polymer solar cells (United States)

    Jen, Kwan-Yue; Yip, Hin-Lap; Li, Chang-Zhi


    Fullerene surfactant compounds useful as interfacial layer in polymer solar cells to enhance solar cell efficiency. Polymer solar cell including a fullerene surfactant-containing interfacial layer intermediate cathode and active layer.

  18. Synthesis of mesoporous nano-hydroxyapatite by using zwitterions surfactant (United States)

    Mesoporous nano-hydroxyapatite (mn-HAP) was successfully synthesized via a novel micelle-templating method using lauryl dimethylaminoacetic acid as zwitterionic surfactant. The systematic use of such a surfactant in combination with microwave energy inputenables the precise contr...

  19. Polyelectrolyte surfactant aggregates and their deposition on macroscopic surfaces

    CERN Document Server

    Voisin, D


    Oppositely charged surfactant and polyelectrolyte are present in hair shampoos and conditioners, together with particles (e.g. anti-dandruff agents for scalp) and droplets (e.g. silicone oil for the hair). These are normally formulated at high surfactant concentrations, beyond the flocculation region for the polyelectrolyte concentration used. However, on dilution with water, during application, flocs are formed which carry the particles and droplets to the scalp and hair. The addition of an anionic surfactant to an aqueous solution of cationic polyelectrolyte, at a given concentration, can lead to the formation of polyelectrolyte-surfactant 'particles', in which the surfactant 'binds' to the polyelectrolyte. This occurs from the critical association concentration (CAC), up to the surfactant concentration corresponding to maximum binding. Within this range of surfactant concentrations, the surfactant bound to the polyelectrolyte is thought to associate to form what might be termed 'internal micelles'. Each po...

  20. Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Gogarten Johann


    Full Text Available Abstract Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

  1. Recent food applications of microbial surfactants. (United States)

    Nitschke, Marcia; Silva, Sumária Sousa E


    Owing to their natural origin and environmental compatibility, interest in microbial surfactants or biosurfactants has gained attention during last few years. These characteristics fulfill the demand of regulatory agencies and society to use more sustained and green chemicals. Microbial-derived surfactants can replace synthetic surfactants in a great variety of industrial applications as detergents, foaming, emulsifiers, solubilizers, and wetting agents. Change in the trend of consumers toward natural from synthetic additives and the increasing health and environmental concerns have created demand for new "green" additives in foods. Apart from their inherent surface-active properties, biosurfactants have shown antimicrobial and anti-biofilm activities against food pathogens; therefore, biosurfactants can be versatile additives or ingredients of food processing. These interesting applications will be discussed in this review.

  2. Natural surfactants used in cosmetics: glycolipids. (United States)

    Lourith, N; Kanlayavattanakul, M


    Cosmetic surfactant performs detergency, wetting, emulsifying, solubilizing, dispersing and foaming effects. Adverse reactions of chemical synthesis surfactant have an effect on environment and humans, particularly severe in long term. Biodegradability, low toxicity and ecological acceptability which are the benefits of naturally derived surfactant that promises cosmetic safety are, therefore, highly on demand. Biosurfactant producible from microorganisms exhibiting potential surface properties suitable for cosmetic applications especially incorporate with their biological activities. Sophorolipids, rhamnolipids and mannosylerythritol lipids are the most widely used glycolipids biosurfactant in cosmetics. Literatures and patents relevant to these three glycolipids reviewed were emphasizing on the cosmetic applications including personal care products presenting the cosmetic efficiency, efficacy and economy benefits of glycolipids biosurfactant.


    Directory of Open Access Journals (Sweden)

    Pirog T. P.


    Full Text Available The literature and own experimental data on the synthesis of microbial surfactants of different chemical nature (rhamnolipids, sophorolipids, manozylerythritollipids, lipopeptides at various waste (vegetable oil and fat, sugar, dairy industry, agriculture, forestry, biodiesel, as well as waste — fried vegetable oils are presented. Most suitable substrates for the synthesis of microbial surfactants are oil containing waste that, unlike, for example, lignocellulose, whey, technical glycerol do not require pre-treatment and purification. Replacing traditional substrates for the biosynthesis of surfactant with industrial waste will help to reduce the cost of technology by several times, dispose of unwanted waste, solve the problem of storage or disposal of large amounts of waste from the food industry, agricultural sector and companies that produce biodiesel, which spent large amount of energy and money for such needs

  4. Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy. (United States)

    Yeung, Heidi O; Förster, Andreas; Bebeacua, Cecilia; Niwa, Hajime; Ewens, Caroline; McKeown, Ciarán; Zhang, Xiaodong; Freemont, Paul S


    The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.

  5. Vacuolar H(+)-ATPase and plasma membrane H(+)-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae. (United States)

    Watanabe, Taisuke; Furukawa, Soichi; Kitamoto, Katsuhiko; Takatsuki, Akira; Hirata, Ryogo; Ogihara, Hirokazu; Yamasaki, Makari


    As a non-thermal sterilization process, high-pressure carbon dioxide treatment (HPCT) is considered to be promising. The main sterilizing effect of HPCT is thought to be acidification in cytoplasm of microorganisms. We investigated the tolerance mechanism of Saccharomyces cerevisiae to HPCT with special reference to vacuolar and plasma membrane H(+)-ATPases. HPCT was imposed at 35 degrees C, 4 to 10 MPa, for 10 min. slp1 mutant defective in vacuole morphogenesis was more sensitive to HPCT than its isogenic parent. Concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), at 10 microM rendered the parent more HPCT-sensitive to the level of slp1. To confirm further the contribution of V-ATPase to the tolerance against HPCT in S. cerevisiae, we compared vma1 mutant of V-ATPase with its isogenic parent for their HPCT sensitivity. vma1 mutant was more sensitive to HPCT than its parent. Addition of 10 microM vanadate, an inhibitor of plasma membrane H(+)-ATPase (P-ATPase), to the wild type strains also increased the inactivation ratio. These results clearly show that V- and P-ATPases contribute to the tolerance against HPCT in S. cerevisiae by accumulating excess H(+) from cytoplasm to vacuole and excluding H(+) outside of the cell, respectively.

  6. Activation of Na+-K+-ATPase with DRm217 attenuates oxidative stress-induced myocardial cell injury via closing Na+-K+-ATPase/Src/Ros amplifier. (United States)

    Yan, Xiaofei; Xun, Meng; Dou, Xiaojuan; Wu, Litao; Zhang, Fujun; Zheng, Jin


    Reduced Na+-K+-ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na+-K+-ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na+-K+-ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H2O2)-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H2O2 on inhibition of Na+-K+-ATPase activity, Na+-K+-ATPase cell surface expression, and Src phosphorylation. H2O2-treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca2+, mitochondrial Ca2+ overload. DRm217 closed Na+-K+-ATPase/ROS amplifier, alleviated Ca2+ accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na+-K+-ATPase in oxidative stress and oxidative stress-related disease.

  7. Oscillations in glycolysis in Saccharomyces cerevisiae: the role of autocatalysis and intracellular ATPase activity. (United States)

    Kloster, Antonina; Olsen, Lars Folke


    We have investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F(0)F(1) ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations also decreases by stimulating the ATPase activity, e.g. by FCCP or Amphotericin B. Thus, ATPase activity strongly affects the glycolytic oscillations. We discuss these data in relation to a simple autocatalytic model of glycolysis which can reproduce the experimental data and explain the role of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although this model faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Microstructure, morphology and lifetime of armored bubbles exposed to surfactants


    Subramaniam, Anand Bala; Mejean, Cecile; Abkarian, Manouk; Stone, Howard A.


    We report the behavior of particle-stabilized bubbles (armored bubbles) when exposed to various classes and concentrations of surfactants. The bubbles are non-spherical due to the jamming of the particles on the interface and are stable to dissolution prior to the addition of surfactant. We find that the dissolving bubbles exhibit distinct morphological, microstructural, and lifetime changes, which correlate with the concentration of surfactant employed. For low concentrations of surfactant a...

  9. Nonequilibrium Energetics of a Single F1-ATPase Molecule


    Toyabe, Shoichi; Watanabe-Nakayama, Takahiro; Okamoto, Tetsuaki; Kudo, Seishi; Muneyuki, Eiro


    Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method. We show that the sum of the heat flow thr...

  10. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening


    Elbatsh, Ahmed M.O.; Haarhuis, Judith H.I.; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H.; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, Rene; Nasmyth, Kim; Rowland, Benjamin D.


    textabstractCohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sor...

  11. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and IP6 in mRNA export (United States)

    Montpetit, Ben; Thomsen, Nathan D.; Helmke, Kara J.; Seeliger, Markus A.; Berger, James M.; Weis, Karsten


    Superfamily 1 (SF1) and superfamily 2 (SF2) RNA helicases are ubiquitous mRNA-protein (mRNP) remodelling enzymes that play critical roles in all aspects of RNA metabolism1, 2. The SF2 DEAD-box ATPase Dbp5/Ddx19 functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC)3–8. Dbp5 is localized to the NPC via an interaction with Nup159/Nup2143–5, 8, 9 and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (IP6) 10, 11. Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo11, 12; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1IP6 and Nup159 regulate the activity of Dbp5. Here we report structures of Dbp5 in complex with Gle1IP6, Nup159/Gle1IP6, and RNA. These structures reveal that IP6 functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1IP6-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1IP6 and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1IP6 relieves Dbp5 auto- regulation and cooperates with Nup159 in stabilizing an open Dbp5-intermediate that precludes RNA binding. These findings explain how Gle1IP6, Nup159, and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators. PMID:21441902

  12. The Leishmania donovani lipophosphoglycan excludes the vesicular proton-ATPase from phagosomes by impairing the recruitment of synaptotagmin V.

    Directory of Open Access Journals (Sweden)

    Adrien F Vinet


    Full Text Available We recently showed that the exocytosis regulator Synaptotagmin (Syt V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galbeta1,4Manalpha1-PO(4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification.


    Directory of Open Access Journals (Sweden)

    P. Pirog


    Full Text Available The aim of the work was to study the effect of calcium and magnesium cations on NADP+-dependent glutamate dehydrogenase activity (key enzyme of biosynthesis of Acinetobacter calcoaceticus ІMV B-7241 surface-active aminolipids followed by modification of medium composition and determining antimicrobial and antiadhesive activity of synthesized surfactants. The strain IMV B-7241 was grown in medium with ethanol. NADP+-dependent glutamate dehydrogenase activity of the cell-free extract was analyzed using the formation of glutamate in the oxidation of NADPH. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1. Antimicrobial against bacteria properties of the surfactants were determined by index of the minimal inhibitory concentration. The number of attached cells and the degree of biofilm destruction were analyzed spectrophotometrically. It was established that in the presence of 10 mM Cа2+ and Mg2 NADP+-dependent glutamate dehydrogenase activity in the cell-free extract increased to 1.5 times in comparison with that without cations. Increasing concentration of magnesium sulfate to 0.2 g/l, or adding CaCl2 (0.1 g/l into cultivation medium of IMV B-7241 strain was accompanied by rise of NADP+-dependent glutamate dehydrogenase activity in 2.4 and 3.0 times respectively, as well as increasing antimicrobial and antiadhesive activity of synthesized surfactants. Minimal inhibitory concentration of surfactants synthesized in modified media against some bacteria was in 1.3−3.5 times, adhesion on abiotic surfaces treated with such surfactants in an average of 7−13% lower, and the degree of biofilm destruction in 7−13% higher as compared to indicators for the surfactant produced in the base medium. The obtained results indicated the possibility of regulating antimicrobial and anti-adhesive activity of surfactants under producer cultivation.

  14. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex. (United States)

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo


    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

    Energy Technology Data Exchange (ETDEWEB)

    Mathur, Chhavi [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India); Savithri, Handanahal S., E-mail: [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India)


    Highlights: Black-Right-Pointing-Pointer Pepper vein banding potyvirus VPg harbors Walker motifs. Black-Right-Pointing-Pointer VPg exhibits ATPase activity in the presence of NIa-Pro. Black-Right-Pointing-Pointer Plausible structural and functional interplay between VPg and NIa-Pro. Black-Right-Pointing-Pointer Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  16. The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism. (United States)

    Zhang, Chen-Song; Jiang, Bin; Li, Mengqi; Zhu, Mingjiang; Peng, Yongying; Zhang, Ya-Lin; Wu, Yu-Qing; Li, Terytty Yang; Liang, Yu; Lu, Zailian; Lian, Guili; Liu, Qing; Guo, Huiling; Yin, Zhenyu; Ye, Zhiyun; Han, Jiahuai; Wu, Jia-Wei; Yin, Huiyong; Lin, Shu-Yong; Lin, Sheng-Cai


    AMPK and mTOR play principal roles in governing metabolic programs; however, mechanisms underlying the coordination of the two inversely regulated kinases remain unclear. In this study we found, most surprisingly, that the late endosomal/lysosomal protein complex v-ATPase-Ragulator, essential for activation of mTORC1, is also required for AMPK activation. We also uncovered that AMPK is a residential protein of late endosome/lysosome. Under glucose starvation, the v-ATPase-Ragulator complex is accessible to AXIN/LKB1 for AMPK activation. Concurrently, the guanine nucleotide exchange factor (GEF) activity of Ragulator toward RAG is inhibited by AXIN, causing dissociation from endosome and inactivation of mTORC1. We have thus revealed that the v-ATPase-Ragulator complex is also an initiating sensor for energy stress and meanwhile serves as an endosomal docking site for LKB1-mediated AMPK activation by forming the v-ATPase-Ragulator-AXIN/LKB1-AMPK complex, thereby providing a switch between catabolism and anabolism. Our current study also emphasizes a general role of late endosome/lysosome in controlling metabolic programs. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L. (United States)

    Chen, Dan; Deng, Yingtian; Zhao, Jie


    Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.

