WorldWideScience

Sample records for cell atp leaking

  1. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  2. Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria.

    Science.gov (United States)

    Affourtit, Charles; Brand, Martin D

    2006-01-01

    Pancreatic beta cells respond to rising blood glucose concentrations by increasing their oxidative metabolism, which leads to an increased ATP/ADP ratio, closure of K(ATP) channels, depolarization of the plasma membrane potential, influx of calcium and the eventual secretion of insulin. Such a signalling mechanism implies that the ATP/ADP ratio is flexible in beta cells (beta-cells), which is in contrast with other cell types (e.g. muscle and liver) that maintain a stable ATP/ADP poise while respiring at widely varying rates. To determine whether this difference in flexibility is accounted for by mitochondrial peculiarities, we performed a top-down metabolic control analysis to quantitatively assess how ATP/ADP is controlled in mitochondria isolated from rat skeletal muscle and cultured beta cells. We show that the ATP/ADP ratio is more strongly controlled (approx. 7.5-fold) by proton leak in beta cells than in muscle. The comparatively high importance of proton leak in beta cell mitochondria (relative to phosphorylation) is evidenced furthermore by its relatively high level of control over membrane potential and overall respiratory activity. Modular-kinetic analysis of oxidative phosphorylation reveals that these control differences can be fully explained by a higher relative leak activity in beta cell mitochondria, which results in a comparatively high contribution of proton leak to the overall respiratory activity in this system.

  3. Duchenne muscular dystrophy: normal ATP turnover in cultured cells

    International Nuclear Information System (INIS)

    Fox, I.H.; Bertorini, T.; Palmieri, G.M.A.; Shefner, R.

    1986-01-01

    This paper examines ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients and there was no difference in ATP synthesis or degradation. The ATP synthesis was measured by the incorporation of C 14-U-adenine into aTP and ADP. although there was a significant decrease in radioactively labelled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration of ADP metabolism

  4. Extracellular ATP is internalized by macropinocytosis and induces intracellular ATP increase and drug resistance in cancer cells.

    Science.gov (United States)

    Qian, Yanrong; Wang, Xuan; Liu, Yi; Li, Yunsheng; Colvin, Robert A; Tong, Lingying; Wu, Shiyong; Chen, Xiaozhuo

    2014-09-01

    ATP plays central roles in cancer metabolism and the Warburg effect. Intratumoral ATP concentrations are up to 10(4) times higher than those of interstitial ATP in normal tissues. However, extracellular ATP is not known to enter cancer cells. Here we report that human A549 lung cancer cells internalized extracellular ATP by macropinocytosis as demonstrated by colocalization of a nonhydrolyzable fluorescent ATP and a macropinocytosis tracer high-molecular-weight dextran, as well as by a macropinocytosis inhibitor study. Extracellular ATP also induced increase of intracellular ATP levels, without involving transcription and translation at significant levels, and cancer cells' resistance to ATP-competitor anticancer drugs, likely through the mechanism of ATP internalization. These findings, described for the first time, have profound implications in ATP-sharing among cancer cells in tumors and highlight a novel anticancer target. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Extracellular ATP, as an energy and phosphorylating molecule, induces different types of drug resistances in cancer cells through ATP internalization and intracellular ATP level increase.

    Science.gov (United States)

    Wang, Xuan; Li, Yunsheng; Qian, Yanrong; Cao, Yanyang; Shriwas, Pratik; Zhang, Haiyun; Chen, Xiaozhuo

    2017-10-20

    Cancer cells are able to uptake extracellular ATP (eATP) via macropinocytosis to elevate intracellular ATP (iATP) levels, enhancing their survival in drug treatment. However, the involved drug resistance mechanisms are unknown. Here we investigated the roles of eATP as either an energy or a phosphorylating molecule in general drug resistance mediated by ATP internalization and iATP elevation. We report that eATP increased iATP levels and promoted drug resistance to various tyrosine kinase inhibitors (TKIs) and chemo-drugs in human cancer cell lines of five cancer types. In A549 lung cancer cells, the resistance was downregulated by macropinocytosis inhibition or siRNA knockdown of PAK1, an essential macropinocytosis enzyme. The elevated iATP upregulated the efflux activity of ABC transporters in A549 and SK-Hep-1 cells as well as phosphorylation of PDGFRα and proteins in the PDGFR-mediated Akt-mTOR and Raf-MEK signaling pathways in A549 cells. Similar phosphorylation upregulations were found in A549 tumors. These results demonstrate that eATP induces different types of drug resistance by eATP internalization and iATP elevation, implicating the ATP-rich tumor microenvironment in cancer drug resistance, expanding our understanding of the roles of eATP in the Warburg effect and offering new anticancer drug resistance targets.

  6. ATP7B detoxifies silver in ciliated airway epithelial cells

    International Nuclear Information System (INIS)

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-01-01

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B -/- mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag + /Cu + transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  7. 105-KE Isolation Barrier leak rate acceptance test procedure WHC-SD-SNF-ATP-005

    International Nuclear Information System (INIS)

    Whitehurst, R.; McCracken, K.; Papenfuss, J.N.

    1994-01-01

    This document establishes the procedure for performing the acceptance test on the two isolation barriers being installed in K East basin. This acceptance test procedure shall be used to : First establish a basin water loss rate prior to installation of the two isolation barriers between the main basin and the discharge chute in K-Basin East. Second, perform an acceptance test to verify an acceptable leakage rate through the barrier seals. This Acceptance Test Procedure (ATP) has been prepared in accordance with CM-6-1 EP 4.2, Standard Engineering Practices

  8. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

    Full Text Available Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30, which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1 cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin reduced both the spontaneous and hypotonically (80%-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1 and vesicular transport (nocodazole. These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ∼90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50% or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8% and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

  9. Quantal release of ATP from clusters of PC12 cells.

    Science.gov (United States)

    Fabbro, Alessandra; Skorinkin, Andrei; Grandolfo, Micaela; Nistri, Andrea; Giniatullin, Rashid

    2004-10-15

    Although ATP is important for intercellular communication, little is known about the mechanism of endogenous ATP release due to a dearth of suitable models. Using PC12 cells known to express the P2X2 subtype of ATP receptors and to store ATP with catecholamines inside dense-core vesicles, we found that clusters of PC12 cells cultured for 3-7 days generated small transient inward currents (STICs) after an inward current elicited by exogenous ATP. The amplitude of STICs in individual cells correlated with the peak amplitude of ATP-induced currents. STICs appeared as asynchronous responses (approximately 20 pA average amplitude) for 1-20 s and were investigated with a combination of patch clamping, Ca2+ imaging, biochemistry and electron microscopy. Comparable STICs were produced by focal KCl pulses and were dependent on extracellular Ca2+. STICs were abolished by the P2X antagonist PPADS and potentiated by Zn2+, suggesting they were mediated by P2X2 receptor activation. The highest probability of observing STICs was after the peak of intracellular Ca2+ increase caused by KCl. Biochemical measurements indicated that KCl application induced a significant release of ATP from PC12 cells. Electron microscopy studies showed narrow clefts without 'synaptic-like' densities between clustered cells. Our data suggest that STICs were caused by quantal release of endogenous ATP by depolarized PC12 cells in close juxtaposition to the recorded cell. Thus, STICs may be a new experimental model to characterize the physiology of vesicular release of ATP and to study the kinetics and pharmacology of P2X2 receptor-mediated quantal currents.

  10. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our...... dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting...

  11. Bioenergetics in chicken embryo fibroblast cells: evidence of lower proton leak in spontaneously immortalized chicken embryo fibroblasts compared to young and senescent primary chicken embryo fibroblast cells.

    Science.gov (United States)

    Lassiter, Kentu; Dridi, Sami; Piekarski, Alissa; Greene, Elizabeth; Hargis, Billy; Kong, Byung-Whi; Bottje, Walter

    2014-09-01

    A spontaneously immortalized chicken embryo fibroblast (CEF) cell line (DF-1) is known to exhibit faster growth rate and greater sensitivity to oxidative stress compared to the primary parent CEF (pCEF1°) cells. Thus, major objectives of this study were to assess cell bioenergetics in pCEF1° and DF-1 cells under control conditions and in response to 4-hydroxy 2-nonenal (4-HNE) induced oxidative challenge. Cell bioenergetics were assessed by flux analysis of oxygen consumption rate (OCR). Under control conditions, DF-1 cells had higher OCR associated with ATP synthase activity and mitochondrial oxygen reserve capacity as well as lower OCR due to proton leak and non-mitochondrial cytochrome c oxidase activity. In response to 4-HNE (0 to 30 μM), DF-1 cells were more sensitive to oxidant challenge than both young (passage 8) and senescent (passage 19) pCEF1° cells. Both passages 8 and 19 pCEF1° cells exhibited higher proton leak in response to 4-HNE, but this was not observed in DF-1 cells. Inducible proton leak occurs by 4-HNE stimulated activation of uncoupling protein (UCP) and adenine nucleotide translocase (ANT). From mRNA expression data indicated that ANT and avian UCP were down-regulated and up-regulated, respectively, in DF-1 compared to pCEF1° cells. Thus, we hypothesize that DF-1 cells are unable to increase proton leak due to lower expression of ANT, but not avian UCP, and this inability to increase proton leak contributes to greater susceptibility to oxidative stress of DF-1 cells compared to pCEF1° cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Alternative mitochondrial functions in cell physiopathology: beyond ATP production

    Directory of Open Access Journals (Sweden)

    Kowaltowski A.J.

    2000-01-01

    Full Text Available It is well known that mitochondria are the main site for ATP generation within most tissues. However, mitochondria also participate in a surprising number of alternative activities, including intracellular Ca2+ regulation, thermogenesis and the control of apoptosis. In addition, mitochondria are the main cellular generators of reactive oxygen species, and may trigger necrotic cell death under conditions of oxidative stress. This review concentrates on these alternative mitochondrial functions, and their role in cell physiopathology.

  13. Characterization of the effects of 2-methylthio-ATP and 2-chloro-ATP on brain capillary endothelial cells: similarities to ADP and differences from ATP.

    OpenAIRE

    Vigne, P.; Feolde, E.; Breittmayer, J. P.; Frelin, C.

    1994-01-01

    1. Brain capillary endothelial cells responded to 2-methylthio-ATP (2MeSATP) by large increases in [Ca2+]i (EC50 = 27 nM) that were partially dependent on the presence of extracellular Ca2+ and that were not associated with a measurable production of inositol phosphates. 2. 2-chloro-ATP (2ClATP) raised [Ca2+]i in a biphasic manner. At low concentrations, intracellular Ca2+ mobilization was not associated with a measurable production of inositol phosphates. At concentrations > 30 microM, 2ClAT...

  14. The involvement of cation leaks in the storage lesion of red blood cells.

    Directory of Open Access Journals (Sweden)

    Joanna F Flatt

    2014-06-01

    Full Text Available Stored blood components are a critical life-saving tool provided to patients by health services worldwide. Red cells may be stored for up to 42 days, allowing for efficient blood bank inventory management, but with prolonged storage comes an unwanted side-effect known as the ‘storage lesion’, which has been implicated in poorer patient outcomes. This lesion is comprised of a number of processes that are inter-dependent. Metabolic changes include a reduction in glycolysis and ATP production after the first week of storage. This leads to an accumulation of lactate and drop in pH. Longer term damage may be done by the consequent reduction in anti-oxidant enzymes, which contributes to protein and lipid oxidation via reactive oxygen species. The oxidative damage to the cytoskeleton and membrane is involved in increased vesiculation and loss of cation gradients across the membrane. The irreversible damage caused by extensive membrane loss via vesiculation alongside dehydration is likely to result in immediate splenic sequestration of these dense, spherocytic cells. Although often overlooked in the literature, the loss of the cation gradient in stored cells will be considered in more depth in this review as well as the possible effects it may have on other elements of the storage lesion. It has now become clear that blood donors can exhibit quite large variations in the properties of their red cells, including microvesicle production and the rate of cation leak. Further study of stored red blood cells from donors known to have a high or low-rate of cation leak will shed more light on the relationship between cation gradients and the manifestation of the various elements of the storage lesion.

  15. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

    Directory of Open Access Journals (Sweden)

    Romina Baaske

    2016-12-01

    Full Text Available Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla. This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L, which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin.

  16. Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network.

    Science.gov (United States)

    Xie, L; Wang, D I

    1996-12-05

    A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH(2). A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH(2) accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand.

  17. Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

    International Nuclear Information System (INIS)

    Steinberg, T.H.; Silverstein, S.C.

    1987-01-01

    Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86 Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86 Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86 Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86 Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86 Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86 Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-

  18. ATP-independent DNA synthesis in Vaccinia-infected L cells

    International Nuclear Information System (INIS)

    Berger, N.A.; Kauff, R.A.; Sikorski, G.W.

    1978-01-01

    Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

  19. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes.

    Directory of Open Access Journals (Sweden)

    M Florencia Leal Denis

    Full Text Available The peptide mastoparan 7 (MST7 triggered in human erythrocytes (rbcs the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe, interacting with P (purinergic receptors, can affect cell volume (Vr, we explored the dynamic regulation between Vr and ATPe.We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors.In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40-50% and swelling by 40-60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%.Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop underlying ATP-induced ATP release of rbcs.

  20. Modular, High-Volume Fuel Cell Leak-Test Suite and Process

    Energy Technology Data Exchange (ETDEWEB)

    Ru Chen; Ian Kaye

    2012-03-12

    Fuel cell stacks are typically hand-assembled and tested. As a result the manufacturing process is labor-intensive and time-consuming. The fluid leakage in fuel cell stacks may reduce fuel cell performance, damage fuel cell stack, or even cause fire and become a safety hazard. Leak check is a critical step in the fuel cell stack manufacturing. The fuel cell industry is in need of fuel cell leak-test processes and equipment that is automatic, robust, and high throughput. The equipment should reduce fuel cell manufacturing cost.