  18. Surfactant therapy in late preterm infants

    Directory of Open Access Journals (Sweden)

    Murat Yurdakök


    Full Text Available Late preterm (LPT neonates are at a high risk for respiratory distress soon after birth due to respiratory distress syndrome (RDS, transient tachypnea of the newborn, persistent pulmonary hypertension, and pneumonia along with an increased need for surfactant replacement therapy, continuous positive airway pressure, and ventilator support when compared with the term neonates. In the past, studies on outcomes of infants with respiratory distress have primarily focused on extremely premature infants, leading to a gap in knowledge and understanding of the developmental biology and mechanism of pulmonary diseases in LPT neonates. Surfactant deficiency is the most frequent etiology of RDS in very preterm and moderately preterm infants, while cesarean section and lung infection play major roles in RDS development in LPT infants. The clinical presentation and the response to surfactant therapy in LPT infants may be different than that seen in very preterm infants. Incidence of pneumonia and occurrence of pneumothorax are significantly higher in LPT and term infants. High rates of pneumonia in these infants may result in direct injury to the type II alveolar cells of the lung with decreasing synthesis, release, and processing of surfactant. Increased permeability of the alveolar capillary membrane to both fluid and solutes is known to result in entry of plasma proteins into the alveolar hypophase, further inhibiting the surface properties of surfactant. However, the oxygenation index value do not change dramatically after ventilation or surfactant administration in LPT infants with RDS compared to very preterm infants. These finding may indicate a different pathogenesis of RDS in late preterm and term infants. In conclusion, surfactant therapy may be of significant benefit in LPT infants with serious respiratory failure secondary to a number of insults. However, optimal timing and dose of administration are not so clear in this group. Additional

  19. The Inhibiting or Accelerating Effect of Different Surfactants on ...

    African Journals Online (AJOL)

    The course of differential capacity curves of the electric double layer at the mercury electrode/surfactant solution interface was described for three different surfactants from different groups. Using square-wave voltammetry (SWV) it was found that the surfactants had a varying effect on the kinetics of electroreduction of Zn2+ ...

  20. Surfactant phosphatidylcholine metabolism in preterm infants studied with stable isotopes

    NARCIS (Netherlands)

    J.E.H. Bunt (Jan Erik)


    textabstractAIM OF THE STUDIES 1. To develop and use a novel method to study surfactant metabolism in preterm and older infants. (chapters 3 and 4). 2. To study endogenous surfactant synthesis in relation to prenatal glucocorticosteroids. (chapters 5 and 6). 3. To study the influence of surfactant


    NARCIS (Netherlands)

    BRACKMAN, JC; ENGBERTS, JBFN; Herb, CA; Prudhomme, RK


    The influence of the chemical structure and charge of the surfactant on polymer-micelle interaction has been studied using various ionic and non-ionic surfactants, and surfactants of which the charge can be varied by (de)protonation. Octylthioglucoside, dodecyldimethylamine-oxide, alkylphosphates,

  2. V-ATPase as an effective therapeutic target for sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Perut, Francesca, E-mail: [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)


    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

  3. Rotary ATPases: models, machine elements and technical specifications. (United States)

    Stewart, Alastair G; Sobti, Meghna; Harvey, Richard P; Stock, Daniela


    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual "machine elements" to the requirement of the right "fuel" and "oil" for different types of motors.

  4. Biophysical properties of protein-free, totally synthetic pulmonary surfactants, ALEC and Exosurf, in comparison with surfactant TA. (United States)

    Takahashi, A; Nemoto, T; Fujiwara, T


    An artificial pulmonary surfactant prepared from chloroform-methanol extract of bovine pulmonary surfactant (surfactant TA) has been shown to be effective in both the prevention and the treatment of respiratory distress syndrome in premature babies. Recently, two types of protein-free totally synthetic surfactants, artificial lung expanding compound (ALEC) and Exosurf, have been evaluated in clinical trials of surfactant therapy. Artificial lung expanding compound was used initially as a dry powder, but is now prepared as a crystalline suspension in saline at 4 degrees C. In this study we compared the biophysical properties of three different forms of ALEC (dry powder, crystalline suspension at 4 degrees C and 37 degrees C), Exosurf and surfactant TA (Surfacten) using a modified Wilhelmy surface balance and a pulsating bubble surfactometer. Surface activity of a crystalline suspension of ALEC in cold saline was no better than the dry powder of ALEC. Surfactant activity of ALEC was improved by addition of hydrophobic surfactant protein B and C (SP-B, SP-C) which are important constituents of surfactant TA. Surface properties of ALEC in any form and Exosurf were not superior to those of surfactant TA. These results suggest that a surfactant which contains SP-B and SP-C does not necessarily have to be dry or crystalline for an effective exogenous surfactant.

  5. Efficient Interfacially Driven Vehiculization of Corticosteroids by Pulmonary Surfactant. (United States)

    Hidalgo, Alberto; Salomone, Fabrizio; Fresno, Nieves; Orellana, Guillermo; Cruz, Antonio; Perez-Gil, Jesus


    Pulmonary surfactant is a crucial system to stabilize the respiratory air-liquid interface. Furthermore, pulmonary surfactant has been proposed as an effective method for targeting drugs to the lungs. However, few studies have examined in detail the mechanisms of incorporation of drugs into surfactant, the impact of the presence of drugs on pulmonary surfactant performance at the interface under physiologically meaningful conditions, or the ability of pulmonary surfactant to use the air-liquid interface to vehiculise drugs to long distances. This study focuses on the ability of pulmonary surfactant to interfacially vehiculize corticosteroids such as beclomethasone dipropionate (BDP) or Budesonide (BUD) as model drugs. The main objectives have been to (a) characterize the incorporation of corticosteroids into natural and synthetic surfactants, (b) evaluate whether the presence of corticosteroids affects surfactant functionality, and (c) determine whether surfactant preparations enable the efficient spreading and distribution of BDP and BUD along the air-liquid interface. We have compared the performance of a purified surfactant from porcine lungs and two clinical surfactants: Poractant alfa, a natural surfactant of animal origin extensively used to treat premature babies, and CHF5633, a new synthetic surfactant preparation currently under clinical trials. Both, natural and clinical surfactants spontaneously incorporated corticosteroids up to at least 10% by mass with respect to phospholipid content. The presence of the drugs did not interfere with their ability to efficiently adsorb into air-liquid interfaces and form surface active films able to reach and sustain very low surface tensions (<2 mN/m) under compression-expansion cycling mimicking breathing dynamics. Furthermore, the combination of clinical surfactant with corticosteroids efficiently promoted the active diffusion of the drug to long distances along the air-liquid interface. This effect could not be

  6. Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum. (United States)

    Lu, Karen W; Pérez-Gil, Jesús; Echaide, Mercedes; Taeusch, H William


    Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum


    Lu, Karen W.; Pérez-Gil, Jesús; Echaide, Mercedes; Taeusch, H. William


    Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfac...

  8. Surfactant-anti-surfactant immune complexes in infants with respiratory distress syndrome.


    Strayer, D. S.; Merritt, T A; Lwebuga-Mukasa, J.; Hallman, M


    The authors sought to determine whether treatment of respiratory distress syndrome (RDS) with human surfactant resulted in the formation of detectable circulating immune complexes. Preterm infants with severe RDS were divided into two groups: one group received human surfactant by intratracheal instillation and the other group did not. Both groups received ventilatory management involving intermittent mandatory ventilation. Plasma samples were drawn from these babies prior to treatment and at...

  9. Palm oil based surfactant products for petroleum industry (United States)

    Permadi, P.; Fitria, R.; Hambali, E.


    In petroleum production process, many problems causing reduced production are found. These include limited oil recovery, wax deposit, asphaltene deposit, sludge deposit, and emulsion problem. Petroleum-based surfactant has been used to overcome these problems. Therefore, innovation to solve these problems using surfactant containing natural materials deserves to be developed. Palm oil-based surfactant is one of the potential alternatives for this. Various types of derivative products of palm oil-based surfactant have been developed by SBRC IPB to be used in handling problems including surfactant flooding, well stimulation, asphaltene dissolver, well cleaning, and wax removal found in oil and gas industry.

  10. Dimeric Surfactants: Promising Ingredients of Cosmetics and Toiletries

    Directory of Open Access Journals (Sweden)

    Naveen Kumar


    Full Text Available Surfactants are an essential ingredient for cosmetic, toiletries and personal care products for enhancing their performance. Dimeric surfactants demonstrate superiority compared to conventional surfactants in all areas of application. Dimeric surfactants are extremely promising for utilization in various cosmetic formulations viz. shampoo, lotions, creams, conditioners etc. These surfactants possess extremely unique surface properties viz. lower surface tension, unique micellization, low critical micelle concentration (CMC and antimicrobial activity, higher solubilization etc. Dimerics enhance the performances of cosmetics in an extraordinary manner and provide eco-friendly preparations for human epidermis.

  11. Thermally stable surfactants and compositions and methods of use thereof (United States)

    Chaiko, David J [Woodridge, IL


    There are provided novel thermally stable surfactants for use with fillers in the preparation of polymer composites and nanocomposites. Typically, surfactants of the invention are urethanes, ureas or esters of thiocarbamic acid having a hydrocarbyl group of from 10 to 50 carbons and optionally including an ionizable or charged group (e.g., carboxyl group or quaternary amine). Thus, there are provided surfactants having Formula I: ##STR00001## wherein the variables are as defined herein. Further provided are methods of making thermally stable surfactants and compositions, including composites and nanocomposites, using fillers coated with the surfactants.

  12. Physicochemical characteristics of PFC surfactants for dry decontamination

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Won Jin; Lee, Chi Woo [Korea University, Seoul (Korea)


    Even the trace amount of the used nuclear fuels of high radioactivity are hazardous to the earth and humans. Perfluorocarbons and perfluorocarbon surfactants are emerging to be efficient chemicals in the dry decontamination process of the used fuels of high radioactivity. The theme was undertaken to increase the knowledge on perfluorocarbon surfactants to develop the perfluorocarbon system in the dry decontamination process in Korea. Several cationic and anionic pfc surfactants were synthesized. Effects of pfc surfactants on electrochemical etching of silicon were investigated to form porous silicons. Forces were measured between silicon surfaces and AFM tip in the absence and presence of pfc surfactants. 7 refs., 10 figs. (Author)

  13. enhanced ultrafiltration at low surfactant concentrations

    African Journals Online (AJOL)


    Apr 24, 2006 ... Micellar-enhanced ultrafiltration (MEUF) of zinc ions (Zn2+) from aqueous solutions using single anionic surfactant sodium dodecyl sulphate (SDS) at low critical micelle concentrations (cmc) (0.2×cmc – 3×cmc) was investigated. When the initial. SDS concentration was below the cmc, unexpectedly high ...

  14. Surfactant protein D is proatherogenic in mice

    DEFF Research Database (Denmark)

    Sørensen, Grith Lykke; Madsen, Jens; Kejling, Karin


    Surfactant protein D (SP-D) is an important innate immune defense molecule that mediates clearance of pathogens and modulates the inflammatory response. Moreover, SP-D is involved in lipid homeostasis, and pulmonary accumulation of phospholipids has previously been observed in SP-D-deficient (Spd...


    Energy Technology Data Exchange (ETDEWEB)

    Kishore K. Mohanty


    There are many carbonate reservoirs in US (and the world) with light oil and fracture pressure below its minimum miscibility pressure (or reservoir may be naturally fractured). Many carbonate reservoirs are naturally fractured. Waterflooding is effective in fractured reservoirs, if the formation is water-wet. Many fractured carbonate reservoirs, however, are mixed-wet and recoveries with conventional methods are low (less than 10%). Thermal and miscible tertiary recovery techniques are not effective in these reservoirs. Surfactant flooding (or huff-n-puff) is the only hope, yet it was developed for sandstone reservoirs in the past. The goal of this research is to evaluate dilute (hence relatively inexpensive) surfactant methods for carbonate formations and identify conditions under which they can be effective. We have acquired field oil and core samples and field brine compositions from Marathon. We have conducted preliminary adsorption and wettability studies. Addition of Na{sub 2}CO{sub 3} decreases anionic surfactant adsorption on calcite surface. Receding contact angles increase with surfactant adsorption. Plans for the next quarter include conducting adsorption, phase behavior and wettability studies.

  16. Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase. (United States)

    Zhang, Z; Chen, D; Wheatly, M G


    The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of

  17. Surfactant for pulmonary haemorrhage in neonates. (United States)

    Aziz, Abdul; Ohlsson, Arne


    In the 1960s and 1970s, pulmonary haemorrhage (PH) occurred mainly in full-term infants with pre-existing illness with an incidence of 1.3 per 1000 live births. Risk factors for PH included severity of illness, intrauterine growth restriction, patent ductus arteriosus (PDA), coagulopathy and the need for assisted ventilation. Presently, PH occurs in 3% to 5% of preterm ventilated infants with severe respiratory distress syndrome (RDS) who often have a PDA and have received surfactant. The cause of PH is thought to be due to rapid lowering of intrapulmonary pressure, which facilitates left to right shunting across a PDA and an increase in pulmonary blood flow. Retrospective case reports and one prospective uncontrolled study have shown promising results for surfactant in treating PH. To evaluate the effect of surfactant treatment compared to placebo or no intervention on mortality and morbidities in neonates with PH. For this update The Cochrane Library, Issue 2, 2012; MEDLINE; EMBASE; CINAHL;;; proceedings (2000 to 2011) of the Annual Meetings of the Pediatric Academic Societies (Abstracts2View) and Web of Science were searched on 8 February 2012. Randomised or quasi-randomised controlled trials that evaluated the effect of surfactant in the treatment of PH in intubated term or preterm (neonates with PH. Infants were included up to 44 weeks' postmenstrual age. The interventions studied were intratracheal instillation of surfactant (natural or synthetic, regardless of dose) versus placebo or no intervention. If studies were identified by the literature search, the planned analyses included risk ratio, risk difference, number needed to treat to benefit or to harm for dichotomous outcomes, and mean difference for continuous outcomes, with their 95% confidence intervals. A fixed-effect model would be used for meta-analyses. The risk of bias for included trials would be assessed. Heterogeneity tests, including the I(2) statistic

  18. Synthesis and properties evaluation of sulfobetaine surfactant with double hydroxyl (United States)

    Zhou, Ming; Luo, Gang; Zhang, Ze; Li, Sisi; Wang, Chengwen


    A series of sulfobetaine surfactants {N-[(3-alkoxy-2-hydroxyl)propoxy] ethyl-N,N-dimethyl-N-(2-hydroxyl)propyl sulfonate} ammonium chloride were synthesized with raw materials containing linear saturated alcohol, N,N-dimethylethanolamine, sodium 3-chloro-2-hydroxyl propane sulfonic acid and epichlorohydrin. The molecule structures of sulfobetaine surfactants were characterized by FTIR, 1HNMR and elemental analysis. Surface tension measurements can provide us information about the surface tension at the CMC (γCMC), pC20, Γmax and Amin. The pC20 values of sulfobetaine surfactants increase with the hydrophobic chain length increasing. Amin values of the surfactants decrease with increasing hydrophobic chain length from 10 to 14. The critical micelle concentration (CMC) and surface tension (γCMC) values of the sulfobetaine surfactants decrease with increasing hydrophobic chain length from 10 to 16. The lipophilicity of surfactant was enhanced with the increase of the carbon chain, however, the ability of anti-hard water was weakened. The minimum oil/water interfacial tension of four kinds of sulfobetaine surfactants is 10-2-10-3 mN/m magnitude, which indicates that the synthesized bis-hydroxy sulfobetaine surfactants have a great ability to reduce interfacial tension in the surfactant flooding system. The surface tension (γCMC) values of synthesized surfactants were lower compared with conventional anionic surfactant sodium dodecyl sulfonate.

  19. Comparative study of clinical pulmonary surfactants using atomic force microscopy (United States)

    Zhang, Hong; Fan, Qihui; Wang, Yi E.; Neal, Charles R.; Zuo, Yi Y.


    Clinical pulmonary surfactant is routinely used to treat premature newborns with respiratory distress syndrome, and has shown great potential in alleviating a number of neonatal and adult respiratory diseases. Despite extensive study of chemical composition, surface activity, and clinical performance of various surfactant preparations, a direct comparison of surfactant films is still lacking. In this study, we use atomic force microscopy to characterize and compare four animal-derived clinical surfactants currently used throughout the world, i.e., Survanta, Curosurf, Infasurf and BLES. These modified-natural surfactants are further compared to dipalmitoyl phosphatidylcholine (DPPC), a synthetic model surfactant of DPPC:palmitoyl-oleoyl phosphatidylglycerol (POPG) (7:3), and endogenous bovine natural surfactant. Atomic force microscopy reveals significant differences in the lateral structure and molecular organization of these surfactant preparations. These differences are discussed in terms of DPPC and cholesterol contents. We conclude that all animal-derived clinical surfactants assume a similar structure of multilayers of fluid phospholipids closely attached to an interfacial monolayer enriched in DPPC, at physiologically relevant surface pressures. This study provides the first comprehensive survey of the lateral structure of clinical surfactants at various surface pressures. It may have clinical implications on future application and development of surfactant preparations. PMID:21439262

  20. F-actin reorganization by V-ATPase inhibition in prostate cancer. (United States)

    Licon-Munoz, Yamhilette; Michel, Vera; Fordyce, Colleen A; Parra, Karlett J


    The vacuolar ATPase (V-ATPase) proton pump sustains cellular pH homeostasis, and its inhibition triggers numerous stress responses. However, the cellular mechanisms involved remain largely elusive in cancer cells. We studied V-ATPase in the prostate cancer (PCa) cell line PC-3, which has characteristics of highly metastatic PCa. V-ATPase inhibitors impaired endo-lysosomal pH, vesicle trafficking, migration, and invasion. V-ATPase accrual in the Golgi and recycling endosomes suggests that traffic of internalized membrane vesicles back to the plasma membrane was particularly impaired. Directed movement provoked co-localization of V-ATPase containing vesicles with F-actin near the leading edge of migrating cells. V-ATPase inhibition prompted prominent F-actin cytoskeleton reorganization. Filopodial projections were reduced, which related to reduced migration velocity. F-actin formed novel cytoplasmic rings. F-actin rings increased with extended exposure to sublethal concentrations of V-ATPase inhibitors, from 24 to 48 h, as the amount of alkalinized endo-lysosomal vesicles increased. Studies with chloroquine indicated that F-actin rings formation was pH-dependent. We hypothesize that these novel F-actin rings assemble to overcome widespread traffic defects caused by V-ATPase inhibition, similar to F-actin rings on the surface of exocytic organelles. © 2017. Published by The Company of Biologists Ltd.

  1. [Effects of cocaine on activities of ATPase, LDH and SDH in mouse splenocytes]. (United States)

    Sun, Wen-ping; Lu, Yan-xu; Zhang, Xiao-yu; Tang, Wei-wei; Huang, Qing-yu


    To examine the effects of cocaine on the activities of ATPase, LDH and SDH in cultured mouse splenocytes in vitro. The ATPase, LDH and SDH activities in mouse splenocytes were detected at day 7 after continuous culturing the mouse cells exposed to cocaine hydrochloride in final concentration of 10, 20 and 100 microg/mL in vitro. The activities of ATPase, LDH and SDH in mouse splenocytes exposed to cocaine hydrochloride in final concentration of 10, 20 and 100 microg/mL were significantly decreased after continuous culturing for 7 days. The present study demonstrated that cocaine could inhibit the activities of ATPase, LDH and SDH in cultured splenocytes in vitro.

  2. Surfactant Replacement Therapy Beyond Respiratory Distress Syndrome in Neonates. (United States)

    Jasani, Bonny; Kabra, Nandkishor; Nanavati, Ruchi


    Surfactant replacement therapy is an established modality of treatment in preterm neonates with respiratory distress syndrome. In addition, there are various neonatal respiratory disorders which are characterized by surfactant deficiency in which surfactant therapy can be a feasible and safe option. To collate the literature on the use of surfactant replacement therapy in neonates beyond respiratory distress syndrome and examine the evidence and newer developments. We searched the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), MEDLINE, and EMBASE up to June 2015; and previous reviews, including cross-references, abstracts, and conference proceedings. Evidence supports surfactant administration via bolus route in neonates with meconium aspiration syndrome, but additional robust evidence is required before its adoption in clinical practice. There is limited evidence to support surfactant therapy in neonates with pneumonia, pulmonary hemorrhage and bronchopulmonary dysplasia. Large multicenter randomized trials are needed to cement or refute the role of surfactant therapy in these disorders.


    Energy Technology Data Exchange (ETDEWEB)

    George J. Hirasaki; Clarence A. Miller; Gary A. Pope; Richard E. Jackson


    Surfactant flooding has the potential to significantly increase recovery over that of conventional waterflooding. The availability of a large number of surfactant structures makes it possible to conduct a systematic study of the relation between surfactant structure and its efficacy for oil recovery. Also, the addition of an alkali such as sodium carbonate makes possible in situ generation of surfactant and significant reduction of surfactant adsorption. In addition to reduction of interfacial tension to ultra-low values, surfactants and alkali can be designed to alter wettability to enhance oil recovery. An alkaline surfactant process is designed to enhance spontaneous imbibition in fractured, oil-wet, carbonate formations. It is able to recover oil from dolomite core samples from which there was no oil recovery when placed in formation brine.

  4. Hereditary Parkinsonism-Associated Genetic Variations in PARK9 Locus Lead to Functional Impairment of ATPase Type 13A2. (United States)

    Park, Jin-Sung; Sue, Carolyn M


    Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of Parkinson's disease (PD) with juvenile onset of parkinsonism, often accompanied by extra clinical features such as supranuclear gaze palsy, dementia and generalised brain atrophy. Mutations in ATP13A2, associated with the PARK9 locus (chromosome 1p36) have been identified in KRS patients. ATP13A2 encodes a lysosomal P5B-type ATPase which has functional domains similar to other P-type ATPases which mainly transport cations. Consistently, recent studies suggest that human ATP13A2 may preferably regulate Zn2+, while ATP13A2 from other species have different substrate selectivity. Until now, fourteen mutations in ATP13A2 have been associated with KRS, while other mutations have been reported in association with neuronal ceroid lipofuscinosis (NCL) and early-onset PD. Experimentally, these disease- associated ATP13A2 mutations have been shown to confer loss-of-function to the protein by disrupting its protein structure and function to varying degrees, ranging from impairment in ATPase function to total loss of protein, confirming their pathogenicity. Loss of functional ATP13A2 has been shown to induce Zn2+ dyshomeostasis. Disturbances in Zn2+ homeostasis impair mitochondrial and lysosomal function which leads to loss of mitochondrial bioenergetic capacity and accumulation of lysosomal substrates such as α-synuclein and lipofuscin. Additionally, ATP13A2 appears to be involved in α-synuclein externalisation through its Zn2+-regulating activity. In this review, we will discuss all the reported KRS/NCL-associated ATP13A2 mutations along with several PD-associated mutations which have been experimentally assessed, in respect to their impact on the protein structure and function of ATP13A2. Copyright© Bentham Science Publishers; For any queries, please email at

  5. Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds. (United States)

    Darginaviciene, Jūrate; Pasakinskiene, Izolda; Maksimov, Gemir; Rognli, Odd Arne; Jurkoniene, Sigita; Sveikauskas, Vaidevutis; Bareikiene, Nijole


    Changes in plasmalemma K(+)Mg(2+)-ATPase dephosphorylating activity and H(+) transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and DeltamuH(+) were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K(+)Mg(2+)-ATPase activity was found throughout the seasonal growth of the plants (i). The K(+)Mg(2+)-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K(+)Mg(2+)-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K(+)Mg(2+)-ATPase activity than in that of H(+) transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4-2 degrees C) and at freezing (-8 degrees C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.

  6. Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle. (United States)

    Bowman, B J; Berenski, C J; Jung, C Y


    Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.

  7. The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport

    DEFF Research Database (Denmark)

    Sahoo, Sanjaya K; Shaikh, Sana A; Sopariwala, Danesh H


    The sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is responsible for intracellular Ca(2+) homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca(2+)-bound SERCA, SLN continues...