  1. Extracellular ATP: a modulator of cell death and pathogen defense in plants.

    Science.gov (United States)

    Chivasa, Stephen; Tomé, Daniel F A; Murphy, Alex M; Hamilton, John M; Lindsey, Keith; Carr, John P

    2009-11-01

    Living organisms acquire or synthesize high energy molecules, which they frugally conserve and use to meet their cellular metabolic demands. Therefore, it is surprising that ATP, the most accessible and commonly utilized chemical energy carrier, is actively secreted to the extracellular matrix of cells. It is now becoming clear that in plants this extracellular ATP (eATP) is not wasted, but harnessed at the cell surface to signal across the plasma membrane of the secreting cell and neighboring cells to control gene expression and influence plant development. Identification of the gene/protein networks regulated by eATP-mediated signaling should provide insight into the physiological roles of eATP in plants. By disrupting eATP-mediated signaling, we have identified pathogen defense genes as part of the eATP-regulated gene circuitry, leading us to the discovery that eATP is a negative regulator of pathogen defense in plants.(1) Previously, we reported that eATP is a key signal molecule that modulates programmed cell death in plants.(2) A complex picture is now emerging, in which eATP-mediated signaling cross-talks with signaling mediated by the major plant defense hormone, salicylic acid, in the regulation of pathogen defense and cell death.

  2. Effect of ATP sulfurylase overexpression in bright yellow 2 tobacco cells: regulation of ATP sulfurylase and SO4(-2) transport activities

    International Nuclear Information System (INIS)

    Hatzfeld, Y.; Cathala, N.; Grignon, C.; Davidian, J.C.

    1998-01-01

    To determine if the ATP sulfurylase reaction is a regulatory step for the SO4(2-)-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 355 promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO4(2-) influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO4(2-) (a toxic SO4(2-) analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism

  3. Intracellular Ca2+ and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells

    Science.gov (United States)

    Huang, Yijen A; Roper, Stephen D

    2010-01-01

    ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca2+ and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca2+ release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca2+ has a dual role: (1) Ca2+ opens TRPM5 channels to depolarize receptor cells and (2) Ca2+ plus membrane depolarization opens ATP-permeable gap junction hemichannels. PMID:20498227

  4. Intracellular Ca(2+) and TRPM5-mediated membrane depolarization produce ATP secretion from taste receptor cells.

    Science.gov (United States)

    Huang, Yijen A; Roper, Stephen D

    2010-07-01

    ATP is a transmitter secreted from taste bud receptor (Type II) cells through ATP-permeable gap junction hemichannels most probably composed of pannexin 1. The elevation of intracellular Ca(2+) and membrane depolarization are both believed to be involved in transmitter secretion from receptor cells, but their specific roles have not been fully elucidated. In the present study, we show that taste-evoked ATP secretion from mouse vallate receptor cells is evoked by the combination of intracellular Ca(2+) release and membrane depolarization. Unexpectedly, ATP secretion is not blocked by tetrodotoxin, indicating that transmitter release from these cells still takes place in the absence of action potentials. Taste-evoked ATP secretion is absent in receptor cells isolated from TRPM5 knockout mice or in taste cells from wild type mice where current through TRPM5 channels has been eliminated. These findings suggest that membrane voltage initiated by TRPM5 channels is required for ATP secretion during taste reception. Nonetheless, even in the absence of TRPM5 channel activity, ATP release could be triggered by depolarizing cells with KCl. Collectively, the findings indicate that taste-evoked elevation of intracellular Ca(2+) has a dual role: (1) Ca(2+) opens TRPM5 channels to depolarize receptor cells and (2) Ca(2+) plus membrane depolarization opens ATP-permeable gap junction hemichannels.

  5. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  6. Quantitative requirement for ATP for active transport in isolated renal cells

    International Nuclear Information System (INIS)

    Tessitore, N.; Sakhrani, L.M.; Massry, S.G.

    1986-01-01

    We investigated the quantitative relationship between cellular ATP concentration and Na+-K+-ATPase activity as measured by ouabain-sensitive 86Rb influx in rabbit proximal renal cells. Cellular ATP was reduced in a stepwise manner by rotenone (10(-7) to 10(-5) M) and was increased by 10 mM adenosine. During these maneuvers, ouabain-sensitive 86Rb influx was linearly related to cellular ATP and did not saturate up to 9.9 mM ATP. In contrast, Na+-K+-ATPase activity in membranes prepared from these cells saturated at 2.0 mM ATP at various sodium (10-100 mM) and potassium (4-100 mM) concentrations. Sodium-dependent phosphate uptake and alpha-methylglucoside (alpha-MG) uptake were both inhibited to a similar degree when cellular ATP was reduced. We conclude that 1) the ATP requirement for saturation of Na+-K+-ATPase is higher in intact renal cells than in the membranes, and 2) the uptake of phosphate and alpha-MG are similarly influenced by reduction in ATP. This effect of ATP on phosphate and AMG uptake is most likely an indirect one and is secondary to changes in the sodium gradient across the cell

  7. Measurement of adenosine triphosphate (ATP) content in single red blood cells using the firefly bioluminescent reaction

    Energy Technology Data Exchange (ETDEWEB)

    Kostuk, R.K.; Muhs, A.G.; Kirkpatrick, F.H.; Gabel, C.W.

    1977-01-01

    A unique optical instrument is described which uses the firefly bioluminscent reaction to measure adenosine triphosphate (ATP) levels in single red blood cells. The method allows chemical content level to be associated with individual cell features. The optical instrument consists of a phase contrast microscope to view cells, a pulsed argon-ion laser to rupture the cell membrane, and a photon counting system to measure the bioluminescent yield. The technique has been calibrated against a standard ATP measurement using bulk analysis methods. The ATP loss mechanism for blood cells in a controlled depletion experiment was also investigated.

  8. Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

    International Nuclear Information System (INIS)

    Liu, P.-S.; Chen, C.-Y.

    2010-01-01

    Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in human osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.

  9. ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

    Science.gov (United States)

    De Col, Valentina; Fuchs, Philippe; Nietzel, Thomas; Elsässer, Marlene; Voon, Chia Pao; Candeo, Alessia; Seeliger, Ingo; Fricker, Mark D; Grefen, Christopher; Møller, Ian Max; Bassi, Andrea; Lim, Boon Leong; Zancani, Marco; Meyer, Andreas J; Costa, Alex; Wagner, Stephan; Schwarzländer, Markus

    2017-01-01

    Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant. DOI: http://dx.doi.org/10.7554/eLife.26770.001 PMID:28716182

  10. ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology.

    Science.gov (United States)

    De Col, Valentina; Fuchs, Philippe; Nietzel, Thomas; Elsässer, Marlene; Voon, Chia Pao; Candeo, Alessia; Seeliger, Ingo; Fricker, Mark D; Grefen, Christopher; Møller, Ian Max; Bassi, Andrea; Lim, Boon Leong; Zancani, Marco; Meyer, Andreas J; Costa, Alex; Wagner, Stephan; Schwarzländer, Markus

    2017-07-18

    Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP 2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP 2- changes in planta. A MgATP 2- map of the Arabidopsis seedling highlights different MgATP 2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

  11. ATP released by injured neurons activates Schwann cells

    Directory of Open Access Journals (Sweden)

    Samuele eNegro

    2016-05-01

    Full Text Available Injured nerve terminals of neuromuscular junctions (NMJs can regenerate. This remarkable and complex response is governed by molecular signals that are exchanged among the cellular components of this synapse: motor axon nerve terminal (MAT, perisynaptic Schwann cells (PSCs, and muscle fibre. The nature of signals that govern MAT regeneration is ill-known. In the present study the spider toxin α-Latrotoxin has been used as tool to investigate the mechanisms underlying peripheral neuroregeneration. Indeed this neurotoxin induces an acute, specific, localized and fully reversible damage of the presynaptic nerve terminal, and its action mimics the cascade of events that leads to nerve terminal degeneration in injured patients and in many neurodegenerative conditions. Here we provide evidence of an early release by degenerating neurons of ATP as alarm messenger, that contributes to the activation of a series of intracellular pathways within SCs that are crucial for nerve regeneration: Ca2+, cAMP, ERK1/2, and CREB. These results contribute to define the cross-talk taking place among degenerating nerve terminals and PSCs, involved in the functional recovery of the NMJ.

  12. Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells.

    Science.gov (United States)

    Deng, Jingjing; Wang, Kai; Wang, Ming; Yu, Ping; Mao, Lanqun

    2017-04-26

    Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn 2+ and imidazole-2-carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

  13. ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells.

    Science.gov (United States)

    Takada, Naoto; Takatsu, Hiroyuki; Miyano, Rie; Nakayama, Kazuhisa; Shin, Hye-Won

    2015-11-01

    Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.

    Science.gov (United States)

    O'Grady, Scott M; Patil, Nandadavi; Melkamu, Tamene; Maniak, Peter J; Lancto, Cheryl; Kita, Hirohito

    2013-09-15

      Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) that was dependent on the coordinated activation of specific purinergic receptor (P2Y2 and P2X7) subtypes. In the present study, pretreatment with a cell-permeable Ca2+-chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca2+]i. Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca2+]i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in [Ca2+]i typically observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.

  15. The effect of gamma radiation and neocarzinostatin on NAD and ATP levels in mouse leukaemia cells

    International Nuclear Information System (INIS)

    Goodwin, P.M.; Lewis, P.J.; Davies, M.I.; Skidmore, C.J.; Shall, S.

    1978-01-01

    When mouse leukaemia cells are treated with γ-radiation or neocarzinostatin the intracelluiar NAD and ATP levels fall rapidly. It is shown that the ATP response is a consequence of the decreased NAD level. It is suggested that this low NAD level results in decreased glycolytic activity and that there is a subsequent accumulation of phosphorylated sugars associated with the fall in ATP. Under these extreme conditions, therefore, the NAD level probably regulates the rate of glycolysis in cells which are utilising a rapidly metabolisable sugar as their energy source. (Auth.)

  16. Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps.

    Science.gov (United States)

    Chu, Haiyan; Puchulu-Campanella, Estela; Galan, Jacob A; Tao, W Andy; Low, Philip S; Hoffman, Joseph F

    2012-07-31

    The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal-membrane complex. These pools of ATP are known to directly fuel both the Na(+)/K(+) and Ca(2+) pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na(+)/K(+) or Ca(2+) pump phosphoproteins (E(Na)-P and E(Ca)-P, respectively) from bulk [γ-(32)P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N(3)-ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N(3)-ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool's architecture can be photolabeled. With the aid of an antibody to N(3)-ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.

  17. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

    International Nuclear Information System (INIS)

    Kalayda, Ganna V; Wagner, Christina H; Buß, Irina; Reedijk, Jan; Jaehde, Ulrich

    2008-01-01

    Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant. In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn

  18. Effect of Inflammatory Mediators on ATP Release of Human Urothelial RT4 Cells

    Directory of Open Access Journals (Sweden)

    Kylie J. Mansfield

    2014-01-01

    Full Text Available Inflammation is an important contributor to the aetiology of a number of bladder dysfunctions including interstitial cystitis, painful bladder syndrome, and overactive bladder. The aim of this study was to examine the effects of inflammatory mediators on urothelial ATP release. Human urothelial RT4 cells were exposed to normal buffer or varying concentrations of inflammatory mediators (bradykinin, histamine, and serotonin in the presence or absence of hypotonic stretch stimuli (1 : 2 dilution of Krebs-Henseleit buffer. Others have demonstrated that bradykinin increased stretch-induced ATP release; however, we observed no change in control or stretch-induced ATP release with bradykinin. Pretreatment of RT4 cells with histamine or serotonin decreased stretch-induced ATP release (P=0.037, P=0.040, resp.. Previous studies have demonstrated increased ATP release in response to inflammation utilising whole bladder preparations in contrast to our simple model of cultured urothelial cells. The current study suggests that it is unlikely that there is a direct interaction between the release of inflammatory mediators and increased ATP release, but rather more complex interactions occurring in response to inflammation that lead to increased bladder sensation.

  19. Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Haanes, Kristian Agmund; Christensen, Nynne

    2015-01-01

    ) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells......BACKGROUND: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic...... increase [Ca(2+)]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca(2+) conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects...

  20. ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

    Science.gov (United States)

    Song, Shanshan; Jacobson, Krista N.; McDermott, Kimberly M.; Reddy, Sekhar P.; Cress, Anne E.; Tang, Haiyang; Dudek, Steven M.; Black, Stephen M.; Garcia, Joe G. N.; Makino, Ayako

    2015-01-01

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

  1. ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

    Science.gov (United States)

    Song, Shanshan; Jacobson, Krista N; McDermott, Kimberly M; Reddy, Sekhar P; Cress, Anne E; Tang, Haiyang; Dudek, Steven M; Black, Stephen M; Garcia, Joe G N; Makino, Ayako; Yuan, Jason X-J

    2016-01-15

    Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. Copyright

  2. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

    Science.gov (United States)

    Birket, Matthew J; Orr, Adam L; Gerencser, Akos A; Madden, David T; Vitelli, Cathy; Swistowski, Andrzej; Brand, Martin D; Zeng, Xianmin

    2011-02-01

    Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1β and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

  3. Computation of Pump-Leak Flux Balance in Animal Cells

    Directory of Open Access Journals (Sweden)

    Igor A. Vereninov

    2014-11-01

    Full Text Available Background/Aims: Many vital processes in animal cells depend on monovalent ion transport across the plasma membrane via specific pathways. Their operation is described by a set of nonlinear and transcendental equations that cannot be solved analytically. Previous computations had been optimized for certain cell types and included parameters whose experimental determination can be challenging. Methods: We have developed a simpler and a more universal computational approach by using fewer kinetic parameters derived from the data related to cell balanced state. A file is provided for calculating unidirectional Na+, K+, and Cl- fluxes via all major pathways (i.e. the Na/K pump, Na+, K+, Cl- channels, and NKCC, KC and NC cotransporters under a balanced state and during transient processes. Results: The data on the Na+, K+, and Cl- distribution and the pump flux of K+ (Rb+ are obtained on U937 cells before and after inhibiting the pump with ouabain. There was a good match between the results of calculations and the experimentally measured dynamics of ion redistribution caused by blocking the pump. Conclusion: The presented approach can serve as an effective tool for analyzing monovalent ion transport in the whole cell, determination of the rate coefficients for ion transfer via major pathways and studying their alteration under various conditions.

  4. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.