  8. Evolution of pulmonary surfactants for the treatment of neonatal respiratory distress syndrome and paediatric lung diseases. (United States)

    Mazela, Jan; Merritt, T Allen; Gadzinowski, Janusz; Sinha, Sunil


    This review documents the evolution of surfactant therapy, beginning with observations of surfactant deficiency in respiratory distress syndrome, the basis of exogenous surfactant treatment and the development of surfactant-containing novel peptides patterned after SP-B. We critically analyse the molecular interactions of surfactant proteins and phospholipids contributing to surfactant function. Peptide-containing surfactant provides clinical efficacy in the treatment of respiratory distress syndrome and offers promise for treating other lung diseases in infancy.

  9. Cysteine residues 244 and 458–459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

    Directory of Open Access Journals (Sweden)

    Irina Yu. Petrushanko


    Full Text Available Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD and nucleotide binding domain (NBD of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines’ thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG. Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459 were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor

  10. Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions. (United States)

    Petrushanko, Irina Yu; Mitkevich, Vladimir A; Lakunina, Valentina A; Anashkina, Anastasia A; Spirin, Pavel V; Rubtsov, Peter M; Prassolov, Vladimir S; Bogdanov, Nikolay B; Hänggi, Pascal; Fuller, William; Makarov, Alexander A; Bogdanova, Anna


    Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase

  11. Pulmonary haptoglobin (pHp) is part of the surfactant system in the human lung. (United States)

    Abdullah, Mahdi; Goldmann, Torsten


    Since the existence of pHp was demonstrated, it has been shown that this molecule and its receptor CD163 are regulated by different stimuli. Furthermore, a comparably fast secretion of pHp was described as well as the immuno-stimulatory effects. The intention of this study was to elucidate the role of pHp in the human lungs further. Here we show, by means of confocal microscopy and immune-electron-microscopy, a clear co-localization of pHp with surfactant protein-B in lamellar bodies of alveolar epithelial cells type II. These results are underlined by immunohistochemical stainings in differently fixed human lung tissues, which show pHp in vesicular and released form. The images of the released form resemble the intended position of surfactant in the human alveolus. pHp is secreted by Alveolar epithelial cells type II as previously shown. Moreover, pHp is co-localized with Surfactant protein-B. We conclude that the presented data shows that pHp is a native part of the surfactant system in the human lung.

  12. Photo-Fenton degradation of non-ionic surfactant and its mixture with cationic or anionic surfactant. (United States)

    Ono, Erina; Tokumura, Masahiro; Kawase, Yoshinori


    The oxidative degradation of non-ionic surfactants by the photo-Fenton process has been examined. The photo-Fenton degradation kinetics of mixtures of non-ionic surfactant and other type surfactants has been also investigated since mixtures of non-ionic and ionic surfactants are commonly used to utilize their synergistic effects in many practices. Effects of operating parameters such as dosages of Fenton reagents (iron and hydrogen peroxide) and UV light intensity on the degradation of commercial non-ionic surfactant Sannonic SS-90 (polyoxyethylene alkyl ether) were studied. Although the dosages of the Fenton reagents increased the degradation rate up to the optimum dosages, further addition of the reagents could not enhance the degradation rate. Excess dosages of Fe and H(2)O(2) caused excess OH radicals which could be a scavenger of OH radicals and as a result could not enhance the degradation of the surfactant. The increase in UV light intensity resulting in the faster photo-Fenton process or the enhancement of OH radical formation rate led to the increase in degradation rate of non-ionic surfactant. Although the existence of the anionic surfactant (sodium dodecylbenzene sulphonate) would inhibit the degradation of the non-ionic surfactant due to the formation of complex with Fe ion, the existence of cationic surfactant (dodecyltrimethyl ammonium chloride) affected insignificantly the photo-Fenton degradation process of the non-ionic surfactant.

  13. The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly

    Energy Technology Data Exchange (ETDEWEB)

    Her, Joonyoung [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of); Chung, In Kwon, E-mail: [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of)


    Highlights: Black-Right-Pointing-Pointer Identification of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. Black-Right-Pointing-Pointer NVL2 associates with catalytically active telomerase via an interaction with hTERT. Black-Right-Pointing-Pointer NVL2 is a telomerase component essential for holoenzyme assembly. Black-Right-Pointing-Pointer ATP-binding activity of NVL2 is required for hTERT binding and telomerase assembly. -- Abstract: Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.

  14. An Overview of Pulmonary Surfactant in the Neonate: Genetics, Metabolism, and the Role of Surfactant in Health and Disease (United States)

    Nkadi, Paul O.; Merritt, T. Allen; Pillers, De-Ann M.


    Pulmonary surfactant is a complex mixture of phospholipids (PL) and proteins (SP) that reduce surface tension at the air-liquid interface of the alveolus. It is made up of about 70% to 80% PL, mainly dipalmitoylphosphatidylcholine (DPPC), 10% SP-A, B, C and D, and 10% neutral lipids, mainly cholesterol. Surfactant is synthesized, assembled, transported and secreted into the alveolus where it is degraded and then recycled. Metabolism of surfactant is slower in newborns, especially preterm, than in adults. Defective pulmonary surfactant metabolism results in respiratory distress with attendant morbidity and mortality. This occurs due to accelerated breakdown by oxidation, proteolytic degradation, inhibition or inherited defects of surfactant metabolism. Prenatal corticosteroids, surfactant replacement, whole lung lavage and lung transplantation have yielded results in managing some of these defects. Gene therapy could prove valuable in treating inherited defects of surfactant metabolism. PMID:19299177

  15. An enzyme-linked immunosorbent assay (ELISA) for quantification of mouse surfactant protein D (SP-D)

    DEFF Research Database (Denmark)

    Hansen, Soren; Schmidt, Vivi; Steffensen, Maria Abildgaard


    Surfactant protein D (SP-D) is a pattern recognition molecule of the collectin family of C-type lectins. It is found in the airways and at mucosal surfaces. SP-D is part of the innate immune system where it neutralizes and leads to elimination of microorganisms. It regulates the functions of othe...

  16. Surfactant for pulmonary hemorrhage in neonates. (United States)

    Aziz, Abdul; Ohlsson, Arne


    In the late 1960's and 1970's, pulmonary hemorrhage (PH) occurred mainly in full term infants with severe pre-existing illness. The incidence of PH was quoted as 1.3 per 1,000 live births. In the older medical literature, the risk factors associated with PH included the severity of the associated illness, intrauterine growth restriction, patent ductus arteriosus (PDA), coagulopathy, and the need for assisted ventilation. Presently, PH occurs mainly in preterm ventilated infants with severe respiratory distress syndrome (RDS) who often have a PDA and have received surfactant. Currently, PH complicates the hospital course of 3-5% of preterm infants with RDS. Although not clear, the cause of PH is thought to be due to a rapid lowering of intrapulmonary pressure, which facilitates left to right shunting across a patent ductus arteriosus and an increase in pulmonary blood flow. Retrospective case reports and one prospective uncontrolled study that used surfactant for PH in neonates have shown promising results in treating PH. To evaluate the effect of surfactant treatment compared to placebo or no intervention on mortality in neonates with pulmonary hemorrhage. In addition, the review will evaluate the effect of surfactant treatment on neonatal morbidities associated with PH compared to placebo or no intervention. The following databases were searched in January 2008: The Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 4, 2007) and MEDLINE from 1966 and EMBASE from 1980 to the time of the full review using the OVID interface. The proceedings of the Annual Meetings of the Pediatric Academic Societies and the European Society of Pediatric Research published in Pediatric Research or electronically on their web sites were searched from 1994 to the time of the full review. Science citation index (Web of Science) was searched for authors quoting key references of observational studies. Randomised or quasi-randomised controlled trials that

  17. Human decidua-derived mesenchymal stem cells differentiate into functional alveolar type II-like cells that synthesize and secrete pulmonary surfactant complexes.

    Directory of Open Access Journals (Sweden)

    Alejandro Cerrada

    Full Text Available Lung alveolar type II (ATII cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs have been differentiated into Alveolar Type II- Like Cells (ATII-LCs, which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

  18. A new role for P2X4 receptors as modulators of lung surfactant secretion

    Directory of Open Access Journals (Sweden)

    Pika eMiklavc


    Full Text Available In recent years P2X receptors have attracted increasing attention as regulators of exocytosis and cellular secretion. In various cell types P2X receptors have been found to stimulate vesicle exocytosis directly via Ca2+ influx and elevation of the intracellular Ca2+ concentration. Recently, a new role for P2X4 receptors as regulators of secretion emerged. Exocytosis of lamellar bodies (LBs, large storage organelles for lung surfactant, results in a local, fusion-activated Ca2+ entry (FACE in alveolar type II epithelial cells. FACE is mediated via P2X4 receptors that are located on the limiting membrane of LBs and inserted into the plasma membrane upon exocytosis of LBs. The localized Ca2+ influx at the site of vesicle fusion promotes fusion pore expansion and facilitates surfactant release. In addition, this inward-rectifying cation current across P2X4 receptors mediates fluid resorption from lung alveoli. It is hypothesized that the concomitant reduction in the alveolar lining fluid facilitates insertion of surfactant into the air-liquid interphase thereby activating it. These findings constitute a novel role for P2X4 receptors in regulating vesicle content secretion as modulators of the secretory output during the exocytic post-fusion phase.

  19. Application of peptide gemini surfactants as novel solubilization surfactants for photosystems I and II of cyanobacteria. (United States)

    Koeda, Shuhei; Umezaki, Katsunari; Noji, Tomoyasu; Ikeda, Atsushi; Kawakami, Keisuke; Kondo, Masaharu; Yamamoto, Yasushi; Shen, Jian-Ren; Taga, Keijiro; Dewa, Takehisa; Ito, Shigeru; Nango, Mamoru; Tanaka, Toshiki; Mizuno, Toshihisa


    We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PG-surfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 °C compared with that using β-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV(2+)) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the light-irradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PG-surfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

  20. Effects of phenol on ATPase activities in crude gill homogenates of rainbow trout (Salmo gairdneri Richardson)

    Energy Technology Data Exchange (ETDEWEB)

    Poston, T.M.


    The ATPase specific activities from crude gill homogenates of rainbow trout were lower than those from microsomal preparations reported in the literature. Sodium pump activity (ouabain sensitive NaK-ATPase) was demonstrable at 37/sup 0/C. An ouabain insensitive NaK-ATPase was demonstrable at temperatures below 30/sup 0/C and may represent a Na-ATPase activity reported by others. Energy of activation at 25/sup 0/C for total NaK-ATPase ws 10,500 cal.mole/sup -1/. Mg-baseline activity had an energy of activation at 25/sup 0/C of 15,600 cal.mole/sup -1/. Mg-baseline activity was thermally labile at temperatures in excess of 30/sup 0/C. Concentrations of Mg/sup +2/ in excess of 5 mM appeared to inhibit total NaK-ATPase activity. At 37/sup 0/C, Na/sup +/ and K/sup +/ exerted little, if any, stimulatory effect on ATPase activities, in spite of the fact that 37/sup 0/C was the only temperature at which sodium pump activity was demonstrable. MS-222 failed to produce any discernible changes in any of the demonstrable ATPase activities in crude gill homogenates. Total NaK-ATPase activities were more sensitive than Mg-baseline activities to in vitro inhibition by phenol. Concentrations of phenol which produce 50% inhibition in total NaK-ATPase produced only 35% inhibition in Mg-baseline activity. The nature of in vitro inhibition was uncompetitive. Sodium pump activity was unaffected by phenol at concentrations as high as 25 mM. An effort was made to demonstrate an in vivo effects of phenol on rainbow trout gill ATPase activites. An infestation of a parasite (Gyrodactilus) on the experimental fish precludes any definative assessment of in vivo effects.

  1. Peran Enzim Ca2+ - ATPase pada Membran Sel Darah Merah

    Directory of Open Access Journals (Sweden)

    Ferry P. Gultom


    Full Text Available Ca2+ - ATP ase enzyme is transmembrane protein which is found in membrane cell. This protein works as a pump in many cases such as thalassemia which causes leakage of the cell as there is oxidation of sulphidril chain from amino acid in membrane. The calcium-ion intake must be pumped out to get red blood cell homeostatic condition. The aim of this paper is to determine the activity of Ca2+ - ATPase enzyme as a pump of Ca ion in membrane cell.

  2. Surfactant Based Enhanced Oil Recovery and Foam Mobility Control

    Energy Technology Data Exchange (ETDEWEB)

    George J. Hirasaki; Clarence A. Miller


    Surfactant flooding has the potential to significantly increase recovery over that of conventional waterflooding. The availability of a large number of surfactant structures makes it possible to conduct a systematic study of the relation between surfactant structure and its efficacy for oil recovery. A mixture of two surfactants was found to be particularly effective for application in carbonate formations at low temperature. The mixture is single phase for higher salinity or calcium concentrations than that for either surfactant used alone. This makes it possible to inject the surfactant slug with polymer close to optimal conditions and yet be single phase. A formulation has been designed for a particular field application. It uses partially hydrolyzed polyacrylamide for mobility control. The addition of an alkali such as sodium carbonate makes possible in situ generation of naphthenic soap and significant reduction of synthetic surfactant adsorption. The design of the process to maximize the region of ultra-low IFT takes advantage of the observation that the ratio of soap to synthetic surfactant is a parameter in the conditions for optimal salinity. Even for a fixed ratio of soap to surfactant, the range of salinity for low IFT was wider than that reported for surfactant systems in the literature. Low temperature, forced displacement experiments in dolomite and silica sandpacks demonstrate that greater than 95% recovery of the waterflood remaining oil is possible with 0.2% surfactant concentration, 0.5 PV surfactant slug, with no alcohol. Compositional simulation of the displacement process demonstrates the role of soap/surfactant ratio on passage of the profile through the ultralow IFT region, the importance of a wide salinity range of low IFT, and the importance of the viscosity of the surfactant slug. Mobility control is essential for surfactant EOR. Foam is evaluated to improve the sweep efficiency of surfactant injected into fractured reservoirs as well as a

  3. Pulmonary surfactant adsorption is increased by hyaluronan or polyethylene glycol. (United States)

    Taeusch, H William; Dybbro, Eric; Lu, Karen W


    In acute lung injuries, inactivating agents may interfere with transfer (adsorption) of pulmonary surfactants to the interface between air and the aqueous layer that coats the interior of alveoli. Some ionic and nonionic polymers reduce surfactant inactivation in vitro and in vivo. In this study, we tested directly whether an ionic polymer, hyaluronan, or a nonionic polymer, polyethylene glycol, enhanced adsorption of a surfactant used clinically. We used three different methods of measuring adsorption in vitro: a modified pulsating bubble surfactometer; a King/Clements device; and a spreading trough. In addition we measured the effects of both polymers on surfactant turbidity, using this assay as a nonspecific index of aggregation. We found that both hyaluronan and polyethylene glycol significantly increased the rate and degree of surfactant material adsorbed to the surface in all three assays. Hyaluronan was effective in lower concentrations (20-fold) than polyethylene glycol and, unlike polyethylene glycol, hyaluronan did not increase apparent aggregation of surfactant. Surfactant adsorption in the presence of serum was also enhanced by both polymers regardless of whether hyaluronan or polyethylene glycol was included with serum in the subphase or added to the surfactant applied to the surface. Therefore, endogenous polymers in the alveolar subphase, or exogenous polymers added to surfactant used as therapy, may both be important for reducing inactivation of surfactant that occurs with various lung injuries.


    Pirog, T P; Beregova, K A; Savenko, I V; Shevchuk, T A; Iutynska, G O


    To study the effect of Nocardia vaccinii IMV B-7405 surfactants on some bacteria (including pathogens of genera Proteus, Staphylococcus, Enterobacter), yeast of Candida species and fungi (Aspergillus niger R-3, Fusarium culmorum T-7). The antimi- crobial properties of surfactant were determined in suspension culture by Koch method and also by index of the minimum inhibitory concentration. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). It is shown that the antimicrobial properties of N. vaccinii IMV B-7405 surfactant depended on the degree of purification (supernatant, solution of surfactant), concentration and exposure. Survival of Escherichia coli IEM-1 and Bacillus subtilis BT-2 (both vegetative cells and spores) after treatment for 1-2 hours with surfactants solution and the supernatant (the surfactant concentration 21 µg/ml) was 3-28%. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants on studied bacteria, yeast and micromycetes were 11.5-85.0; 11.5-22.5 and 165.0-325.0 µ/ml respectively. Minimum inhibitory concentrations of N. vaccinii IMV B-7405 surfactants are comparable to those of the known microbial surfactants. The possibility of using the supernatant of culture liquid as an effective antimicrobial agent noticeably simplifies and reduces the cost of the technology of its obtaining.


    Energy Technology Data Exchange (ETDEWEB)

    George J. Hirasaki; Clarence A. Miller; Gary A. Pope; Richard E. Jackson


    Surfactant flooding has the potential to significantly increase recovery over that of conventional waterflooding. The availability of a large number of surfactants makes it possible to conduct a systematic study of the relation between surfactant structure and its efficacy for oil recovery. Also, the addition of an alkali such as sodium carbonate makes possible in situ generation of surfactant and significant reduction of surfactant adsorption. In addition to reduction of interfacial tension to ultra-low values, surfactants and alkali can be designed to alter wettability to enhance oil recovery. An alkaline surfactant process is designed to enhance spontaneous imbibition in fractured, oil-wet, carbonate formations. It is able to recover oil from dolomite core samples from which there was no oil recovery when placed in formation brine. Mobility control is essential for surfactant EOR. Foam is evaluted to improve the sweep efficiency of surfactant injected into fractured reservoirs. UTCHEM is a reservoir simulator specially designed for surfactant EOR. A dual-porosity version is demonstrated as a potential scale-up tool for fractured reservoirs.

  6. Next Generation Surfactants for Improved Chemical Flooding Technology

    Energy Technology Data Exchange (ETDEWEB)

    Laura Wesson; Prapas Lohateeraparp; Jeffrey Harwell; Bor-Jier Shiau


    The principle objective of this project was to characterize and test current and next generation high performance surfactants for improved chemical flooding technology, focused on reservoirs in the Pennsylvanian-aged (Penn) sands. In order to meet this objective the characteristic curvatures (Cc) of twenty-eight anionic surfactants selected for evaluation for use in chemical flooding formulations were determined. The Cc values ranged from -6.90 to 2.55 with the majority having negative values. Crude oil samples from nine Penn sand reservoirs were analyzed for several properties pertinent to surfactant formulation for EOR application. These properties included equivalent alkane carbon numbers, total acid numbers, and viscosity. The brine samples from these same reservoirs were analyzed for several cations and for total dissolved solids. Surfactant formulations were successfully developed for eight reservoirs by the end of the project period. These formulations were comprised of a tertiary mixture of anionic surfactants. The identities of these surfactants are considered proprietary, but suffice to say the surfactants in each mixture were comprised of varying chemical structures. In addition to the successful development of surfactant formulations for EOR, there were also two successful single-well field tests conducted. There are many aspects that must be considered in the development and implementation of effective surfactant formulations. Taking into account these other aspects, there were four additional studies conducted during this project. These studies focused on the effect of the stability of surfactant formulations in the presence of polymers with an associated examination of polymer rheology, the effect of the presence of iron complexes in the brine on surfactant stability, the potential use of sacrificial agents in order to minimize the loss of surfactant to adsorption, and the effect of electrolytes on surfactant adsorption. In these last four studies

  7. Surfactant protein B inhibits secretory phospholipase A2 hydrolysis of surfactant phospholipids (United States)

    Grier, Bonnie L.; Waite, B. Moseley; Veldhuizen, Ruud A.; Possmayer, Fred; Yao, Li-Juan; Seeds, Michael C.


    Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A2 (sPLA2) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA2 exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA2-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA2-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1–5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (DPPC:POPC:POPG, 40:40:20) prepared as vesicles and monomolecular films in the presence or absence of SP-B. Hydrolysis of PG and PC by Group IB sPLA2 (PLA2G1A) was significantly lower in the extracted OS, which contains SP-B, compared with Survanta (P = 0.005), which is SP-B poor. Hydrolysis of PG and PC in nonextracted OS, which contains all SPs, was lower than both Survanta and extracted OS. When Survanta was supplemented with 1% SP-B, PG and PC hydrolysis by PLA2G1B was significantly lower (P hydrolysis by both PLA2G1B and Group IIA sPLA2 (PLA2G2A). In films, PLA2G1B hydrolyzed surfactant PL monolayers at surface pressures ≤30 mN/m (P hydrolysis can occur. These results suggest the hydrophobic SP, SP-B, protects alveolar surfactant PL from hydrolysis mediated by multiple sPLA2 in both vesicles (alveolar subphase) and monomolecular films (air-liquid interface). PMID:22037357

  8. Microemulsion-based lycopene extraction: Effect of surfactants, co-surfactants and pretreatments. (United States)

    Amiri-Rigi, Atefeh; Abbasi, Soleiman


    Lycopene is a potent antioxidant that has received extensive attention recently. Due to the challenges encountered with current methods of lycopene extraction using hazardous solvents, industry calls for a greener, safer and more efficient process. The main purpose of present study was application of microemulsion technique to extract lycopene from tomato pomace. In this respect, the effect of eight different surfactants, four different co-surfactants, and ultrasound and enzyme pretreatments on lycopene extraction efficiency was examined. Experimental results revealed that application of combined ultrasound and enzyme pretreatments, saponin as a natural surfactant, and glycerol as a co-surfactant, in the bicontinuous region of microemulsion was the optimal experimental conditions resulting in a microemulsion containing 409.68±0.68 μg/glycopene. The high lycopene concentration achieved, indicates that microemulsion technique, using a low-cost natural surfactant could be promising for a simple and safe separation of lycopene from tomato pomace and possibly from tomato industrial wastes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Hydrogels of sodium alginate in cationic surfactants: Surfactant dependent modulation of encapsulation/release toward Ibuprofen. (United States)

    Jabeen, Suraya; Chat, Oyais Ahmad; Maswal, Masrat; Ashraf, Uzma; Rather, Ghulam Mohammad; Dar, Aijaz Ahmad


    The interaction of cetyltrimethylammoium bromide (CTAB) and its gemini homologue (butanediyl-1,4-bis (dimethylcetylammonium bromide), 16-4-16 with biocompatible polymer sodium alginate (SA) has been investigated in aqueous medium. Addition of K2CO3 influences viscoelastic properties of surfactant impregnated SA via competition between electrostatic and hydrophobic interactions. Viscosity of these polymer-surfactant systems increases with increase in concentration of K2CO3, and a cryogel is formed at about 0.5M K2CO3 concentration. The thermal stability of gel (5% SA+0.5M K2CO3) decreases with increase in surfactant concentration, a minimum is observed with increase in 16-4-16 concentration. The impact of surfactant addition on the alginate structure vis-à-vis its drug loading capability and release thereof was studied using Ibuprofen (IBU) as the model drug. The hydrogel with 16-4-16 exhibits higher IBU encapsulation and faster release in comparison to the one containing CTAB. This higher encapsulation-cum-faster release capability has been related to micelle mediated solubilization and greater porosity of the hydrogel with gemini surfactant. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. The effect of postadded ethylene glycol surfactants on DNA-cationic surfactant/water mesophases. (United States)

    Leal, Cecilia; Bilalov, Azat; Lindman, Björn


    The addition of amphiphiles grafted with polyethylene glycol units to constructs of DNA-amphiphiles is of most relevance for applications demanding colloidal stability. In this work, we study the self-assembly behavior of a true ternary mixture comprising (i) an electroneutral complex of DNA and a cationic surfactant (dodecyltrimethylammonium, DTA), (ii) water, and (iii) nonionic surfactant (dodecyl tetraethylene glycol, C12EO4; and dodecyl octaethylene glycol, C12EO8). The phase diagrams of the two systems: DNA-DTA/C12EO4/water and DNA-DTA/C12EO8/water were carried out using 2H NMR, and small-angle X-ray scattering (SAXS). In both mixtures, the DNA-DTA complex incorporates the postadded nonionic surfactant and large liquid crystalline regions were found. The supramolecular assemblies evolve from a 2D hexagonal structure of the normal type to a lamellar phase as the content of nonionic surfactant is increased. The effect of ethylene glycol unit size in the phase behavior is discussed. We suggest that when longer ethylene glycol units (C12EO8 vs C12EO4) are used, the DNA-DTA aggregate gets saturated with the nonionic surfactant and there exists a coexistence of a fully swollen mesophase phase of C12EO8 alone presumably of the normal hexagonal type with the lamellar and hexagonal phases of DNA-DTA/C12EO8/water.

  11. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

    Directory of Open Access Journals (Sweden)

    Geisler Markus


    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  12. Topological transformation of a surfactant bilayer

    DEFF Research Database (Denmark)

    Le, T.D.; Olsson, U.; Mortensen, K.


    Surfactant lamellar phases are often complicated by the formation of multilamellar (onions) under shear, which can originate simply by shaking the sample. A systematic study has been performed on the C10E3-D2O system in which different bilayer structures under a steady shear flow were investigate......>(b), in the transformation from onion to plane to multiply connected bilayer structure as a function of temperature. (C) 2000 Elsevier Science B.V. All rights reserved.......Surfactant lamellar phases are often complicated by the formation of multilamellar (onions) under shear, which can originate simply by shaking the sample. A systematic study has been performed on the C10E3-D2O system in which different bilayer structures under a steady shear flow were investigated...