    Science.gov (United States)

    Uekawa, Atsushi; Yamanaka, Hitoki; Lieben, Liesbet; Kimira, Yoshifumi; Uehara, Mariko; Yamamoto, Yoko; Kato, Shigeaki; Ito, Kosei; Carmeliet, Geert; Masuyama, Ritsuko

    2018-01-05

    Extracellular low phosphate strongly enhances intestinal calcium absorption independently of active vitamin D [1,25(OH) 2 D 3 ] signaling, but the underlying mechanisms remain poorly characterized. To elucidate the phosphate-dependent regulation of calcium transport, we investigated part of the enteral environment that is involved in 1,25(OH) 2 D 3 -independent calcium absorption, which responds to dietary phosphate levels in mice that lack intestinal vitamin D receptor ( Vdr) activity. Impaired calcium absorption in intestinal Vdr-null mice was improved by dietary phosphate restriction. Accordingly, calcium transport in cultured intestinal epithelial cells was increased when the apical side was exposed to low phosphate levels (0.5 mM) compared with normal or high phosphate levels (1.0 or 5.0 mM, respectively). Mechanistically, low phosphate increased ATP in the apical side medium and allowed calcium entry into epithelial cells via the P2X7 purinoreceptor, which results in increased calcium transport. We found that luminal ATP was regulated by the release and degradation of ATP at the epithelium, and phosphate restriction increased ATP release from epithelial cells via connexin-43 hemichannels. Furthermore, ATP degradation by ectonucleotide pyrophosphatase-1 was reduced, which was caused by the reduction of the MAPK cascade. These findings indicate that luminal ATP metabolism regulates transcellular calcium transport in the intestine by an 1,25(OH) 2 D 3 -independent mechanism in response to dietary phosphate levels.-Uekawa, A., Yamanaka, H., Lieben, L., Kimira, Y., Uehara, M., Yamamoto, Y., Kato, S., Ito, K., Carmeliet, G., Masuyama, R. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.

  5. Changes in intracellular ATP-content of CHO cells as response to hyperosmolality.

    Science.gov (United States)

    Pfizenmaier, Jennifer; Matuszczyk, Jens-Christoph; Takors, Ralf

    2015-01-01

    A variety of approaches has been published to enhance specific productivity (qp) of recombinant Chinese hamster ovary (CHO) cells. Changes in culture conditions, e. g. temperature shifts, sodium butyrate treatment and hyperosmolality, were shown to improve qp . To contribute to a better understanding of the correlation between hyperosmolality and enhanced qp , we analyzed cellular kinetics and intracellular adenine nucleotide pools during osmotic shift periods. Known phenotypes like increased formation rates for lactate and product (anti-IL-8 antibody; qlactate, qp) as well as increased cell specific uptake rate for glucose (qglucose ) were found--besides inhibition of cell growth and G1-arrest occurred during batch cultivations with osmotic shift. The analysis of intracellular AXP pools revealed enlarged ATP amounts for cells as response to hyperosmolality while energy charges remained unchanged. Enhanced ATP-pools coincided with severely increased ATP formation rates (qATP ) which outweighed by far the putative requirements attributed to regulatory volume increase. Therefore elevated qATP mirrored an increased cellular demand for energy while experiencing hyperosmotic shift. © 2015 American Institute of Chemical Engineers.

  6. ATP-sensitive K(+-channels in muscle cells: features and physiological role

    Directory of Open Access Journals (Sweden)

    O. B. Vadzyuk

    2014-08-01

    Full Text Available ATP-sensitive K+-channels of plasma membranes belong to the inward rectifier potassium channels type. They are involved in coupling of electrical activity of muscle cell with its metabolic­ state. These channels are heterooctameric and consist of two types of subunits: four poreforming (Kir 6.х and four regulatory (SUR, sulfonylurea receptor. The Kir subunits contain highly selective K+ filter and provide for high-velocity K+ currents. The SUR subunits contain binding sites for activators and blockers and have metabolic sensor, which enables channel activation under conditions of metabolic stress. ATP blocks K+ currents through the ATP-sensitive K+-channels in the most types of muscle cells. However, functional activity of these channels does not depend on absolute concentration of ATP but on the АТР/ADP ratio and presence of Mg2+. Physiologically active substances, such as phosphatidylinositol bisphosphate and fatty acid esters can regulate the activity of these structures in muscle cells. Activation of these channels under ischemic conditions underlies their cytoprotective action, which results in prevention of Ca2+ overload in cytosol. In contrast to ATP-sensitive K+-channels of plasma membranes, the data regarding the structure and function of ATP-sensitive K+-channels of mitochondrial membrane are contradictory. Pore-forming subunits of this channel have not been firmly identified yet. ATP-sensitive K+ transport through the mitochondrial­ membrane is easily tested by different methods, which are briefly reviewed in this paper. Interaction of mitoKATP with physiological and pharmacological ligands is discussed as well.

  7. Increased ATP generation in the host cell is required for efficient vaccinia virus production

    Directory of Open Access Journals (Sweden)

    Hsu Che-Fang

    2009-09-01

    Full Text Available Abstract To search for cellular genes up-regulated by vaccinia virus (VV infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

  8. ATP-Evoked Intracellular Ca(2+) Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea

    NARCIS (Netherlands)

    Horvath, T.; Polony, G.; Fekete, A.; Aller, M.; Halmos, G.; Lendvai, B.; Heinrich, A.; Sperlagh, B.; Vizi, E. S.; Zelles, T.

    Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different

  9. ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage.

    Science.gov (United States)

    Cao, Cong; Healey, Sarah; Amaral, Ashley; Lee-Couture, Avery; Wan, Shu; Kouttab, Nicola; Chu, Wenming; Wan, Yinsheng

    2007-07-01

    Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.

  10. Mitochonic Acid 5 (MA-5 Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases

    Directory of Open Access Journals (Sweden)

    Tetsuro Matsuhashi

    2017-06-01

    Full Text Available Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015. MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model “Mitomouse” (JASN, 2016. To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96% were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.

  11. Dependence of structure stability and integrity of aerobic granules on ATP and cell communication.

    Science.gov (United States)

    Jiang, Bo; Liu, Yu

    2013-06-01

    Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules.

  12. Distinct cell stress responses induced by ATP restriction in quiescent human fibroblasts

    Directory of Open Access Journals (Sweden)

    Nirupama Yalamanchili

    2016-10-01

    Full Text Available Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated transcription factors and altered transcription factor subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

  13. Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

    Science.gov (United States)

    Anwar, Zubeya; Albert, Jennifer L; Gubby, Sharon E; Boyle, John P; Roberts, Jonathon A; Webb, Tania E; Boarder, Michael R

    1999-01-01

    In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists.These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors. PMID:10510459

  14. Simple catalytic cell for restoring He leak detector sensitivity on vacuum systems with high D2 backgrounds

    International Nuclear Information System (INIS)

    Busath, J.; Chiu, H.K.

    1998-12-01

    The DIII-D National Fusion Facility at General Atomics focuses on plasma physics and fusion energy science. The DIII-D tokamak is a 35 m 3 toroidal vacuum vessel with over 200 ports for diagnostic instrumentation, cryogenics, microwave heating, and four large neutral beam injectors. Maintaining vacuum in the 10 -8 Torr range is crucial for producing high performance plasma discharges. He leak checking the DIII-D tokamak and the neutral beamlines has historically been difficult. D 2 is used as the fuel gas in most plasma discharges and neutral beams. After plasma operations, D 2 out-gassing from the torus walls and internal beamline components can exceed 10 -4 std cc/s. The mass of the D 2 molecule (4.028 u) is indistinguishable from that of the He atom (4.003 u) to a standard mass spectrometer leak detector. High levels of D 2 reduce leak detector sensitivity and effectively mask the He trace gas signal rendering normal leak checking techniques ineffective. A simple apparatus was developed at GA to address these problems. It consists of a palladium based catalyst cell and associated valves and piping placed in series with the leak detector. This reduces the D 2 throughput by a factor greater than 10,000, restoring leak detector sensitivity. This paper will briefly discuss the development of the cell, the physical processes involved, the tests performed to quantify and optimize the processes, and the operational results at DIII-D

  15. Extracellular ATP elevates cytoplasmatic free Ca2+ in HeLa cells by the interaction with a 5'-nucleotide receptor

    NARCIS (Netherlands)

    Smit, M J; Leurs, R; Bloemers, S M; Tertoolen, L G; Bast, A; De Laat, S W; Timmerman, H

    1993-01-01

    In the present study we have characterized the effects of ATP and several other nucleotides on the intracellular Ca2+ levels of HeLa cells. Using fura-2 microscopy fluorescence measurements, the ATP-mediated increase in intracellular Ca2+ was shown to consist of a rapid rise which decreased after a

  16. ATP-dependent modification of gamma irradiation effects on red blood cells

    International Nuclear Information System (INIS)

    Galutzov, B.; Ivanov, S.; Ratcheva-Kantcheva, M.

    1987-01-01

    The in vitro gamma irradiation effects on human red blood cell membrane properties were investigated. Osmotic fragility, rate of hemolysis, electrophoretic mobility and cell size distribution were monitored during nutrient-free in vitro storage and postirradiation incubation at the 1st, 5th and 25th hour. Experimental results confirm a time-dependent radiation-induced cell membrane damage. The increase of osmotic fragility, rate of hemolysis and cell size and the decrease in electrophoretic mobility are discussed as compared to membrane destabilization during in vitro ageing. Exogeneous ATP treatment of erythrocytes before and after irradiation results in some dose-dependent membrane protection. (author)

  17. CSF leak

    Science.gov (United States)

    ... fluid leak References Boahene K, Dagi TF, Quiñones-Hinojosa A. Management of cerebrospinal fluid leaks. In: Quiñones-Hinojosa A, ed. Schmidek and Sweet's Operative Neurosurgical Techniques . ...

  18. ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors

    Directory of Open Access Journals (Sweden)

    Selvi Ruthrotha B

    2012-08-01

    Full Text Available Abstract Background Intrinsically fluorescent glucose derived carbon nanospheres (CSP efficiently enter mammalian cells and also cross the blood brain barrier (BBB. However, the mechanistic details of CSP entry inside mammalian cells and its specificity are not known. Results In this report, the biochemical and cellular mechanism of CSP entry into the living cell have been investigated. By employing confocal imaging we show that CSP entry into the mammalian cells is an ATP-dependent clathrin mediated endocytosis process. Zeta potential studies suggest that it has a strong preference for cells which possess high levels of glucose transporters such as the glial cells, thereby enabling it to target individual organs/tissues such as the brain with increased specificity. Conclusion The endocytosis of Glucose derived CSP into mammalian cells is an ATP dependent process mediated by clathrin coated pits. CSPs utilize the surface functional groups to target cells containing glucose transporters on its membrane thereby implicating a potential application for specific targeting of the brain or cancer cells.

  19. Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells.

    Science.gov (United States)

    Salvestrini, Valentina; Orecchioni, Stefania; Talarico, Giovanna; Reggiani, Francesca; Mazzetti, Cristina; Bertolini, Francesco; Orioli, Elisa; Adinolfi, Elena; Di Virgilio, Francesco; Pezzi, Annalisa; Cavo, Michele; Lemoli, Roberto M; Curti, Antonio

    2017-01-24

    Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2X7R is the most consistently expressed by tumors. P2X7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2X7R. Here, we show that P2X7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2X7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo.Overall, our results demonstrate that P2X7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML.

  20. Leak detection

    OpenAIRE

    Hilleret, Noël

    1999-01-01

    This paper will give an introduction to the leak detection of vacuum systems. Various methods to detect leaks as well as the most widely used helium leak detectors and their different applications are presented. Practical examples in the context of accelerator vacuum systems will illustrate the topic.

  1. Leak detection

    CERN Document Server

    Zapfe, K

    2007-01-01

    This paper will give an introduction to the leak detection of vacuum systems. Various methods to detect leaks as well as the most widely used helium leak detectors and their different applications are presented. Practical examples in the context of accelerator vacuum systems will illustrate the topic.

  2. Persistence of biomarker ATP and ATP-generating capability in bacterial cells and spores contaminating spacecraft materials under earth conditions and in a simulated martian environment.

    Science.gov (United States)

    Fajardo-Cavazos, Patricia; Schuerger, Andrew C; Nicholson, Wayne L

    2008-08-01

    Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5'-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m(-2) of UV-C (200 to 280 nm), -10 degrees C, and a Mars gas mix of CO(2) (95.54%), N(2) (2.7%), Ar (1.6%), O(2) (0.13%), and H(2)O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.

  3. [ATP and ACh induced CICR in outer hair cells of the guinea pig cochlea: study of confocal microscopy].

    Science.gov (United States)

    Huang, Li; Yang, Jun

    2009-04-01

    Effects of ATP and acetylcholine (ACh) on intracellular Ca2+ concentrations ([Ca2+]i) and possible mechanism of Ca2+-induced Ca2+ release (CICR) of the isolated outer hair cells (OHCs) in the guinea pig cochlea were studied with confocal microscopy. OHCs were isolated from guinea pig cochlea by enzymatic and mechanical methods. The effects of ATP, ACh, Ryanodine + ATP (or ACh) and Thapsigargin + ATP (or ACh) in the presence or absence of extracellular Ca2+ on [Ca2+]i in OHCs were examined by confocal microscopy. In the presence of ATP, Ryanodine + ATP, Thapsigargin + ATP, ACh, Ryanodine + ACh and Thapsigargin + ACh increased [Ca2+]i and evoked an evident wave, respectively, the relative magnitude of fluorescence were 1.60 +/- 0.01(ATP), 1.644 +/- 0.005 (Ryanodine + ATP), 1.491 +/- 0.005 (Thapsigargin + ATP), 1.43 +/- 0.01 (ACh), 1.58 +/- 0.02 (Ryanodine + ACh), 1.398 +/- 0.003 (Thapsigargin + ACh) in OHCs in the presence of extracellular Ca2+ respectively. In the absence of extracellular Ca2+, ATP and Ryanodine + ATP induced a gradual and small [Ca2+]i wave, the relative magnitude of fluorescence were 1.341 +/- 0.006 and 1.386 +/- 0.008, however, ACh, Ryanodine + ACh, Thapsigargin + ACh and Thapsigargin + ATP can not induce wave but a gradual [Ca2+]i elevation. ACh can not increase [Ca2+]i. In the presence of extracellular Ca2+, ATP and ACh increased [Ca2+]i in OHCs not only by Ca2+ influx through ion channel on cell membrane but also a release of Ca2+ from IP3-sensitive calcium reservoir and CICR. In the absence of extracellular Ca2+, ATP activated IP3 sensitive calcium reservoir and Ca2+ release through IP3 sensitive calcium reservoir, in turn CICR was induced. ACh can not activate IP3 sensitive calcium reservoir and CICR in the absence of extracellular Ca2+, therefore, the effect of ACh was dependent of extracellular Ca2+.