  13. Surfactant protein D in newborn infants

    DEFF Research Database (Denmark)

    Dahl, Marianne; Juvonen, Pekka Olavi; Holmskov, Uffe


    Surfactant protein D (SP-D) is a collectin that plays an important role in the innate immune system. The role of SP-D in the metabolism of surfactant is as yet quite unclear. The aims of this study were to establish normal values of SP-D in the umbilical cord blood and capillary blood of mature...... was measured by enzyme-linked immunosorbent assay. The median concentration of SP-D in umbilical cord blood was 392.1 ng/mL and was found to be influenced by maternal smoking and labor. The median concentration of SP-D in capillary blood was 777.5 ng/mL and was found to be influenced by the mode of delivery...

  14. Tailoring Hierarchical Zeolites with Designed Cationic Surfactants and Their High Catalytic Performance. (United States)

    Zhang, Kai; Li, Chao; Liu, Zewei; Wang, Min; Yan, Xin; Xi, Hongxia


    Three hierarchical porous zeolites (H-*BEA, H-MTW, and H-*MRE) were successfully synthesized with the assistance of designed cationic surfactants under hydrothermal synthesis conditions. The as-synthesized zeolite samples can be easily regulated by changing the number of long hydrophobic n-alkyl chains. Also, we investigated the relationship between the length of the surfactant and the formation of the microporous structure of the zeolite. Furthermore, the alkylation of benzene with propene was performed as a probe reaction to evaluate the catalytic performance of the synthesized hierarchical zeolites. The resulting materials were characterized by using a complementary combination of techniques, that is, X-ray powder diffraction, N 2 adsorption-desorption isotherms, scanning electron microscopy, transmission electron microscopy, Fourier transform IR spectroscopy, 28 Si and 27 Al MAS NMR spectroscopies, thermogravimetric analysis, and computer simulation. These analysis results indicated that quaternary ammonium surfactants acted as organic structure-directing agents (OSDAs) in the formation of these hierarchical zeolite samples, whether the surfactant had long hydrophobic tail groups or not. The simulation results indicated that the organic molecules with no long hydrophobic chain could lead to the synthesis of zeolite through charge control, and the hydrophobic molecules with long hydrophobic chains could form zeolites through orbital control. These hierarchical zeolites showed improved catalytic activity towards the industrially relevant alkylation of benzene with propene compared with conventional zeolites with the same frameworks. More importantly, the success of using quaternary ammonium surfactants with no hydrophobic n-alkyl tail group in the synthesis of hierarchically structured mesoporous zeolites provides a new pathway for the synthesis of hierarchical porous materials by a soft-templating method. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Role of matrix metalloprotease-2 in oxidant activation of Ca ATPase ...

    Indian Academy of Sciences (India)


    Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2. (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth ...

  16. Interaction of the indole class of vacuolar H+-ATPase inhibitors with lipid bilayers

    NARCIS (Netherlands)

    Fernandes, F.; Loura, L.; Koehorst, R.B.M.; Dixon, N.; Kee, T.P.; Hemminga, M.A.; Prieto, M.


    The selective inhibitor of osteoclastic V-ATPase (2Z,4E)-5-(5,6-dichloro-2-indolyi)-2-methoxy-N-(1,2,2,6,6-pentamethylpip eridin-4-yl)-2,4-pentadienamide (SB 242784), member of the indole class of V-ATPase inhibitors, is expected to target the membrane-bound domain of the enzyme. A structural study

  17. Excess capacity of H+ ATPase and inverse respiratory control in Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Westerhoff, Hans V.; Michelsen, Ole


    With succinate as free-energy source, Escherichia coli generating virtually all ATP by oxidative phosphorylation might be expected heavily to tax its ATP generating capacity. To examine this the H+-ATPase (ATP synthase) was modulated over a 30-fold range. Decreasing the amount of H+-ATPase reduced...

  18. Na+,K+-ATPase concentration in rodent and human heart and skeletal muscle

    DEFF Research Database (Denmark)

    Kjeldsen, K; Bjerregaard, P; Richter, Erik


    0.02) and the heart to body weight ratio by 14% (p less than 0.005). The increase in Na+,K+-ATPase concentration was only slowly reversible. After three weeks of deconditioning an increase of 12% (p less than 0.05) was still observed. In comparison skeletal muscle Na+,K+-ATPase concentration...


    DEFF Research Database (Denmark)

    Andersen, A.; Treiman, M.; Poulsen, J. C. J.


    The preparation of a nonionic desoxy-analogue of thapsigargin possessing a Ca2+-ATPase inhibitory potency similar to that of thapsioargin is described.......The preparation of a nonionic desoxy-analogue of thapsigargin possessing a Ca2+-ATPase inhibitory potency similar to that of thapsioargin is described....

  20. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus; Liu, Xiang-Yu; Skjørringe, Tina


    copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B...... proteins associated with Menkes' and Wilson's diseases....

  1. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.


    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  2. A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K. (United States)

    Homareda, Haruo; Otsu, Masahiro; Yamamoto, Sachiko; Ushimaru, Makoto; Ito, Sayaka; Fukutomi, Toshiyuki; Jo, Taeho; Eishi, Yoshinobu; Hara, Yukichi


    The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the β subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

  3. Leptin decreases renal medullary Na(+), K(+)-ATPase activity through phosphatidylinositol 3-kinase dependent mechanism. (United States)

    Bełtowski, J; Marciniak, A; Wójcicka, G


    We examined the effect of leptin on renal function and renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities in the rat. Leptin was infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. Leptin infused at doses of 1 and 10 microg/kg/min increased urine output by 40% and 140%, respectively. Urinary Na(+) excretion increased in rats receiving leptin at doses of 0.1, 1, and 10 microg/kg/min by 57.6%, 124.2% and 163.6%, respectively. Leptin had no effect on creatinine clearance, potassium excretion and phosphate excretion. Na(+),K(+)-ATPase activity in the renal medulla of rats treated with 1 and 10 microg/kg/min leptin was lower than in control animals by 25.5% and 33.2%, respectively. In contrast, cortical Na(+),K(+)-ATPase as well as either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase activities did not differ between leptin-treated and control animals. The effect of leptin on Na(+),K(+)-ATPase activity was abolished by actin depolymerizing agents, cytochalazin D and latrunculin B, and by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002. These results indicate that: 1). natriuretic effect of leptin is mediated, at least in part, by decrease in renal medullary Na(+),K(+)-ATPase activity, 2). inhibition of medullary Na(+),K(+)-ATPase by leptin is mediated by PI3K and requires integrity of actin cytoskeleton.

  4. Hemin reconstitutes proton extrusion in an H+-ATPase-negative mutant of Lactococcus lactis

    DEFF Research Database (Denmark)

    Blank, L.M.; Købmann, Brian Jensen; Michelsen, Ole


    H+-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H+-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells...

  5. Molecular dynamics of surfactant protein C

    DEFF Research Database (Denmark)

    Ramírez, Eunice; Santana, Alberto; Cruz, Anthony


    Surfactant protein C (SP-C) is a membrane-associated protein essential for normal respiration. It has been found that the alpha-helix form of SP-C can undergo, under certain conditions, a transformation from an alpha-helix to a beta-strand conformation that closely resembles amyloid fibrils, which...... occurs at the amino terminal, and despite this unfolding, no transition from alpha-helix to beta-strand was observed....

  6. Relationship between Na(+),K(+)-ATPase and cell attachment. (United States)

    Contreras, R G; Shoshani, L; Flores-Maldonado, C; Lázaro, A; Cereijido, M


    A prolonged ouabain blockade of the Na(+),K(+)-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, alpha-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190(Rho-GAP), a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved. Another clone of MDCK cells whose Na(+),K(+)-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P-->A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.

  7. Examining zinc transporting P-type ATPases by genome editing

    DEFF Research Database (Denmark)

    Østerberg, Jeppe Thulin

    Zinc is an essential micronutrient in plants. It is taken up from the soil by the plant roots and transported from there throughout the plant tissues, as a final destination ending up in the plant progeny, the seed. In this route from soil to seed, several bottlenecks occur. The export of zinc...... into apoplastic gaps is required both for xylem loading in the roots and for loading into the apoplastic space in the seeds, the tissue surrounding the next plant generation. The movement of the positively charged zinc ions out of the symplast is against the electrochemical gradient and thus requires active...... transport. Both in the model plant Arabidopsis thaliana and in the cereals, the P1B2-ATPases have been demonstrated to be involved in the loading of zinc into the xylem, and here we demonstrate that the Arabidopsis P1B2-ATPase AtHMA2 and AtHMA4 are involved in loading of zinc into the seed apoplast. The P1B...

  8. Differential adjustment in gill Na+/K+- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae). (United States)

    Faleiros, Rogério Oliveira; Goldman, Maria Helena S; Furriel, Rosa P M; McNamara, John Campbell


    We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase α-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25‰ S. During a 10-day acclimation period to 25‰ S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase α-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25‰ S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21‰ S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the

  9. Probing Nanoscale Thermal Transport in Surfactant Solutions (United States)

    Cao, Fangyu; Liu, Ying; Xu, Jiajun; He, Yadong; Hammouda, B.; Qiao, Rui; Yang, Bao


    Surfactant solutions typically feature tunable nanoscale, internal structures. Although rarely utilized, they can be a powerful platform for probing thermal transport in nanoscale domains and across interfaces with nanometer-size radius. Here, we examine the structure and thermal transport in solution of AOT (Dioctyl sodium sulfosuccinate) in n-octane liquids using small-angle neutron scattering, thermal conductivity measurements, and molecular dynamics simulations. We report the first experimental observation of a minimum thermal conductivity occurring at the critical micelle concentration (CMC): the thermal conductivity of the surfactant solution decreases as AOT is added till the onset of micellization but increases as more AOT is added. The decrease of thermal conductivity with AOT loading in solutions in which AOT molecules are dispersed as monomers suggests that even the interfaces between individual oleophobic headgroup of AOT molecules and their surrounding non-polar octane molecules can hinder heat transfer. The increase of thermal conductivity with AOT loading after the onset of micellization indicates that the thermal transport in the core of AOT micelles and across the surfactant-oil interfaces, both of which span only a few nanometers, are efficient.

  10. Characterization of the phospholemman knockout mouse heart: depressed left ventricular function with increased Na-K-ATPase activity. (United States)

    Bell, James R; Kennington, Erika; Fuller, William; Dighe, Kushal; Donoghue, Pamela; Clark, James E; Jia, Li-Guo; Tucker, Amy L; Moorman, J Randall; Marber, Michael S; Eaton, Philip; Dunn, Michael J; Shattock, Michael J


    Phospholemman (PLM, FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na-K-ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE and Western blot analysis of ventricular homogenates revealed small, nonsignificant changes in Na- K-ATPase subunit expression, with two-dimensional gel (isoelectric focusing, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate that PLM plays an important role in the contractile function of the normoxic mouse heart. Data are consistent with the hypothesis that PLM modulates Na-K-ATPase activity, indirectly affecting intracellular Ca and hence contractile function.

  11. Effect of Surfactants on the Deformation and Detachment of Oil Droplets in a Model Laminar Flow Cell

    Directory of Open Access Journals (Sweden)

    Fréville V.


    Full Text Available Sugar-based surfactants are increasingly present in the development of eco-friendly detergents due to current regulations and consumer demand. In order to assess the degreasing performance of these new surfactants, the behavior of model oil droplets subjected to the action of a flow of surfactant solutions of different concentrations was studied in a laminar flow cell and related to the physico-chemical properties measured at the liquid/liquid (interfacial tension and solid/liquid/liquid interfaces (contact angle. With the surfactant solutions and the model oils employed in this study, three main behaviors were observed when a critical flow rate was reached: elongation, fragmentation or spontaneous detachment of the droplet. The analysis of the results leads to a correlation between the droplet behavior and the balance of the forces applied on the droplet in its initial position, in particular the gravity force Fg, which tends to move the oil droplet upwards (given the density difference, and the capillary force Fc, which tends to keep the droplet spherical. A state diagram could be established, based on the dimensionless Bond number (Fg/Fc and cosθ, θ being the initial contact angle of the drop on the surface before the establishment of the flow. One can thus predict the droplet behavior as a function of the system initial characteristics. The results allowed the comparison of degreasing performance of the different surfactants used and illustrated the potential of AlkylPolyPentosides (APP for detergent formulations.

  12. Nitric oxide and Na,K-ATPase activity in rat skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten


    activity was depressed by oxidized glutathione. Conclusion: NO and cGMP stimulate the Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely......Aim: It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. Method: The study used...... isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Results: Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles...

  13. Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

    DEFF Research Database (Denmark)

    Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O.Hanlon


    We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles...... inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na......+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model...

  14. Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA. (United States)

    Martinez-Higuera, Aarón; Salas-Casas, Andrés; Calixto-Gálvez, Mercedes; Chávez-Munguía, Bibiana; Pérez-Ishiwara, D Guillermo; Ximénez, Cecilia; Rodríguez, Mario A


    Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Influence of metacide - surfactant complexes on agricultural crops

    Directory of Open Access Journals (Sweden)

    Orynkul Esimova


    Full Text Available The complexes based on surfactants and polyhexamethyleneguanidine hydrochloride (metacide are important for agriculture. This paper considers compositions of known bactericidal metacide with different surfactants: anionic surfactant sodium dodecylsulphate (DDSNa and nonionic surfactant Tween 80 (monooleate of oxyethylenated anhydrosorbitols. The effect of individual components and associates of metacide and surfactants on productivity and infection of cereals was studied. According to the study, the highest productivity and infection rate were shown by the associate of metacide and Tween-80. At concentration of Tween-80 in aqueous solution equal to 0.001% in combination with metacide, efficiency was 98% at 0% infection. The surface tension and the wetting of metacide, DDSNa, Tween-80, and associates of metacide with surfactants were studied. In comparison with individual components, metacide-DDSNa and metacide-Tween-80 associates have higher surface activity.

  16. Gemini imidazolium surfactants: synthesis and their biophysiochemical study. (United States)

    Kamboj, Raman; Singh, Sukhprit; Bhadani, Avinash; Kataria, Hardeep; Kaur, Gurcharan


    New gemini imidazolium surfactants 9-13 have been synthesized by a regioselective epoxy ring-opening reaction under solvent-free conditions. The surface properties of these new gemini surfactants were evaluated by surface tension and conductivity measurements. These surfactants have been found to have low critical micelle concentration (cmc) values as compared to other categories of gemini cationic surfactants and also showed the tendency to form premicellar aggregates in solution at sufficiently low concentration below their cmc values. The thermal degradation of these surfactants was determined by thermograviometry analysis (TGA). These new cationic surfactants have a good DNA binding capability as determined by agarose gel electrophoresis and ethidium bromide exclusion experiments. They have also been found to have low cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on the C6 glioma cell line.

  17. Restoring pulmonary surfactant membranes and films at the respiratory surface. (United States)

    Echaide, Mercedes; Autilio, Chiara; Arroyo, Raquel; Perez-Gil, Jesus


    Pulmonary surfactant is a complex of lipids and proteins assembled and secreted by the alveolar epithelium into the thin layer of fluid coating the respiratory surface of lungs. There, surfactant forms interfacial films at the air-water interface, reducing dramatically surface tension and thus stabilizing the air-exposed interface to prevent alveolar collapse along respiratory mechanics. The absence or deficiency of surfactant produces severe lung pathologies. This review describes some of the most important surfactant-related pathologies, which are a cause of high morbidity and mortality in neonates and adults. The review also updates current therapeutic approaches pursuing restoration of surfactant operative films in diseased lungs, mainly through supplementation with exogenous clinical surfactant preparations. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. [Pulmonary surfactant homeostasis associated genetic abnormalities and lung diseases]. (United States)

    Jiang, Xiaojing; Sun, Xiuzhu; Du, Weihua; Hao, Haisheng; Zhao, Xueming; Wang, Dong; Zhu, Huabin; Liu, Yan


    Pulmonary surfactant (PS) is synthesized and secreted by alveolar epithelial type II (AEII) cells, which is a complex compound formed by proteins and lipids. Surfactant participates in a range of physiological processes such as reducing the surface tension, keeping the balance of alveolar fluid, maintaining normal alveolar morphology and conducting host defense. Genetic disorders of the surfactant homeostasis genes may result in lack of surfactant or cytotoxicity, and lead to multiple lung diseases in neonates, children and adults, including neonatal respiratory distress syndrome, interstitial pneumonia, pulmonary alveolar proteinosis, and pulmonary fibrosis. This paper has provided a review for the functions and processes of pulmonary surfactant metabolism, as well as the connection between disorders of surfactant homeostasis genes and lung diseases.

  19. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: [Department of Physiology, University of Tuebingen (Germany)


    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  20. Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.

    Directory of Open Access Journals (Sweden)

    Margaret Clarke


    Full Text Available The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

  1. Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

    Directory of Open Access Journals (Sweden)

    Jianhua Zhao


    Full Text Available Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

  2. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.


    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

  3. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness

    NARCIS (Netherlands)

    Karet, FE; Finberg, KE; Nelson, RD; Nayir, A; Mocan, H; Sanjad, SA; Rodriguez-Soriano, J; Santos, F; Cremers, CWRJ; di Pietro, A; Hoffbrand, BI; Winiarski, J; Bakkaloglu, A; Ozen, S; Dusunsel, R; Goodyer, P; Hulton, SA; Wu, DK; Skvorak, AB; Morton, CC; Cunningham, MJ; Jha, [No Value; Lifton, RP

    H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause

  4. 75 FR 1798 - Prospective Grant of Exclusive License: Development of V-ATPase Inhibitor Compounds for the... (United States)


    ... use will specifically exclude rheumatoid arthritis and other osteo-specific autoimmune diseases. DATES... Alzheimer's diseases. Briefly, vacuolar type (H+) ATPase (V-ATPase) has been described as ``a universal...-ATPase Inhibitor Compounds for the Treatment of Human Cancers and Osteoclastic Bone Diseases Excluding...

  5. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene

    National Research Council Canada - National Science Library

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K


    .... The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation...

  6. Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene: e87235

    National Research Council Canada - National Science Library

    Nidhi Thakur; Santosh Kumar Upadhyay; Praveen C Verma; Krishnappa Chandrashekar; Rakesh Tuli; Pradhyumna K Singh


    .... The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation...

  7. A disorder of surfactant metabolism without identified genetic mutations


    Montella, Silvia; Vece, Timothy J.; Langston, Claire; Carrera, Paola; Nogee, Lawrence M.; Hamvas, Aaron; Manna, Angelo; Cervasio, Mara; Santamaria, Francesca


    Background Surfactant metabolism disorders may result in diffuse lung disease in children. Case presentation We report a 3-years-old boy with dry cough, progressive hypoxemia, dyspnea and bilateral ground glass opacities at chest high-resolution computed tomography (HRCT) who had no variants in genes encoding surfactant proteins or transcription factors. Lung histology strongly suggested an abnormality of surfactant protein. A 7-month course of pulse intravenous high-dose methylprednisolone p...

  8. Surfactant development for enhanced oil recover. Final report

    Energy Technology Data Exchange (ETDEWEB)



    The general objective of the project is to develop novel surfactants for tertiary recovery of light oil at elevated temperatures and high brine concentrations. Specific objectives are: to design, synthesize and characterize new surfactants capable of forming microemulsions of high stability at high temperatures and high salinity; to select microemulsions that will yield optimum efficiency and effectiveness in oil solubilization; to characterize the physico-chemical properties of selected microemulsion; to correlate surfactant efficacy with physico-chemical variables of selected reservoirs.

  9. Surfactant Based Enhanced Oil Recovery and Foam Mobility Control

    Energy Technology Data Exchange (ETDEWEB)

    George J. Hirasaki; Clarence A. Miller; Gary A. Pope


    Surfactant flooding has the potential to significantly increase recovery over that of conventional waterflooding. The availability of a large number of surfactant structures makes it possible to conduct a systematic study of the relation between surfactant structure and its efficacy for oil recovery. A combination of two surfactants was found to be particularly effective for application in carbonate formations at low temperature. A formulation has been designed for a particular field application. The addition of an alkali such as sodium carbonate makes possible in situ generation of surfactant and significant reduction of surfactant adsorption. In addition to reduction of interfacial tension to ultra-low values, surfactants and alkali can be designed to alter wettability to enhance oil recovery. The design of the process to maximize the region of ultra-low IFT is more challenging since the ratio of soap to synthetic surfactant is a parameter in the conditions for optimal salinity. Compositional simulation of the displacement process demonstrates the interdependence of the various components for oil recovery. An alkaline surfactant process is designed to enhance spontaneous imbibition in fractured, oil-wet, carbonate formations. It is able to recover oil from dolomite core samples from which there was no oil recovery when placed in formation brine. Mobility control is essential for surfactant EOR. Foam is evaluated to improve the sweep efficiency of surfactant injected into fractured reservoirs. UTCHEM is a reservoir simulator specially designed for surfactant EOR. It has been modified to represent the effects of a change in wettability. Simulated case studies demonstrate the effects of wettability.

  10. Seipin promotes adipose tissue fat storage through the ER Ca²⁺-ATPase SERCA. (United States)

    Bi, Junfeng; Wang, Wei; Liu, Zhonghua; Huang, Xiahe; Jiang, Qingqing; Liu, George; Wang, Yingchun; Huang, Xun


    Adipose tissue is central to the regulation of lipid metabolism. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most severe lipodystrophy diseases, is caused by mutation of the Seipin gene. Seipin plays an important role in adipocyte differentiation and lipid homeostasis, but its exact molecular functions are still unknown. Here, we show that Seipin physically interacts with the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in both Drosophila and man. SERCA, an endoplasmic reticulum (ER) calcium pump, is solely responsible for transporting cytosolic calcium into the ER lumen. Like dSeipin, dSERCA cell-autonomously promotes lipid storage in Drosophila fat cells. dSeipin affects dSERCA activity and modulates intracellular calcium homeostasis. Adipose tissue-specific knockdown of the ER-to-cytosol calcium release channel ryanodine receptor (RyR) partially restores fat storage in dSeipin mutants. Our results reveal that Seipin promotes adipose tissue fat storage by regulating intracellular calcium homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. The Pulmonary Surfactant: Impact of Tobacco Smoke and Related Compounds on Surfactant and Lung Development

    Directory of Open Access Journals (Sweden)

    Scott J Elliott


    Full Text Available Abstract Cigarette smoking, one of the most pervasive habits in society, presents many well established health risks. While lung cancer is probably the most common and well documented disease associated with tobacco exposure, it is becoming clear from recent research that many other diseases are causally related to smoking. Whether from direct smoking or inhaling environmental tobacco smoke (ETS, termed secondhand smoke, the cells of the respiratory tissues and the lining pulmonary surfactant are the first body tissues to be directly exposed to the many thousands of toxic chemicals in tobacco. Considering the vast surface area of the lung and the extreme attenuation of the blood-air barrier, it is not surprising that this organ is the primary route for exposure, not just to smoke but to most environmental contaminants. Recent research has shown that the pulmonary surfactant, a complex mixture of phospholipids and proteins, is the first site of defense against particulates or gas components of smoke. However, it is not clear what effect smoke has on the surfactant. Most studies have demonstrated that smoking reduces bronchoalveolar lavage phospholipid levels. Some components of smoke also appear to have a direct detergent-like effect on the surfactant while others appear to alter cycling or secretion. Ultimately these effects are reflected in changes in the dynamics of the surfactant system and, clinically in changes in lung mechanics. Similarly, exposure of the developing fetal lung through maternal smoking results in postnatal alterations in lung mechanics and higher incidents of wheezing and coughing. Direct exposure of developing lung to nicotine induces changes suggestive of fetal stress. Furthermore, identification of nicotinic receptors in fetal lung airways and corresponding increases in airway connective tissue support a possible involvement of nicotine in postnatal asthma development. Finally, at the level of the alveoli of the lung

  12. Infrared investigation of organo-montmorillonites prepared from different surfactants (United States)

    Ma, Yuehong; Zhu, Jianxi; He, Hongping; Yuan, Peng; Shen, Wei; Liu, Dong


    In this paper, a series of organoclays were prepared from montmorillonites with different CEC and surfactants with different alkyl chain numbers and chain length. Then, FTIR spectroscopy using ATR, DRIFT and KBr pressed disk techniques was used to characterize the local environments of surfactant and host clays in various surfactants modified montmorillonites under wet and dry states. The present study demonstrates that the alkyl chain length and chain number have significant influences on the local environment of the intercalated surfactants. Also, this study indicates that the surface property of the resulting organoclays is affected by the loading and configuration of the intercalated surfactants. In wet state, more gauche conformers are introduced into the alkyl chains in the organoclays with low surfactant loading, evidenced by the shift of CH 2 vibration to higher frequency. Meanwhile, in the case of the organo-montmorillonites with high surfactant loading, the interaction between the surfactant and silicate surface results in a re-arrangement of SiO 4 tetrahedral sheets and a splitting of Si-O stretching vibration. The KBr pressed disk technique is suitable to probe the conformational ordering of the confined amine chains and the reflectance spectroscopy with ATR and/or DRIFT technique is more suitable to probe the water in organoclays. These findings are of high importance to the preparation of organoclays with proper surfactants and investigation of the microstructure of the resulting organoclays using suitable techniques.