  4. Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

    Directory of Open Access Journals (Sweden)

    Quadros Edward V

    2009-05-01

    Full Text Available Abstract Background Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration. Methods Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM, or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2. Results There was a high correlation of cell growth with ATP concentration (r = 0.948 in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p Conclusion Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies.

  5. Development of New Openers of ATP-Sensitive Potassium Channels of the Cell Membranes

    Directory of Open Access Journals (Sweden)

    Strutynskyi, R.B.

    2016-07-01

    Full Text Available Two innovative libraries (413 cyclosulfamides and 709 orthopyridine sulfamides of potential new openers of ATP-sensitive potassium channels of cell membranes were developed. It is shown experimentally that at least ten new original compounds have properties of pharmacological openers of the channels. Seven compounds, namely Z851154982, Z56762024, Z1269122570, Z31153162, Z45679561, Z756371174 and Z649723638, open channels of both types — sarcoplasmic as well as mitochondrial membranes: Simultaneously, Z734043408 compound is a potent activator of aforementioned channels of sarcolemmal membrane only. Z31197374 and Z666664306 compounds show the affinity onlyto КATP-channels of mitochondrial type. The results of the work can be used by pharmaceutical companies and scientific research institutes.

  6. ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells.

    Science.gov (United States)

    Lecciso, Mariangela; Ocadlikova, Darina; Sangaletti, Sabina; Trabanelli, Sara; De Marchi, Elena; Orioli, Elisa; Pegoraro, Anna; Portararo, Paola; Jandus, Camilla; Bontadini, Andrea; Redavid, Annarita; Salvestrini, Valentina; Romero, Pedro; Colombo, Mario P; Di Virgilio, Francesco; Cavo, Michele; Adinolfi, Elena; Curti, Antonio

    2017-01-01

    Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC) cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML), ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs) with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1), was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1), which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1 + CD39 + DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

  7. ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Mariangela Lecciso

    2017-12-01

    Full Text Available Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML, ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1, was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1, which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1+CD39+ DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

  8. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

    Science.gov (United States)

    Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, Mark; Bewersdorf, Joerg; Ježek, Petr

    2016-05-01

    The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J

  9. Effect of angiotensin II, ATP, and ionophore A23187 on potassium efflux in adrenal glomerulosa cells

    International Nuclear Information System (INIS)

    Lobo, M.V.; Marusic, E.T.

    1986-01-01

    Angiotensin II stimulus on perifused bovine adrenal glomerulosa cells elicited an increase in 86Rb efflux from cells previously equilibrated with the radioisotope. When 45Ca fluxes were measured under similar conditions, it was observed that Ca and Rb effluxes occurred within the first 30 s of the addition of the hormone and were independent of the presence of external Ca. The 86Rb efflux due to angiotensin II was inhibited by quinine and apamin. The hypothesis that the angiotensin II response is a consequence of an increase in the K permeability of the glomerulosa cell membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated 86Rb or K loss (as measured by an external K electrode). This increased K conductance was also seen with 10(-4) M ATP. Quinine and apamin greatly reduced the effect of ATP or A23187 on 86Rb or K release in adrenal glomerulosa cells. The results suggest that Ca-dependent K channels or carriers are present in the membranes of bovine adrenal glomerulosa cells and are sensitive to hormonal stimulus

  10. Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

    Science.gov (United States)

    Sumi, Daigo; Asao, Masashi; Okada, Hideta; Yogi, Kuniko; Miyataka, Hideki; Himeno, Seiichiro

    2016-06-01

    Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity.

  11. Sildenafil protects neuronal cells from mitochondrial toxicity induced by β-amyloid peptide via ATP-sensitive K+ channels.

    Science.gov (United States)

    Son, Yonghae; Kim, Koanhoi; Cho, Hyok-Rae

    2018-06-02

    To understand the molecular mechanisms underlying the beneficial effects of sildenafil in animal models of neurological disorders, we investigated the effects of sildenafil on the mitochondrial toxicity induced by β-amyloid (Aβ) peptide. Treatment of HT-22 hippocampal neuronal cells with Aβ 25∼35 results in increased mitochondrial Ca 2+ load, which is subsequently suppressed by sildenafil as well as by diazoxide, a selective opener of the ATP-sensitive K + channels (K ATP ). However, the suppressive effects of sildenafil and diazoxide are significantly attenuated by 5-hydroxydecanoic acid (5-HD), a K ATP inhibitor. The increased mitochondrial Ca 2+ overload is accompanied by decrease in the intracellular ATP concentration, increase in intracellular ROS generation, occurrence of mitochondrial permeability transition, and activation of caspase-9 and cell death. Exposure to sildenafil inhibited the mitochondria-associated changes and cell death induced by Aβ. However, the inhibitory effects of sildenafil are abolished or weakened in the presence of 5-HD, suggesting that opening of the mitochondrial K ATP is required for sildenafil to exert these effects. Taken together, these results indicate that at the mitochondrial levels, sildenafil plays a protective role towards neuronal cell in an environment rich in Aβ, and exerts its effects via the mitochondrial K ATP channels-dependent mechanisms. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    Science.gov (United States)

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

  13. Low energy costs of F1Fo ATP synthase reversal in colon carcinoma cells deficient in mitochondrial complex IV.

    Science.gov (United States)

    Zhdanov, Alexander V; Andreev, Dmitry E; Baranov, Pavel V; Papkovsky, Dmitri B

    2017-05-01

    Mitochondrial polarisation is paramount for a variety of cellular functions. Under ischemia, mitochondrial membrane potential (ΔΨm) and proton gradient (ΔpH) are maintained via a reversal of mitochondrial F1Fo ATP synthase (mATPase), which can rapidly deplete ATP and drive cells into energy crisis. We found that under normal conditions in cells with disassembled cytochrome c oxidase complex (COX-deficient HCT116), mATPase maintains ΔΨm at levels only 15-20% lower than in WT cells, and for this utilises relatively little ATP. For a small energy expenditure, mATPase enables mitochondrial ΔpH, protein import, Ca 2+ turnover, and supports free radical detoxication machinery enlarged to protect the cells from oxidative damage. Whereas in COX-deficient cells the main source of ATP is glycolysis, the ΔΨm is still maintained upon inhibition of the adenine nucleotide translocators with bongkrekic acid and carboxyatractyloside, indicating that the role of ANTs is redundant, and matrix substrate level phosphorylation alone or in cooperation with ATP-Mg/P i carriers can continuously support the mATPase activity. Intriguingly, we found that mitochondrial complex III is active, and it contributes not only to free radical production, but also to ΔΨm maintenance and energy budget of COX-deficient cells. Overall, this study demonstrates that F1Fo ATP synthase can support general mitochondrial and cellular functions, working in extremely efficient 'energy saving' reverse mode and flexibly recruiting free radical detoxication and ATP producing / transporting pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. ATP and UTP responses of cultured rat aortic smooth muscle cells revisited: dominance of P2Y2 receptors

    Science.gov (United States)

    Kumari, Rajendra; Goh, Gareth; Ng, Leong L; Boarder, Michael R

    2003-01-01

    It has previously been shown that ATP and UTP stimulate P2Y receptors in vascular smooth muscle cells (VSMCs), but the nature of these receptors, in particular the contribution of P2Y2 and P2Y4 subtypes, has not been firmly established. Here we undertake a further pharmacological analysis of [3H]inositol polyphosphate responses to nucleotides in cultured rat VSMCs. ATP generated a response that was partial compared to UTP, as reported earlier. In the presence of a creatine phosphokinase (CPK) system for regenerating nucleoside triphosphates, the response to ATP was increased, the response to UTP was unchanged, and the difference between UTP and ATP concentration–response curves disappeared. Chromatographic analysis showed that ATP was degraded slightly faster than UTP. The response to UDP was always smaller than that to UTP, but with a shallow slope and a high potency component. In the presence of hexokinase (which prevents the accumulation of ATP/UTP from ADP/UDP), the maximum response to UDP was reduced and the high-potency component of the curve was retained. By contrast, the response to ADP was weaker throughout in the presence of hexokinase. ATPγS was an effective agonist with a similar EC50 to UTP, but with a lower maximum. ITP was a weak agonist compared with UTP. Suramin was an effective antagonist of the response to UTP (pA2=4.48), but not when ATP was the agonist. However, suramin was an effective antagonist (pA2=4.45) when stimulation with ATP was in the presence of the CPK regenerating system. Taken together with the results of others, these findings indicate that the response of cultured rat VSMCs to UTP and to ATP is predominantly at the P2Y2 receptor, and that there is also a response to UDP at the P2Y6 receptor. PMID:14597595

  15. A Loose Coupling Mechanism of Synthesis of ATP by Proton Flux in the Molecular Machine of Living Cells

    Science.gov (United States)

    Oosawa, Fumio; Hayashi, Shigeru

    1984-04-01

    A loose coupling mechanism is proposed for the molecular machine of living cells which performs the synthesis of ATP from ADP and inorganic phosphate by a proton flux across the membrane. The basic assumption is that a torque or rotational movement is produced in protein molecules by the proton flux and the ATP synthesis is promoted by the torque or rotational movement in the molecules. The first process is carried out by a mechanism similar to the bacterial flagellar motor where the coupling between the flow of protons and the rotation of the motor is loose. A structural model for the second process is also presented. The threshold of the potential difference of proton for the ATP synthesis can be low and the number of protons to synthesize one ATP molecule is not an integer but a variable number.

  16. Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

    Directory of Open Access Journals (Sweden)

    Boriana K Tchernookova

    Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

  17. Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells

    Directory of Open Access Journals (Sweden)

    JoAnn S. Roberts

    2017-07-01

    Full Text Available Porphyromonas gingivalis, a major opportunistic pathogen in the etiology of chronic periodontitis, successfully survives in human gingival epithelial cells (GECs. P. gingivalis abrogates the effects of a host danger molecule, extracellular ATP (eATP/P2X7 signaling, such as the generation of reactive oxygen species (ROS via the mitochondria and NADPH oxidases (NOX from primary GECs. However, antimicrobial functions of ROS production are thoroughly investigated in myeloid-lineage immune cells and have not been well-understood in epithelial cells. Therefore, this study characterizes antibacterial NOX2 generated ROS and host downstream effects in P. gingivalis infected human primary GECs. We examined the expression of NOX isoforms in the GECs and demonstrate eATP stimulation increased the mRNA expression of NOX2 (p < 0.05. Specific peptide inhibition of NOX2 significantly reduced eATP-mediated ROS as detected by DCFDA probe. The results also showed P. gingivalis infection can temporally modulate NOX2 pathway by reorganizing the localization and activation of cytosolic molecules (p47phox, p67phox, and Rac1 during 24 h of infection. Investigation into downstream biocidal factors of NOX2 revealed an eATP-induced increase in hypochlorous acid (HOCl in GECs detected by R19-S fluorescent probe, which is significantly reduced by a myeloperoxidase (MPO inhibitor. MPO activity of the host cells was assayed and found to be positively affected by eATP treatment and/or infection. However, P. gingivalis significantly reduced the MPO product, bactericidal HOCl, in early times of infection upon eATP stimulation. Analysis of the intracellular levels of a major host-antioxidant, glutathione during early infection revealed a substantial decrease (p < 0.05 in reduced glutathione indicative of scavenging of HOCl by P. gingivalis infection and eATP treatment. Examination of the mRNA expression of key enzymes in the glutathione synthesis pathway displayed a marked

  18. T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP

    OpenAIRE

    Perruzza, Lisa; Gargari, Giorgio; Proietti, Michele; Fosso, Bruno; D’Erchia, Anna Maria; Faliti, Caterina Elisa; Rezzonico-Jost, Tanja; Scribano, Daniela; Mauri, Laura; Colombo, Diego; Pellegrini, Giovanni; Moregola, Annalisa; Mooser, Catherine; Pesole, Graziano; Nicoletti, Mauro

    2017-01-01

    Summary The ATP-gated ionotropic P2X7 receptor regulates T?follicular helper (Tfh) cell abundance in the Peyer?s patches (PPs) of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem ...

  19. The Warburg Hypothesis and the ATP Supply In Cancer Cells Is Oxidative Phosphorylation impaired in malignant neoplasias?

    Science.gov (United States)

    Rodríguez-Enríquez, Sara; Gallardo-Pérez, Juan Carlos; Marín-Hernández, Alvaro; Moreno-Sánchez, Rafael

    2012-06-01

    Since Warburg proposed that cancer cells exhibit increased glycolysis due to apparent mitochondrial damage, numerous researchers have assumed that glycolysis is the predominant ATP supplier for cancer cell energy-dependent processes. However, chemotherapeutic strategies using glycolytic inhibitors have been unsuccessful in arresting tumor proliferation indicating that the original Warburg proposal may not be applicable to all existing neoplasias. This review analyzes recent information on mitochondrial metabolism in several malignant neoplasias emphasizing that, although tumor cells maintain a high glycolytic rate, the principal ATP production may derive from active oxidative phosphorylation. Thus, anti-mitochondrial drug therapy may be an adequate strategy to arrest proliferation of oxidative phosphorylation-dependent neoplasias.

  20. The bioenergetics of cancer: is glycolysis the main ATP supplier in all tumor cells?

    Science.gov (United States)

    Moreno-Sánchez, Rafael; Rodríguez-Enríquez, Sara; Saavedra, Emma; Marín-Hernández, Alvaro; Gallardo-Pérez, Juan Carlos

    2009-01-01

    The molecular mechanisms by which tumor cells achieve an enhanced glycolytic flux and, presumably, a decreased oxidative phosphorylation are analyzed. As the O(2) concentration in hypoxic regions of tumors seems not limiting for oxidative phosphorylation, the role of this mitochondrial pathway in the ATP supply is re-evaluated. Drugs that inhibit glycoysis and oxidative phosphorylation are analyzed for their specificity toward tumor cells and effect on proliferation. The energy metabolism mechanisms involved in the use of positron emission tomography are revised and updated. It is proposed that energy metabolism may be an alternative therapeutic target for both hypoxic (glycolytic) and oxidative tumors. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

  1. ATP Release from Dying Autophagic Cells and Their Phagocytosis Are Crucial for Inflammasome Activation in Macrophages

    Science.gov (United States)

    Ayna, Gizem; Krysko, Dmitri V.; Kaczmarek, Agnieszka; Petrovski, Goran; Vandenabeele, Peter; Fésüs, László

    2012-01-01

    Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P2X7 purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses. PMID:22768222

  2. Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death

    Directory of Open Access Journals (Sweden)

    Ying Xue

    2016-01-01

    Full Text Available P2X7 receptor (P2X7R, an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis, as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL, which was reversed by coinjection of brilliant blue G (BBG, a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.

  3. Arsenic Alters ATP-Dependent Ca2+ Signaling in Human Airway Epithelial Cell Wound Response

    Science.gov (United States)

    Sherwood, Cara L.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2011-01-01

    Arsenic is a natural metalloid toxicant that is associated with occupational inhalation injury and contaminates drinking water worldwide. Both inhalation of arsenic and consumption of arsenic-tainted water are correlated with malignant and nonmalignant lung diseases. Despite strong links between arsenic and respiratory illness, underlying cell responses to arsenic remain unclear. We hypothesized that arsenic may elicit some of its detrimental effects on the airway through limitation of innate immune function and, specifically, through alteration of paracrine ATP (purinergic) Ca2+ signaling in the airway epithelium. We examined the effects of acute (24 h) exposure with environmentally relevant levels of arsenic (i.e., arsenic reduces purinergic Ca2+ signaling in a dose-dependent manner and results in a reshaping of the Ca2+ signaling response to localized wounds. We next examined arsenic effects on two purinergic receptor types: the metabotropic P2Y and ionotropic P2X receptors. Arsenic inhibited both P2Y- and P2X-mediated Ca2+ signaling responses to ATP. Both inhaled and ingested arsenic can rapidly reach the airway epithelium where purinergic signaling is essential in innate immune functions (e.g., ciliary beat, salt and water transport, bactericide production, and wound repair). Arsenic-induced compromise of such airway defense mechanisms may be an underlying contributor to chronic lung disease. PMID:21357385

  4. The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP.

    Science.gov (United States)

    James, Andrew D; Patel, Waseema; Butt, Zohra; Adiamah, Magretta; Dakhel, Raga; Latif, Ayse; Uggenti, Carolina; Swanton, Eileithyia; Imamura, Hiromi; Siriwardena, Ajith K; Bruce, Jason I E

    2015-10-09

    Evidence suggests that the plasma membrane Ca(2+)-ATPase (PMCA), which is critical for maintaining a low intracellular Ca(2+) concentration ([Ca(2+)]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca(2+)]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca(2+)]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca(2+)]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

    Science.gov (United States)

    ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

  6. ATP Release from Mast Cells by Physical Stimulation: A Putative Early Step in Activation of Acupuncture Points

    Directory of Open Access Journals (Sweden)

    Lina Wang

    2013-01-01

    Full Text Available In Chinese medicine acupuncture points are treated by physical stimuli to counteract various diseases. These stimuli include mechanical stress as applied during the needle manipulation or tuina, high temperatures as applied during moxibustion, and red laser light applied during laser acupuncture. This study aimed to investigate cellular responses to stimuli that might occur in the tissue of acupuncture points. Since they have a characteristically high density of mast cells that degranulate in response to acupuncture, we asked whether these processes lead to ATP release. We tested in in vitro experiments on mast cells of the human mast-cell line HMC-1 the effects of the physical stimuli; mechanical stress was applied by superfusion of the cells with hypotonic solution, heat was applied by incubation of the cells at 52°C, and red laser light of 657 nm was used for irradiation. We demonstrate that all the stimuli induce ATP release from model human mast HMC-1 cells, and this release is associated with an intracellular free Ca2+ rise. We hypothesize that ATP released from mast cells supplements the already known release of ATP from keratinocytes and, by acting on P2X receptors, it may serve as initial mediator of acupuncture-induced analgesia.

  7. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  8. Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP.

    Science.gov (United States)

    Sander, Philip; Mostafa, Haouraa; Soboh, Ayman; Schneider, Julian M; Pala, Andrej; Baron, Ann-Kathrin; Moepps, Barbara; Wirtz, C Rainer; Georgieff, Michael; Schneider, Marion

    2017-05-23

    Glioblastomas (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutic options are urgently needed. A novel drug, Vacquinol-1 (Vac), a quinolone derivative, displays promising properties by inducing rapid cell death in GBM but not in non-transformed tissues. Features of this type of cell death are compatible with a process termed methuosis. Here we tested Vac on a highly malignant glioma cell line observed by long-term video microscopy. Human dental-pulp stem cells (DPSCs) served as controls. A major finding was that an exogenous ATP concentration of as little as 1 μM counter regulated the Vac-induced cell death. Studies using carvacrol, an inhibitor of transient receptor potential cation channel, subfamily M, member 7 (TRPM7), demonstrated that the ATP-inducible inhibitory effect is likely to be via TRPM7. Exogenous ATP is of relevance in GBM with large necrotic areas. Our results support the use of GBM cultures with different grades of malignancy to address their sensitivity to methuosis. The video-microscopy approach presented here allows decoding of signaling pathways as well as mechanisms of chemotherapeutic resistance by long-term observation. Before implementing Vac as a novel therapeutic drug in GBM, cells from each individual patient need to be assessed for their ATP sensitivity. In summary, the current investigation supports the concept of methuosis, described as non-apoptotic cell death and a promising approach for GBM treatment. Tissue-resident ATP/necrosis may interfere with this cell-death pathway but can be overcome by a natural compound, carvacrol that even penetrates the blood-brain barrier.

  9. ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment.

    Science.gov (United States)

    Bianchi, G; Vuerich, M; Pellegatti, P; Marimpietri, D; Emionite, L; Marigo, I; Bronte, V; Di Virgilio, F; Pistoia, V; Raffaghello, L

    2014-03-20

    Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b(+)/Gr-1(+) cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1(+) population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

  10. Histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to Ca2+ mobilization and cell proliferation.

    Science.gov (United States)

    Pinheiro, Ana Rita; Paramos-de-Carvalho, Diogo; Certal, Mariana; Costa, Maria Adelina; Costa, Cristina; Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Sévigny, Jean; Correia-de-Sá, Paulo

    2013-09-20

    Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca(2+) ([Ca(2+)]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca(2+)]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca(2+)]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with (10)Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca(2+)]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca(2+)]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

  11. Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation*

    Science.gov (United States)

    Pinheiro, Ana Rita; Paramos-de-Carvalho, Diogo; Certal, Mariana; Costa, Maria Adelina; Costa, Cristina; Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Sévigny, Jean; Correia-de-Sá, Paulo

    2013-01-01

    Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors. PMID:23918924

  12. Nucleotide receptors stimulation by extracellular ATP controls Hsp90 expression through APE1/Ref-1 in thyroid cancer cells: a novel tumorigenic pathway.

    Science.gov (United States)

    Pines, Alex; Bivi, Nicoletta; Vascotto, Carlo; Romanello, Milena; D'Ambrosio, Chiara; Scaloni, Andrea; Damante, Giuseppe; Morisi, Roberta; Filetti, Sebastiano; Ferretti, Elisabetta; Quadrifoglio, Franco; Tell, Gianluca

    2006-10-01

    Nucleotide receptors signaling affects cell proliferation, with possible implications on tumorigenic processes. However, molecular targets and action mechanisms of the extracellular nucleotides are still poorly elucidated. We have previously shown in ARO cells that APE1/Ref-1, a transcriptional coactivator responsible for the maintenance of the cellular proliferative rate, is functionally controlled by P2-mediated signaling. Here, we demonstrate that extracellular ATP has a mitogenic effect on ARO cells, increasing ERK phosphorylation, AP1 activation, and cyclin D1 expression. Using the ATP/ADPase apyrase and the P2 receptor antagonist suramin, we show that the extracellular ATP, physiologically released by ARO cells, exerts mitogenic effects. A differential proteomic approach was used to identify molecular events associated with the ATP-induced cell proliferation. Among other proteins, Hsp90 was found upregulated upon ATP stimulation. Pretreatment with suramin completely blocked the ATP-induced Hsp90 activation, confirming the involvement of cell-surface P2 nucleotide receptors in the ATP-mediated activation of ARO cells. Treatment of proliferating ARO cells with suramin and apyrase significantly reduced the intracellular levels of Hsp90, suggesting an autocrine/paracrine mechanism of control on Hsp90 expression by extracellular ATP. The influence of Hsp90 on ATP-induced cell proliferation was also demonstrated by its specific inhibition with 17-AAG. The molecular pathway by which ATP stimulates cell proliferation was further investigated by siRNA strategies showing that Hsp90 is a target of APE1/Ref-1 functional activation. Stimulation of ARO cells with specific nucleotide receptors agonists evidenced a major involvement of P2Y1 and P2Y2 receptors in controlling the Hsp90 activation. Accordingly, these two receptors resulted significantly upregulated in sample biopsies from different thyroid tumors. Copyright 2006 Wiley-Liss, Inc.

  13. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

    Directory of Open Access Journals (Sweden)

    Randall Marcelo Chin

    2018-03-01

    Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

  14. Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.

    Science.gov (United States)

    Vázquez-Cuevas, Francisco G; Martínez-Ramírez, Angélica S; Robles-Martínez, Leticia; Garay, Edith; García-Carrancá, Alejandro; Pérez-Montiel, Delia; Castañeda-García, Carolina; Arellano, Rogelio O

    2014-11-01

    P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability. © 2014 Wiley Periodicals, Inc.

  15. DNA damage response in renal ischemia-reperfusion and ATP-depletion injury of renal tubular cells.

    Science.gov (United States)

    Ma, Zhengwei; Wei, Qingqing; Dong, Guie; Huo, Yuqing; Dong, Zheng

    2014-07-01

    Renal ischemia-reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia-reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or "reperfusion" of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia-reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular ATP levels and thiol groups

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    The experiments concerned the effect of ethacrynic acid (0.5 mM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 microM). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose....... Furthermore, the inhibition of histamine release occurred mainly through interaction with thiol groups, although inhibition of cellular energy production may have been an additional mechanism....... as well as in presence of 2-deoxyglucose. A23187-induced histamine release was inhibited by ethacrynic acid, and this inhibition was completely reversed by dithiothreitol. These observations may indicate that ethacrynic acid inhibits glycolytic and respiratory energy production in rat mast cells...

  17. T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP.

    Science.gov (United States)

    Perruzza, Lisa; Gargari, Giorgio; Proietti, Michele; Fosso, Bruno; D'Erchia, Anna Maria; Faliti, Caterina Elisa; Rezzonico-Jost, Tanja; Scribano, Daniela; Mauri, Laura; Colombo, Diego; Pellegrini, Giovanni; Moregola, Annalisa; Mooser, Catherine; Pesole, Graziano; Nicoletti, Mauro; Norata, Giuseppe Danilo; Geuking, Markus B; McCoy, Kathy D; Guglielmetti, Simone; Grassi, Fabio

    2017-03-14

    The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh) cell abundance in the Peyer's patches (PPs) of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. T Follicular Helper Cells Promote a Beneficial Gut Ecosystem for Host Metabolic Homeostasis by Sensing Microbiota-Derived Extracellular ATP

    Directory of Open Access Journals (Sweden)

    Lisa Perruzza

    2017-03-01

    Full Text Available The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh cell abundance in the Peyer’s patches (PPs of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help.

  19. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

    Directory of Open Access Journals (Sweden)

    Flavio Alves Lara

    Full Text Available In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA, a well-known inhibitor of ATP binding cassette (ABC transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may

  20. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells

    Science.gov (United States)

    Lara, Flavio Alves; Pohl, Paula C.; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H. F.; Almeida, Igor C.; Vaz, Itabajara da Silva; Oliveira, Pedro L.

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  1. Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

    Science.gov (United States)

    Durand, Maxim; Dubois, Florian; Dejou, Cécile; Durand, Eugénie; Danger, Richard; Chesneau, Mélanie; Brosseau, Carole; Guerif, Pierrick; Soulillou, Jean-Paul; Degauque, Nicolas; Eliaou, Jean-François; Giral, Magali; Bonnefoy, Nathalie; Brouard, Sophie

    2018-05-01

    Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  2. Alkali pH directly activates ATP-sensitive K+ channels and inhibits insulin secretion in beta-cells.

    Science.gov (United States)

    Manning Fox, Jocelyn E; Karaman, Gunce; Wheeler, Michael B

    2006-11-17

    Glucose stimulation of pancreatic beta-cells is reported to lead to sustained alkalization, while extracellular application of weak bases is reported to inhibit electrical activity and decrease insulin secretion. We hypothesize that beta-cell K(ATP) channel activity is modulated by alkaline pH. Using the excised patch-clamp technique, we demonstrate a direct stimulatory action of alkali pH on recombinant SUR1/Kir6.2 channels due to increased open probability. Bath application of alkali pH similarly activates native islet beta-cell K(ATP) channels, leading to an inhibition of action potentials, and hyperpolarization of membrane potential. In situ pancreatic perfusion confirms that these cellular effects of alkali pH are observable at a functional level, resulting in decreases in both phase 1 and phase 2 glucose-stimulated insulin secretion. Our data are the first to report a stimulatory effect of a range of alkali pH on K(ATP) channel activity and link this to downstream effects on islet beta-cell function.

  3. The Role of Platelet-Derived ADP and ATP in Promoting Pancreatic Cancer Cell Survival and Gemcitabine Resistance

    Directory of Open Access Journals (Sweden)

    Omar Elaskalani

    2017-10-01

    Full Text Available Platelets have been demonstrated to be vital in cancer epithelial-mesenchymal transition (EMT, an important step in metastasis. Markers of EMT are associated with chemotherapy resistance. However, the association between the development of chemoresistance, EMT, and the contribution of platelets to the process, is still unclear. Here we report that platelets regulate the expression of (1 human equilibrative nucleoside transporter 1 (hENT1 and (2 cytidine deaminase (CDD, markers of gemcitabine resistance in pancreatic cancer. Human ENT1 (hENT1 is known to enable cellular uptake of gemcitabine while CDD deactivates gemcitabine. Knockdown experiments demonstrate that Slug, a mesenchymal transcriptional factor known to be upregulated during EMT, regulates the expression of hENT1 and CDD. Furthermore, we demonstrate that platelet-derived ADP and ATP regulate Slug and CDD expression in pancreatic cancer cells. Finally, we demonstrate that pancreatic cancer cells express the purinergic receptor P2Y12, an ADP receptor found mainly on platelets. Thus ticagrelor, a P2Y12 inhibitor, was used to examine the potential therapeutic effect of an ADP receptor antagonist on cancer cells. Our data indicate that ticagrelor negated the survival signals initiated in cancer cells by platelet-derived ADP and ATP. In conclusion, our results demonstrate a novel role of platelets in modulating chemoresistance in pancreatic cancer. Moreover, we propose ADP/ATP receptors as additional potential drug targets for treatment of pancreatic cancer.

  4. Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells

    International Nuclear Information System (INIS)

    Kun, Ernest; Kirsten, Eva; Hakam, Alaeddin; Bauer, Pal I.; Mendeleyev, Jerome

    2008-01-01

    Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical 'switchpoint' indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells

  5. Search for compounds able to modulate fof1 atp synthase in switching from life enzyme to cell death executor

    OpenAIRE

    Zancani, Marco; et al.

    2014-01-01

    Mitochondria are the main site of energy power in eukaryotic cells. The enzyme FOF1 synthase is responsible for ATP production driven by the transmembrane proton gradient. The maintenance of a very low permeability of the inner mitochondrial membrane is crucial for this mechanism, since sudden opening of the permeability transition pore (PTP) leads to matrix swelling and outer membrane rupture, with release of proapoptotic factors. Recently, it has been suggested that dimers of AT...

  6. Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

    Science.gov (United States)

    Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

    2007-01-01

    The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures

  7. Shock wave-induced ATP release from osteosarcoma U2OS cells promotes cellular uptake and cytotoxicity of methotrexate.

    Science.gov (United States)

    Qi, Baochang; Yu, Tiecheng; Wang, Chengxue; Wang, Tiejun; Yao, Jihang; Zhang, Xiaomeng; Deng, Pengfei; Xia, Yongning; Junger, Wolfgang G; Sun, Dahui

    2016-10-03

    Osteosarcoma is the most prevalent primary malignant bone tumor, but treatment is difficult and prognosis remains poor. Recently, large-dose chemotherapy has been shown to improve outcome but this approach can cause many side effects. Minimizing the dose of chemotherapeutic drugs and optimizing their curative effects is a current goal in the management of osteosarcoma patients. In our study, trypan blue dye exclusion assay was performed to investigate the optimal conditions for the sensitization of osteosarcoma U2OS cells. Cellular uptake of the fluorophores Lucifer Yellow CH dilithium salt and Calcein was measured by qualitative and quantitative methods. Human MTX ELISA Kit and MTT assay were used to assess the outcome for osteosarcoma U2OS cells in the present of shock wave and methotrexate. To explore the mechanism, P2X7 receptor in U2OS cells was detected by immunofluorescence and the extracellular ATP levels was detected by ATP assay kit. All data were analyzed using SPSS17.0 statistical software. Comparisons were made with t test between two groups. Treatment of human osteosarcoma U2OS cells with up to 450 shock wave pulses at 7 kV or up to 200 shock wave pulses at 14 kV had little effect on cell viability. However, this shock wave treatment significantly promoted the uptake of Calcein and Lucifer Yellow CH by osteosarcoma U2OS cells. Importantly, shock wave treatment also significantly enhanced the uptake of the chemotherapy drug methotrexate and increased the rate of methotrexate-induced apoptosis. We found that shock wave treatment increased the extracellular concentration of ATP and that KN62, an inhibitor of P2X7 receptor reduced the capacity methotrexate-induced apoptosis. Our results suggest that shock wave treatment promotes methotrexate-induced apoptosis by altering cell membrane permeability in a P2X7 receptor-dependent manner. Shock wave treatment may thus represent a possible adjuvant therapy for osteosarcoma.

  8. Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells

    OpenAIRE

    Blum, Andrew E.; Corbett Walsh, B.; Dubyak, George R.

    2009-01-01

    We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755–763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion...

  9. Extracellular ATP-induced nuclear Ca{sup 2+} transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic {beta}-cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zheng; Li, Zhengzheng; Peng, Gong; Chen, Xiaoli; Yin, Wenxuan [National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd., Beijing 100101 (China); Kotlikoff, Michael I. [Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (United States); Yuan, Zeng-qiang, E-mail: zqyuan@sun5.ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd., Beijing 100101 (China); Ji, Guangju, E-mail: gj28@ibp.ac.cn [National Laboratory of Biomacromolecules, Institute of Biophysics of Chinese Academy of Sciences, 15 Datun Rd., Beijing 100101 (China)

    2009-05-01

    Extracellular ATP (eATP) induces an intracellular Ca{sup 2+} transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca{sup 2+} release from intracellular stores in mouse pancreatic {beta}-cells. Using laser scanning confocal microscopy, Ca{sup 2+} indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca{sup 2+} release in pancreatic {beta}-cell nuclei. eATP induced a higher nuclear Ca{sup 2+} transient in pancreatic {beta}-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca{sup 2+}-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca{sup 2+} transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca{sup 2+}-ATPase (PMCA) or the plasma membrane Na{sup +}/Ca{sup 2+} exchanger (NCX) by LaCl{sub 3} or by replacement of Na{sup +} with N-Methyl-Glucosamine. eATP-induced nuclear Ca{sup 2+} transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic {beta}-cells. These results indicate that eATP triggers nuclear Ca{sup 2+} transients by mobilizing a nuclear Ca{sup 2+} store via nuclear IP3Rs.

  10. Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

    Science.gov (United States)

    Chiesa, Scott T.; Trangmar, Steven J.; Ali, Leena; Lotlikar, Makrand D.; González‐Alonso, José

    2017-01-01

    New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP

  11. Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature-dependent ATP release from human blood and endothelial cells.

    Science.gov (United States)

    Kalsi, Kameljit K; Chiesa, Scott T; Trangmar, Steven J; Ali, Leena; Lotlikar, Makrand D; González-Alonso, José

    2017-02-01

    What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature-sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min -1  °C -1 and twofold, respectively, (all P tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9-fold from 33 to 39°C (P tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP signalling generators for

  12. HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

    Energy Technology Data Exchange (ETDEWEB)

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa, E-mail: nebojsa.andric@dbe.uns.ac.rs

    2015-01-01

    Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATP level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig

  13. Dose-dependent ATP depletion and cancer cell death following calcium electroporation, relative effect of calcium concentration and electric field strength

    DEFF Research Database (Denmark)

    Hansen, Emilie Louise; Sozer, Esin Bengisu; Romeo, Stefania

    2015-01-01

    death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. METHODS: In three human cell lines--H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability...... was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. RESULTS: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (p...-dependently reduced cell survival and intracellular ATP. Increasing extracellular calcium allows the use of a lower electric field. GENERAL SIGNIFICANCE: This study supports the use of calcium electroporation for treatment of cancer and possibly lowering the applied electric field in future trials....

  14. Ins(1,4,5)P3 facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells

    International Nuclear Information System (INIS)

    Sun, Jing; Ogata, Shigenori; Segawa, Masaru; Usune, Sadaharu; Zhao, Yumei; Katsuragi, Takeshi

    2010-01-01

    So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5 μg) after 3 days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1 μM inositol(1,4,5)trisphosphate (Ins(1,4,5)P 3 ) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca 2+ -removal from ICM and by adding thapsigargin, a Ca 2+ -ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P 3 receptor antagonists. Administration of 500 μM adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P 3 .

  15. Electrochemical fuel cell generator having an internal and leak tight hydrocarbon fuel reformer

    Science.gov (United States)

    Dederer, Jeffrey T.; Hager, Charles A.

    1998-01-01

    An electrochemical fuel cell generator configuration is made having a generator section which contains a plurality of axially elongated fuel cells, each cell containing a fuel electrode, air electrode, and solid oxide electrolyte between the electrodes, in which axially elongated dividers separate portions of the fuel cells from each other, and where at least one divider also reforms a reformable fuel gas mixture prior to electricity generation reactions, the at least one reformer-divider is hollow having a closed end and an open end entrance for a reformable fuel mixture to pass to the closed end of the divider and then reverse flow and pass back along the hollowed walls to be reformed, and then finally to pass as reformed fuel out of the open end of the divider to contact the fuel cells, and further where the reformer-divider is a composite structure having a gas diffusion barrier of metallic foil surrounding the external walls of the reformer-divider except at the entrance to prevent diffusion of the reformable gas mixture through the divider, and further housed in an outer insulating jacket except at the entrance to prevent short-circuiting of the fuel cells by the gas diffusion barrier.

  16. Gap-junction coupling and ATP-sensitive potassium channels in human β -cell clusters: Effects on emergent dynamics

    Science.gov (United States)

    Loppini, A.; Pedersen, M. G.; Braun, M.; Filippi, S.

    2017-09-01

    The importance of gap-junction coupling between β cells in pancreatic islets is well established in mouse. Such ultrastructural connections synchronize cellular activity, confine biological heterogeneity, and enhance insulin pulsatility. Dysfunction of coupling has been associated with diabetes and altered β -cell function. However, the role of gap junctions between human β cells is still largely unexplored. By using patch-clamp recordings of β cells from human donors, we previously estimated electrical properties of these channels by mathematical modeling of pairs of human β cells. In this work we revise our estimate by modeling triplet configurations and larger heterogeneous clusters. We find that a coupling conductance in the range 0.005 -0.020 nS/pF can reproduce experiments in almost all the simulated arrangements. We finally explore the consequence of gap-junction coupling of this magnitude between β cells with mutant variants of the ATP-sensitive potassium channels involved in some metabolic disorders and diabetic conditions, translating studies performed on rodents to the human case. Our results are finally discussed from the perspective of therapeutic strategies. In summary, modeling of more realistic clusters with more than two β cells slightly lowers our previous estimate of gap-junction conductance and gives rise to patterns that more closely resemble experimental traces.

  17. Anoctamin 1 is Apically Expressed on Thyroid Follicular Cells and Contributes to ATP- and Calcium-Activated Iodide Efflux

    Directory of Open Access Journals (Sweden)

    Carmela Iosco

    2014-08-01

    Full Text Available Background/Aims: Iodide efflux from thyroid cells into the follicular lumen is essential for the synthesis of thyroid hormones, however, the pathways mediating this transport have only been partially identified. A calcium-activated pathway of iodide efflux has long been recognized, but its molecular identity unknown. Anoctamin 1 (ANO1 is a calcium-activated chloride channel (CaCC, and this study aims to investigate its contribution to iodide fluxes in thyroid cells. Methods: RT-PCR, immunohistochemistry, and live cell imaging with the fluorescent halide biosensor YFP-H148Q/I152L were used to study the expression, localization and function of ANO1 in thyroid cells. Results: ANO1 mRNA was detected in human thyroid tissue and FRTL-5 thyrocytes, and ANO1 protein was localized to the apical membrane of follicular cells. ATP induced a transient loss of iodide from FRTL-5 cells that was dependent on the mobilization of intracellular calcium, and was inhibited by CaCC/ANO1 inhibitors and siRNA against ANO1. Calcium-activated iodide efflux was also observed in CHO cells over-expressing the Sodium Iodide Symporter (NIS and ANO1. Conclusion: ANO1 in thyrocytes functions as a calcium-activated channel mediating iodide efflux, and may contribute to the rapid delivery of iodide into the follicular lumen for the synthesis of thyroid hormones following activation by calcium-mobilizing stimuli.

  18. Cell and molecular biology of ATP-binding cassette proteins in plants.

    Science.gov (United States)

    Yazaki, Kazufumi; Shitan, Nobukazu; Sugiyama, Akifumi; Takanashi, Kojiro

    2009-01-01

    ATP-binding cassette (ABC) proteins constitute a large and diverse superfamily of membrane-bound and soluble proteins, which are involved in a wide range of biological processes in all organisms from prokaryotes to eukaryotes. Genome analyses of model plants, for example, Arabidopsis and rice, have revealed that plants have more than double numbers of this family member in their genomes compared to animals and insects. In recent years, various biochemical and physiological functions of ABC proteins in plants have been reported. Some are relevant for the defense mechanisms to biotic and abiotic stresses, whereas others are involved in the basic functions necessary for maintaining the plant life. Here, we provide an updated inventory of plant ABC proteins and summarize their tissue specificities, membrane localizations, and physiological functions.

  19. ATP-binding cassette G-subfamily transporter 2 regulates cell cycle progression and asymmetric division in mouse cardiac side population progenitor cells.

    Science.gov (United States)

    Sereti, Konstantina-Ioanna; Oikonomopoulos, Angelos; Unno, Kazumasa; Cao, Xin; Qiu, Yiling; Liao, Ronglih

    2013-01-04

    After cardiac injury, cardiac progenitor cells are acutely reduced and are replenished in part by regulated self-renewal and proliferation, which occurs through symmetric and asymmetric cellular division. Understanding the molecular cues controlling progenitor cell self-renewal and lineage commitment is critical for harnessing these cells for therapeutic regeneration. We previously have found that the cell surface ATP-binding cassette G-subfamily transporter 2 (Abcg2) influences the proliferation of cardiac side population (CSP) progenitor cells, but through unclear mechanisms. To determine the role of Abcg2 on cell cycle progression and mode of division in mouse CSP cells. Herein, using CSP cells isolated from wild-type and Abcg2 knockout mice, we found that Abcg2 regulates G1-S cell cycle transition by fluorescence ubiquitination cell cycle indicators, cell cycle-focused gene expression arrays, and confocal live-cell fluorescent microscopy. Moreover, we found that modulation of cell cycle results in transition from symmetric to asymmetric cellular division in CSP cells lacking Abcg2. Abcg2 modulates CSP cell cycle progression and asymmetric cell division, establishing a mechanistic link between this surface transporter and cardiac progenitor cell function. Greater understanding of progenitor cell biology and, in particular, the regulation of resident progenitor cell homeostasis is vital for guiding the future development of cell-based therapies for cardiac regeneration.

  20. Cellular mechanisms of fever and pasteurization: ATP depletion and RNA degradation in mouse L-cells at supra-optimal temperatures.

    Science.gov (United States)

    Lau, L C

    1978-01-01

    Supra-optimal temperatures have been found to reduce ATP concentrations within cells. This reduction depresses energy-dependent processes such as RNA synthesis, the rate of which may fall below that of its simultaneous breakdown resulting in excessive auto-digestion or cell death.

  1. Leak rate models and leak detection

    International Nuclear Information System (INIS)

    1992-01-01

    Leak detection may be carried out by a number of detection systems, but selection of the systems must be carefully adapted to the fluid state and the location of the leak in the reactor coolant system. Computer programs for the calculation of leak rates contain different models to take into account the fluid state before its entrance into the crack, and they have to be verified by experiments; agreement between experiments and calculations is generally not satisfactory for very small leak rates resulting from narrow cracks or from a closing bending moment

  2. PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

    Science.gov (United States)

    Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2015-10-15

    Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

  3. Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells.

    Science.gov (United States)

    Bachmeier, Beatrice E; Iancu, Cristina M; Killian, Peter H; Kronski, Emanuel; Mirisola, Valentina; Angelini, Giovanna; Jochum, Marianne; Nerlich, Andreas G; Pfeffer, Ulrich

    2009-12-23

    Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFkappaB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFkappaB dependent manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFkappaB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

  4. Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells

    Directory of Open Access Journals (Sweden)

    Angelini Giovanna

    2009-12-01

    Full Text Available Abstract Background Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. Results We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFκB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFκB dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFκB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. Conclusion Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

  5. Addition of granulosa cell mass to the culture medium of oocytes derived from early antral follicles increases oocyte growth, ATP content, and acetylation of H4K12.

    Science.gov (United States)

    Sugiyama, Miyako; Sumiya, Mei; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-12-01

    The main aim of the present study was to examine the hypothesis that an increase in the number of granulosa cells surrounding developing bovine oocytes results in both high ATP levels and an increase in the acetylation level of H4K12 in oocytes grown in vitro. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles (EAFs, 0.4-0.7 mm in diameter), and individually cultured on 96-well plates with or without additional granulosa cell mass that had been prepared from other OGCs. After 16 days of culture, we examined: (i) the rate of antrum formation of the OGCs; (ii) the diameter, maturation, and fertilization rate of the oocytes; and (iii) the ATP content and acetylation level of H4K12 in the oocytes grown in vitro. Granulosa cell mass added to the culture medium contributed to the development of OGCs with a higher rate of antrum formation and oocyte growth. Furthermore, the addition of granulosa cells increased the ATP content and acetylation level of H4K12 in oocytes grown in vitro compared with those developed without addition of granulosa cells. In addition, there was a positive correlation between the ATP content in oocytes grown in vitro and the number of granulosa cells in the corresponding OGCs. The results suggest that granulosa cells play a role not only in the development of OGCs and the growth of oocytes, but also in the determination of ATP content and the acetylation of H4K12 in the oocytes developed in vitro.

  6. The Cytoskeleton and ATP in Sulfur Mustard-Medicated Injury to Endothelial Cells and Keratinocytes

    National Research Council Canada - National Science Library

    Hinshaw, Daniel

    1998-01-01

    The major goal of this project is to test the hypothesis that sulfur mustard (SM)-mediated cell death in keratinocytes and endothelial cells is primarily apoptotic in nature, and that several factors...

  7. A novel approach to regulate cell membrane permeability for ATP and NADH formation in Saccharomyces cerevisiae induced by air cold plasma

    Science.gov (United States)

    Dong, Xiaoyu; Liu, Tingting; Xiong, Yuqin

    2017-02-01

    Air cold plasma has been used as a novel method for enhancing microbial fermentation. The aim of this work was to explore the effect of plasma on membrane permeability and the formation of ATP and NADH in Saccharomyces cerevisiae, so as to provide valuable information for large-scale application of plasma in the fermentation industry. Suspensions of S. cerevisiae cells were exposed to air cold plasma for 0, 1, 2, 3, 4 and 5 min, and then subjected to various analyses prior to fermentation (0 h) and at the 9 and 21 h stages of fermentation. Compared with non-exposed cells, cells exposed to plasma for 1 min exhibited a marked increase in cytoplasmic free Ca2+ concentration as a result of the significant increase in membrane potential prior to fermentation. At the same time, the ATP level in the cell suspension decreased by about 40%, resulting in a reduction of about 60% in NADH prior to culturing. However, the levels of ATP and NADH in the culture at the 9 and 21 h fermentation stages were different from the level at 0 h. Taken together, the results indicated that exposure of S. cerevisiae to air cold plasma could increase its cytoplasmic free Ca2+ concentration by improving the cell membrane potential, consequently leading to changes in ATP and NADH levels. Supported by National Natural Science Foundation of China (Nos. 21246012, 21306015 and 21476032).

  8. Inhibition of cell proliferation through an ATP-responsive co-delivery system of doxorubicin and Bcl-2 siRNA

    Science.gov (United States)

    Zhang, Jianxu; Wang, Yudi; Chen, Jiawen; Liang, Xiao; Han, Haobo; Yang, Yan; Li, Quanshun; Wang, Yanbo

    2017-01-01

    Herein, DNA duplex was constructed through the hybridization of adenosine triphosphate (ATP)-responsive aptamer and its cDNA in which GC-rich motif could be used to load doxorubicin (DOX), and then, cationic polymer PEI25K was used as a carrier to simultaneously condense DOX-Duplex and Bcl-2 siRNA to prepare the ternary nanocomplex polyethylenimine (PEI)/DOX-Duplex/siRNA. The ATP concentration gradient between the cytosol and extracellular environment could achieve the stable loading of DOX in duplex and the rapid drug release in an ATP-responsive manner. Using human prostate tumor cell line PC-3 as a model, an obvious induction of cell proliferation could be detected with a cell viability of 53.3%, which was stronger than single cargo delivery, indicating the synergistic effect between these two components. The enhanced anti-proliferative effect of ternary nanocomplex could be attributed to the improved induction of cell apoptosis in a mitochondria-mediated pathway and cell-cycle arrest at the G2 phase. Overall, the ATP-responsive nanocarrier for co-delivering DOX and Bcl-2 siRNA has been demonstrated to be a smart delivery system with favorable anti-proliferative effect, especially for solving the multidrug resistance of tumors. PMID:28740380

  9. ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

    DEFF Research Database (Denmark)

    Jorgensen, N R; Geist, S T; Civitelli, R

    1997-01-01

    mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein...... connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U...

  10. Leak detection : Principles and practice

    International Nuclear Information System (INIS)

    Rama Rao, V.V.K.

    1981-01-01

    Principles of leak detection are explained and various aspects of leak detection techniques and leak detectors are reviewed. The review covers: units for leaks and leak tightness, classification of leaks, timing of leak testing, designing for ease of leak testing of any job, methods of leak detection, their ranges of application and limitations, leak detectors, response time of leak test, minimum detectable concentration of search gas during leak tests, and validity of leak tests. Helium mass spectrometer type leak detector and technique are described in detail. Recent improvements in leak detectors and techniques, particularly mass spectrometer leak detectors using gases other than helium (e.g. hydrogen, argon) are also covered in the review. (M.G.B.)

  11. Autophagy is essential for the maintenance of amino acids and ATP levels during acute amino acid starvation in MDAMB231 cells.

    Science.gov (United States)

    Thomas, Mark; Davis, Tanja; Loos, Ben; Sishi, Balindiwe; Huisamen, Barbara; Strijdom, Hans; Engelbrecht, Anna-Mart

    2018-03-01

    Autophagy plays a major role in the adaptive metabolic response of cancer cells during adverse conditions such as nutrient deprivation. However, specific data that assess metabolite profiles in context with adenosine triphosphate (ATP) availability and cell death susceptibility remain limited. Human breast cancer cells, MDAMB231, and normal breast epithelial cells, MCF12A, were subjected to short-term amino acid starvation and the cellular apoptotic and autophagic responses assessed. The role of autophagy in the control of cellular amino acid, ATP, free fatty acid, and glucose levels during amino acid starvation were compared. We demonstrate that breast cancer cells have an increased metabolic demand contributing to significant amino acid and ATP depletion in a nutrient-poor environment. Upregulation of autophagy was important for the generation of amino acids and free fatty acids and maintenance of cellular ATP levels. In contrast to normal cells, breast cancer cells were unable to maintain the response after 12 hours of amino acid starvation. Regulation of autophagic activity in these environments had indirect consequences on cell death susceptibility. Overall, our data provide support for autophagy as an important survival mechanism capable of providing metabolic substrates when cancer cells are faced with nutrient-deprived environments. The results obtained in this study helps to expand our current knowledge on how cells respond to environmental changes; the biochemical and metabolic consequences and the physiological processes activated in response. The environmental stress applied in this study is relevant to tumour physiology, and results can be translated to cancer therapeutic and clinical research areas, ultimately assisting in the specific targeting of cancer cells while avoiding harm to normal cells. Copyright © 2018 John Wiley & Sons, Ltd.

  12. Octyl gallate reduces ATP levels and Ki67 expression leading HepG2 cells to cell cycle arrest and mitochondria-mediated apoptosis.

    Science.gov (United States)

    Lima, Kelly Goulart; Krause, Gabriele Catyana; da Silva, Elisa Feller Gonçalves; Xavier, Léder Leal; Martins, Léo Anderson Meira; Alice, Laura Manzoli; da Luz, Luiza Bueno; Gassen, Rodrigo Benedetti; Filippi-Chiela, Eduardo Cremonese; Haute, Gabriela Viegas; Garcia, Maria Claudia Rosa; Funchal, Giselle Afonso; Pedrazza, Leonardo; Reghelin, Camille Kirinus; de Oliveira, Jarbas Rodrigues

    2018-04-01

    Octyl gallate (OG) is an antioxidant that has shown anti-tumor, anti-diabetic and anti-amyloidogenic activities. Mitochondria play an important role in hepatocellular carcinoma, mainly by maintaining accelerated cellular proliferation through the production of ATP. Thus, the mitochondria may be a target for antitumor therapies. Here, we investigated the effects of OG in the hepatocarcinoma cell line (HepG2) and the mechanisms involved. We report, for the first time, that treatment with OG for 24h inhibited HepG2 cell growth by decreasing mitochondrial activity and mass, which led to the reduction of ATP levels. This reduction in the energy supply triggered a decrease in Ki67 protein expression, leading cells to cycle arrest. In addition, treatment with two doses of OG for 48h induced loss of mitochondrial functionality, mitochondrial swelling and apoptosis. Finally, we report that HepG2 cells had no resistance to treatment after multiple doses. Collectively, our findings indicate that metabolic dysregulation and Ki67 protein reduction are key events in the initial anti-proliferative action of OG, whereas mitochondrial swelling and apoptosis induction are involved in the action mechanism of OG after prolonged exposure. This suggests that OG targets mitochondria, thus representing a candidate for further research on therapies for hepatocarcinoma. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. ATP-ase positive cells in human oral mucosa transplanted to nude mice

    DEFF Research Database (Denmark)

    Dabelsteen, E; Kirkeby, S

    1981-01-01

    A model to study the differentiation of human oral epithelium in vivo utilizing transplantation of human tissue to nude mice has been described. Previous studies have described the epithelial cells in this model. In this study we demonstrate that 8 d after transplantation, Langerhans cells...

  14. NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV Nonstructural Protein 5A (NS5A via MEK/Extracellular Signal Regulated Kinase (ERK Pathway

    Directory of Open Access Journals (Sweden)

    Xuesong Gao

    2013-05-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein 5A (NS5A is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

  15. Altered Intracellular ATP Production by Activated CD4+ T-Cells in Very Preterm Infants

    Directory of Open Access Journals (Sweden)

    Giulia Aquilano

    2016-01-01

    Full Text Available Background. The neonatal immune system is not fully developed at birth; newborns have adequate lymphocytes counts but these cells lack function. Objective. To assess the activity of T-cells and the influence of the main perinatal factors in very preterm infants (birth weight < 1500 g. Design. Blood samples from 59 preterm infants (21/59 were dizygotic twins were collected at birth and at 30 days of life to measure CD4+ T-cell activity using the ImmuKnow™ assay. Fifteen healthy adults were included as a control group. Results. CD4+ T-cell activity was lower in VLBW infants compared with adults (p<0.001. Twins showed lower immune activity compared to singletons (p=0.005. Infants born vaginally showed higher CD4+ T-cell activity compared to those born by C-section (p=0.031; infants born after prolonged Premature Rupture of Membranes (pPROM showed higher CD4+ T-cell activity at birth (p=0.002 compared to infants born without pPROM. Low CD4+ T-cell activity at birth is associated with necrotizing enterocolitis (NEC in the first week of life (p=0.049. Conclusions. Preterm infants show a lack in CD4+ T-cell activity at birth. Perinatal factors such as intrauterine inflammation, mode of delivery, and zygosity can influence the adaptive immune activation capacity at birth and can contribute to exposing these infants to serious complications such as NEC.

  16. Ins(1,4,5)P{sub 3} facilitates ATP accumulation via phosphocreatine/creatine kinase in the endoplasmic reticulum extracted from MDCK cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jing [Medical Research Center, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Department of Dental Implantology, School of Stomatology, Tongji University, Shanghai 200072 (China); Ogata, Shigenori [Joint Laboratory for Frontier Medical Science, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Segawa, Masaru [Central Laboratory for Pathology and Morphology, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Usune, Sadaharu [Research Laboratory of Biodynamics, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan); Zhao, Yumei [Department of Pediatric Dentistry, School of Dentistry of Shanghai Tongji University, Shanghai 200072 (China); Katsuragi, Takeshi, E-mail: katsurag@fukuoka-u.ac.jp [Medical Research Center, School of Medicine, Fukuoka University, Fukuoka 814-0180 (Japan)

    2010-07-02

    So far, the content and accumulation of ATP in isolated endoplasmic reticulum (ER) are little understood. First, we confirmed using electron microscopic and Western blotting techniques that the samples extracted from MDCK cells are endoplasmic reticulum (ER). The amounts of ATP in the extracted ER were measured from the filtrate after a spinning down of ultrafiltration spin column packed with ER. When the ER sample (5 {mu}g) after 3 days freezing was suspended in intracellular medium (ICM), 0.1% Triton X and ultrapure water (UPW), ATP amounts from the ER with UPW were the highest and over 10 times compared with that from the control with ICM, indicating that UPW is the most effective tool in destroying the ER membrane. After a 10-min-incubation with ICM containing phosphocreatine (PCr)/creatine kinase (CK) of the fresh ER. ATP amounts in the filtrate obtained by spinning down were not changed from that in the control (no PCr/CK). However, ATP amounts in the filtrate from the second spinning down of the ER (treated with PCr/CK) suspended in UPW became over 10-fold compared with the control. When 1 {mu}M inositol(1,4,5)trisphosphate (Ins(1,4,5)P{sub 3}) was added in the incubation medium (ICM with PCr/CK), ATP amounts from the filtrate after the second spinning down were further enhanced around three times. This enhancement was almost canceled by Ca{sup 2+}-removal from ICM and by adding thapsigargin, a Ca{sup 2+}-ATPase inhibitor, but not by 2-APB and heparin, Ins(1,4,5)P{sub 3} receptor antagonists. Administration of 500 {mu}M adenosine to the incubation medium (with PCr/CK) failed to enhance the accumulation of ATP in the ER. These findings suggest that the ER originally contains ATP and ATP accumulation in the ER is promoted by PCr/CK and Ins(1,4,5)P{sub 3}.

  17. Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase.

    Science.gov (United States)

    Guzman, Juan D; Pesnot, Thomas; Barrera, Diana A; Davies, Heledd M; McMahon, Eleanor; Evangelopoulos, Dimitrios; Mortazavi, Parisa N; Munshi, Tulika; Maitra, Arundhati; Lamming, Eleanor D; Angell, Richard; Gershater, Markus C; Redmond, Joanna M; Needham, Deborah; Ward, John M; Cuca, Luis E; Hailes, Helen C; Bhakta, Sanjib

    2015-01-01

    (S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. Biomimetic Pictet-Spengler or Bischler-Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  18. Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2.

    Science.gov (United States)

    Hosokawa, Yuki; Takahashi, Hisaaki; Inoue, Akihiro; Kawabe, Yuya; Funahashi, Yu; Kameda, Kenji; Sugimoto, Kana; Yano, Hajime; Harada, Hironobu; Kohno, Shohei; Ohue, Shiro; Ohnishi, Takanori; Tanaka, Junya

    2015-06-01

    Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma. The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA. Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues. Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells. If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  20. Docosahexaenoic acid and L-Carnitine prevent ATP loss in SH-SY5Y neuroblastoma cells after exposure to silver nanoparticles.

    Science.gov (United States)

    Tan, Joey Wee-Shan; Ho, Christabel Fung-Yih; Ng, Yee-Kong; Ong, Wei-Yi

    2016-02-01

    Silver nanoparticles (AgNPs) are among the most commonly used nanomaterials, but thus far, little is known about ways to mitigate against potential toxic effects of exposure. In this study, we examined the potential effects of AgNPs on mitochondrial function and cellular ATP levels, and whether these could be prevented by treatment with docosahexaenoic acid (DHA) and L-carnitine (LC). Acute exposure of AgNPs for 1 h to SH-SY5Y cells resulted in decreased mitochondrial membrane potential, and decreased ATP and ADP levels, indicating mitochondrial damage and reduced production of ATP. Incubation of cells with DHA partially reduced, while treatment with LC and DHA completely abolished the AgNP induced decreases in ATP and ADP levels. This could be due to a LC-facilitated entry of DHA to mitochondria, for repair of damaged phospholipids. It is postulated that DHA and LC may be useful for treatment of accidental environmental exposure to AgNPs. © 2014 Wiley Periodicals, Inc.

  1. Leak detection/verification

    Energy Technology Data Exchange (ETDEWEB)

    Krhounek, V.; Zdarek, J.; Pecinka, L. [Nuclear Research Institute, Rez (Czech Republic)

    1997-04-01

    Loss of coolant accident (LOCA) experiments performed as part of a Leak Before Break (LBB) analysis are very briefly summarized. The aim of these experiments was to postulate the leak rates of the coolant. Through-wall cracks were introduced into pipes by fatigue cycling and hydraulically loaded in a test device. Measurements included coolant pressure and temperature, quantity of leaked coolant, displacement of a specimen, and acoustic emission. Small cracks were plugged with particles in the coolant during testing. It is believed that plugging will have no effect in cracks with leak rates above 35 liters per minute. The leak rate safety margin of 10 is sufficient for cracks in which the leak rate is more than 5 liters per minute.

  2. Regulation of ATP-sensitive K+ channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    International Nuclear Information System (INIS)

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1989-01-01

    The actions of somatostatin and of the phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and 86 Rb + flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K + channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive 86 Rb + efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K + channels are discussed

  3. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering

    Czech Academy of Sciences Publication Activity Database

    Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, M.; Bewersdorf, J.; Ježek, Petr

    2016-01-01

    Roč. 30, č. 5 (2016), s. 1941-1957 ISSN 0892-6638 R&D Projects: GA ČR(CZ) GA13-02033S; GA ČR GJ15-02022Y Institutional support: RVO:67985823 ; RVO:68378041 Keywords : Mitofilin * Mic60 * OPA1 * dSTORM * 3D immunocytochemistry * electron microscopy * mitochondrial cristae morphology Subject RIV: EA - Cell Biology Impact factor: 5.498, year: 2016

  4. Ultrasensitive leak detection

    International Nuclear Information System (INIS)

    Winkelman, C.R.; Davidson, H.G.

    1978-01-01

    The objective of this investigation was to develop a method of detecting leaks to a sensitivity of 1.0 x 10 -13 std/cm 3 /s in vacuum devices and to develop a qualifiable standard leak to provide system calibration at this leak rate. The development work demonstrated that minimum detectable leak rates of 6.5 x 10 -14 std/cm 3 /s and 5.5 x 10 -15 std/cm 3 /s are possible for respective analog and digital measurement modes

  5. Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.

    Science.gov (United States)

    Jiang, Shuang; Zhang, Yu; Zheng, Jin-Hua; Li, Xia; Yao, You-Li; Wu, Yan-Ling; Song, Shun-Zong; Sun, Peng; Nan, Ji-Xing; Lian, Li-Hua

    2017-03-01

    Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1

  6. Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

    Science.gov (United States)

    Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

    2017-07-01

    Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels

  7. Restoration of Impaired Metabolic Energy Balance (ATP Pool and Tube Formation Potential of Endothelial Cells under “high glucose”, Diabetic Conditions by the Bioinorganic Polymer Polyphosphate

    Directory of Open Access Journals (Sweden)

    Xiaohong Wang

    2017-11-01

    Full Text Available Micro-vascularization is a fast, energy-dependent process that is compromised by elevated glucose concentrations such as in diabetes mellitus disease. Here, we studied the effect of the physiological bioinorganic polymer, polyphosphate (polyP, on the reduced ATP content and impaired function of endothelial cells cultivated under “high glucose” (35 mM diabetes mellitus conditions concentrations. This high-energy biopolymer has been shown to provide a source of metabolic energy, stored in its phosphoanhydride bonds. We show that exposure of human umbilical vein endothelial cells (HUVEC cells to “high glucose” levels results in reduced cell viability, increased apoptotic cell death, and a decline in intracellular ATP level. As a consequence, the ability of HUVEC cells to form tube-like structures in the in vitro cell tube formation assay was almost completely abolished under “high glucose” conditions. Those cells were grown onto a physiological collagen scaffold (collagen/basement membrane extract. We demonstrate that these adverse effects of increased glucose levels can be reversed by administration of polyP to almost normal values. Using Na-polyP, complexed in a stoichiometric (molar ratio to Ca2+ ions and in the physiological concentration range between 30 and 300 µM, an almost complete restoration of the reduced ATP pool of cells exposed to “high glucose” was found, as well as a normalization of the number of apoptotic cells and energy-dependent tube formation. It is concluded that the adverse effects on endothelial cells caused by the metabolic energy imbalance at elevated glucose concentrations can be counterbalanced by polyP, potentially opening new strategies for treatment of the micro-vascular complications in diabetic patients.

  8. Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Hayoz Sébastien

    2012-05-01

    Full Text Available Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP

  9. ATP-binding and -hydrolysis activities of ALDP (ABCD1) and ALDRP (ABCD2), human peroxisomal ABC proteins, overexpressed in Sf21 cells.

    Science.gov (United States)

    Morita, Masashi; Kurisu, Mikinori; Kashiwayama, Yoshinori; Yokota, Sadaki; Imanaka, Tsuneo

    2006-09-01

    The peroxisomal ATP-binding cassette (ABC) proteins, adrenoleukodystrophy protein (ALDP, ABCD1) and ALD-related protein (ALDRP, ABCD2), were expressed in Spodoptera frugiperda 21 (Sf21) insect cells using a baculovirus-mediated expression system. Immunoelectron microscopy and subcellular fractionation revealed that the overexpressed ALDP was distributed in various subcellular organelles including mitochondria, nucleus and peroxisomes. The ALDP was not extractable with Na(2)CO(3) treatment, suggesting that it integrated into membranes. ATPase activity was detected in the membrane fraction expressing ALDP. The nucleotide-binding capacities of the expressed ALDP were estimated by the binding to ATP- or ADP-agarose. ALDP exhibited an affinity to both ADP and ATP. In contrast, ALDRP exhibited an affinity to ADP but scarcely to ATP. The ALDP in the Sf21 membrane fraction was extracted with n-dodecyl-beta-maltoside and successively purified with a chelate column. The nucleotide-binding and ATPase activities of the purified ALDP were, however, not detected. It may be that certain membranous components are required for the activity. We demonstrate for the first time that the peroxisomal ABC proteins can be expressed in Sf21 membranes maintaining their nucleotide-binding abilities and ATPase activities, and the expressed proteins will be of use for further characterization.

  10. ATP depletion and oxidative damage of hepatic cells following acute exposure to malathion in rat: beneficial role of porphyrin–fullerene nanoparticles carrying magnetic magnesium

    Directory of Open Access Journals (Sweden)

    Azam Bakhtiarian

    2015-06-01

    Full Text Available The objective of the present study was to investigate the role of nanocarrier of magnetic isotope of 25-Mg2+ (PMC16 in liver toxicity, ATP content and oxidative stress due to malathion (MAL exposure. PMC16 nanoparticles were administered in different doses (0.05, 0.1 and 0.2 LD50 intravenously (iv 40 minutes after a single MAL (0.25 LD50= 207 mg/kg intraperitoneal (ip injection as a complement to standard therapy of pralidoxime (PAM and atropine (AT. MgSO4 was used as another supplement for comparison with PMC16. ATP/ADP ratio, antioxidant enzymes including catalase (CAT, glutathione peroxidase (GPx, superoxide dismutase (SOD, and oxidative stress biomarker including lipid peroxidation (LPO were evaluated in liver tissue cells. Results indicated that after MAL administration, ADP/ATP ratio had a significant increase in liver tissues in comparison with control but this ratio was improved using various doses of PMC16. LPO was significantly decreased at all doses of PMC16 and MgSO4 when compared with MAL-exposed group. SOD and CAT activities significantly were increased in MAL-treated group as compared to saline group. SOD was reduced by all doses of PMC16 and CAT activity was decreased in PMC16-0.1 group. These results lead us to conclude that PMC16 and MgSO4 are so useful for protection against MAL-induced liver toxicity, ATP depletion and oxidative damages.

  11. Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

    Science.gov (United States)

    Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

    2016-03-01

    Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

  12. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    and an Advance Coupe luminometer. The investigations showed a 60 times higher response of the PCP-kit, making it more suitable for measurement of samples with low ATP content. ATP-standard dilutions prepared in tap water were stable for at least 15 months when stored frozen at -80ºC, and storage of large...... aliquots of standards increase quality control and ease daily operation. The medium (Lumin(PM) buffer, tap water or MilliQ water) for preparation of ATP-standard dilution significantly affected the rlu response of the ATP-standard dilutions (20% difference). The effect of dilution media and of sample...... characteristics can be eliminated by use of internal standard. In strongly coloured biofilm samples the measuring efficiency can be reduced with up to 85%. Extra cellular ATP made up a significant part of the total ATP (>50%) in some samples, so when only intra cellular ATP is of interest the cells need...

  13. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    /or misfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  14. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    aggregation and/or mislfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  15. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES

    International Nuclear Information System (INIS)

    FLANAGAN, J.M.; BEWLEY, M.C.

    2001-01-01

    /or misfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins

  16. Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Francis, Brian R; White, Karen H; Thorsness, Peter E

    2007-04-01

    ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

  17. miR-143 inhibits intracellular salmonella growth by targeting ATP6V1A in macrophage cells in pig.

    Science.gov (United States)

    Huang, Tinghua; Huang, Xiali; Yao, Min

    2018-04-01

    Salmonella infects many vertebrate species, and animals such as pigs can be colonized with Salmonella and become established carriers. Analyzing the roles of microRNA in intracellular proliferation is important for understanding the process of Salmonella infection. The objective of this study is to verify the regulation effect of miR-143 on ATP6V1A and its functions in the intracellular growth of Salmonella. A new miR-143 binding site was discovered in the 3' UTR of ATP6V1A using a newly developed prediction tool. The binding site was confirmed by binding site deletion assay. Real-time PCR results indicated that ATP6V1A was predominantly expressed in bone-marrow-derived macrophages, and the expression of miR-143 in different tissues was negatively correlated with ATP6V1A. The Salmonella proliferation assay showed that the expression of miR-143 could inhibit intracellular Salmonella growth in macrophages by target ATP6V1A. The results strongly suggest that miR-143 plays important regulatory roles in the development of Salmonella infection in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain.

    Science.gov (United States)

    Cintra-Francischinelli, Mariana; Caccin, Paola; Chiavegato, Angela; Pizzo, Paola; Carmignoto, Giorgio; Angulo, Yamileth; Lomonte, Bruno; Gutiérrez, José María; Montecucco, Cesare

    2010-08-10