  13. Surfactant-induced core/shell phase equilibrium in hydrogels. (United States)

    Gernandt, J; Hansson, P


    The formation of core/shell structures in hydrogels upon interaction with surfactants is a well-known phenomenon, but whether they are equilibrium states or not is still under debate. This paper presents an equilibrium theory of phase coexistence in hydrogels meant to answer the question of the stability of core/shell separation. The theory suggests that core/shell separation caused by surfactants can indeed be thermodynamically stable if the amount of added surfactant is not too large, but that the exact phase behaviour is governed by both the volume and concentration of the added surfactant solution.

  14. Surfactants have multi-fold effects on skin barrier function. (United States)

    Lemery, Emmanuelle; Briançon, Stéphanie; Chevalier, Yves; Oddos, Thierry; Gohier, Annie; Boyron, Olivier; Bolzinger, Marie-Alexandrine


    The stratum corneum (SC) is responsible for the barrier properties of the skin and the role of intercorneocyte skin lipids, particularly their structural organization, in controlling SC permeability is acknowledged. Upon contacting the skin, surfactants interact with the SC components leading to barrier damage. To improve knowledge of the effect of several classes of surfactant on skin barrier function at three different levels. The influence of treatments of human skin explants with six non-ionic and four ionic surfactant solutions on the physicochemical properties of skin was investigated. Skin surface wettability and polarity were assessed through contact angle measurements. Infrared spectroscopy allowed monitoring the SC lipid organization. The lipid extraction potency of surfactants was evaluated thanks to HPLC-ELSD assays. One anionic and one cationic surfactant increased the skin polarity by removing the sebaceous and epidermal lipids and by disturbing the organization of the lipid matrix. Another cationic surfactant displayed a detergency effect without disturbing the skin barrier. Several non-ionic surfactants disturbed the lipid matrix organization and modified the skin wettability without any extraction of the skin lipids. Finally two non-ionic surfactants did not show any effect on the investigated parameters or on the skin barrier. The polarity, the organization of the lipid matrix and the lipid composition of the skin allowed describing finely how surfactants can interact with the skin and disturb the skin barrier function.

  15. Inactivation of Herpes Simplex Viruses by Nonionic Surfactants (United States)

    Asculai, Samuel S.; Weis, Margaret T.; Rancourt, Martha W.; Kupferberg, A. B.


    Nonionic surface-active agents possessing ether or amide linkages between the hydrophillic and hydrophobic portions of the molecule rapidly inactivated the infectivity of herpes simplex viruses. The activity stemmed from the ability of nonionic surfactants to dissolve lipid-containing membranes. This was confirmed by observing surfactant destruction of mammalian cell plasma membranes and herpes simplex virus envelopes. Proprietary vaginal contraceptive formulations containing nonionic surfactants also inactivated herpes simplex virus infectivity. This observation suggests that nonionic surfactants in appropriate formulation could effectively prevent herpes simplex virus transmission. Images PMID:208460

  16. Pulmonary Surfactant Function in Alveoli and Conducting Airways

    Directory of Open Access Journals (Sweden)

    Goran Enhorning


    Full Text Available Surface tension plays a very important role in aeration of the neonate's lungs. Pulmonary surfactant, which is inadequate in the premature infant, modifies surface tension during the act of breathing and is necessary for maintenance of alveolar stability. These facts led to the development of the concept that it might be possible to treat the premature infant by supplementing the infant's inadequate surfactant supply. In addition to maintaining alveolar stability, pulmonary surfactant might also be of vital importance for maintenance of small airway patency. Various conditions, most importantly asthma, might be the reason for a surfactant dysfunction to develop. This in turn might cause airway resistance to increase.

  17. Surfactant alteration and replacement in acute respiratory distress syndrome

    Directory of Open Access Journals (Sweden)

    Walmrath Dieter


    Full Text Available Abstract The acute respiratory distress syndrome (ARDS is a frequent, life-threatening disease in which a marked increase in alveolar surface tension has been repeatedly observed. It is caused by factors including a lack of surface-active compounds, changes in the phospholipid, fatty acid, neutral lipid, and surfactant apoprotein composition, imbalance of the extracellular surfactant subtype distribution, inhibition of surfactant function by plasma protein leakage, incorporation of surfactant phospholipids and apoproteins into polymerizing fibrin, and damage/inhibition of surfactant compounds by inflammatory mediators. There is now good evidence that these surfactant abnormalities promote alveolar instability and collapse and, consequently, loss of compliance and the profound gas exchange abnormalities seen in ARDS. An acute improvement of gas exchange properties together with a far-reaching restoration of surfactant properties was encountered in recently performed pilot studies. Here we summarize what is known about the kind and severity of surfactant changes occuring in ARDS, the contribution of these changes to lung failure, and the role of surfactant administration for therapy of ARDS.

  18. Recent clinical trials of surfactant treatment for neonates. (United States)

    Halliday, Henry L


    To search for recent clinical trials of neonatal surfactant treatment and report their findings. Recent was defined as published between 2000 and 2005. An online search on PubMed was made on 30th December 2005 using the following terms: surfactant treatment, clinical trials and neonate, with limits of years 2000 to 2005 and age - newborn from birth to 1 month. Randomised clinical trials (RCTs) and systematic reviews of RCTs were prioritised and studies in children and animals were excluded from further analysis. 175 papers were found in this search. Only about half of these papers were directly related to some aspect of surfactant treatment and of these just over one-half were either RCTs or systematic reviews of RCTs. Of the 34 RCTs of surfactant treatment, 3 were excluded as they involved children or animals rather than neonates. Twenty-nine trials studied preterm babies with respiratory distress syndrome (RDS) and 2 were for meconium aspiration syndrome (MAS) in term infants. The median sample sizes of these studies were RDS (92, range 19-1,361) and MAS (42, range 22-61). Eighteen of the RDS trials compared two or more surfactant preparations, the most frequently studied being Curosurf and Survanta but altogether 11 different surfactants were compared. These new RCTs need to be analysed by meta-analyses in systematic reviews. Twelve systematic reviews were found and these demonstrated the superiority of prophylactic over selective use of surfactant in babies surfactant and the absence of an increase in long-term developmental sequelae. Surfactant for MAS may reduce the severity of respiratory illness and the need for extracorporeal membrane oxygenation. Of the non-randomised trials' novel delivery methods, failure to use evidence-based guidelines and the benefit of surfactant for babies Surfactant remains one of the most effective and safest interventions in neonatology. Prophylactic natural surfactant seems to be the most evidence-based treatment for babies


    The relative environmental toxicities of synthetic and biogenic surfactants used in oil spill remediation efforts are not well understood. Acute and chronic toxicities of three synthetic surfactants and three microbially produced surfactants were determined and compared in this s...

  20. C-reactive protein increases membrane fluidity and distorts lipid lateral organization of pulmonary surfactant. Protective role of surfactant protein A

    DEFF Research Database (Denmark)

    Saenz, Alejandra; Lopez-Sanchez, Almudena; Mojica-Lazaro, Jonas


    The purpose of this study was to investigate how surfactant membranes can be perturbed by C-reactive protein (CRP) and whether surfactant protein A (SP-A) might overcome CRP-induced surfactant membrane alterations. The effect of CRP on surfactant surface adsorption was evaluated in vivo after int...

  1. Bufadienolides from amphibians: A promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na+/K+-ATPase inhibition. (United States)

    Sousa, Lívia Queiroz de; Machado, Kátia da Conceição; Oliveira, Samara Ferreira de Carvalho; Araújo, Lidiane da Silva; Monção-Filho, Evaldo Dos Santos; Melo-Cavalcante, Ana Amélia de Carvalho; Vieira-Júnior, Gerardo Magela; Ferreira, Paulo Michel Pinheiro


    differentiation, angiogenesis inhibition, multidrug resistance reversion, and also regulate immune responses. Then, bufadienolides isolated from amphibians, some of them at risk of extinction, emerge as a natural class of incredible chemical biodiversity, has moderate selectivity against human tumor cells and weak activity on murine cells, probably due to structural differences between subunits of human and mice Na + /K + -ATPases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. MORC2 Signaling Integrates Phosphorylation-Dependent, ATPase-Coupled Chromatin Remodeling during the DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Da-Qiang Li


    Full Text Available Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2, an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1, an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.

  3. Nucleate pool boiling heat transfer in aqueous surfactant solutions (United States)

    Wasekar, Vivek Mahadeorao

    Saturated, nucleate pool boiling in aqueous surfactant solutions is investigated experimentally. Also, the role of Marangoni convection, driven both by temperature and surfactant concentration gradients at the vapor-liquid interface of a nucleating bubble is computationally explored. Experimental measurements of dynamic and equilibrium sigma using the maximum bubble pressure method indicate dynamic sigma to be higher than the corresponding equilibrium value, both at room and elevated temperatures. Also, nonionic surfactants (Triton X-100, Triton X-305) show larger sigma depression than anionic surfactants (SDS, SLES), and a normalized representation of their dynamic adsorption isotherms is shown to be helpful in generalizing the surfactant effectiveness to reduce surface tension. The dynamic sigma has a primary role in the modification of bubble dynamics and associated heat transfer, and is dictated by the adsorption kinetics of the surfactant molecules at boiling temperatures. In general, an enhancement in heat transfer is observed, which is characterized by an early incipience and an optimum boiling performance at or around the critical micelle concentration of the surfactant. The optimum performances, typically in the fully developed boiling regime ( q''w > 100 kW/m2), show a reverse trend with respect to surfactant molecular weights M, i.e., higher molecular weight additives promote lower enhancement. Normalized boiling performance using the respective solution's dynamic sigma correlates heat transfer coefficient by M-0.5 for anionics and M 0 for nonionics. This has been shown to be brought about by the surfactant concentration and its interfacial activity in a concentration sublayer around the growing vapor bubble, which governs the bubble growth behavior through the mechanism of dynamic sigma. The ionic nature of the surfactant influences the thickness and molecular makeup of the enveloping sublayer, thereby affecting the bubble dynamics and boiling heat

  4. Clamp loader ATPases and the evolution of DNA replication machinery (United States)


    Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life. PMID:22520345

  5. Clamp loader ATPases and the evolution of DNA replication machinery

    Directory of Open Access Journals (Sweden)

    Kelch Brian A


    Full Text Available Abstract Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.

  6. Characterization and effect of light on the plasma membrane H(+) -ATPase of bean leaves (United States)

    Linnemeyer, P. A.; Van Volkenburgh, E.; Cleland, R. E.


    Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the Km was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, Km, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.

  7. An extended nomenclature for mammalian V-ATPase subunit genes and splice variants.

    Directory of Open Access Journals (Sweden)

    Kevin C Miranda


    Full Text Available The vacuolar-type H(+-ATPase (V-ATPase is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.

  8. Specialized Functional Diversity and Interactions of the Na,K-ATPase (United States)

    Matchkov, Vladimir V.; Krivoi, Igor I.


    Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations, and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions, and protein kinase signaling pathways. In addition to its “classical” function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids (CTS) triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function. PMID:27252653

  9. Homotypic vacuole fusion in yeast requires organelle acidification and not the V-ATPase membrane domain. (United States)

    Coonrod, Emily M; Graham, Laurie A; Carpp, Lindsay N; Carr, Tom M; Stirrat, Laura; Bowers, Katherine; Bryant, Nia J; Stevens, Tom H


    Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H(+)-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H(+)-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Inhibitory effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent atpase from temporalis muscle. (United States)

    Sánchez, Gabriel A; Casadoumecq, Ana C; Alonso, Guillermo L; Takara, Delia


    Myotoxic effects of local anesthetics on skeletal musclefibers involve the inhibition ofsarcoplasmic reticulum Ca2+ -dependent ATPase activity and Ca2 transport. Lidocaine is a local anesthetic frequently used to relieve the symptoms of trigeminal neuralgia. The aim of this work was to test the inhibitory and/or stimulatory effect of lidocaine on sarcoplasmic reticulum Ca2+ -dependent ATPase isolated from rabbit temporalis muscle. Ca2+ -dependent ATPase activity was determined by a colorimetric method Calcium-binding to the Ca dependent ATPase, Ca2+ transport, and phosphorylation of the enzyme by ATP were determined with radioisotopic techniques. Lidocaine inhibited the Ca2+ -dependent ATPase activity in a concentration-dependent manner. The preincubation of the sarcoplasmic reticulum membranes with lidocaine enhanced the Ca2+ dependent ATPase activity in the absence of calcium ionophore. Lidocaine also inhibited both Ca2+ uptake and enzyme phosphorylation by ATP but had no effect on Ca2+ -binding to the enzyme. We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+ -dependent ATPase from temporalis muscle is due to the drug's direct interaction with the enzyme and the increased permeability of the sarcoplasmic reticulum membrane to Ca.

  11. Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain. (United States)

    Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin


    Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellula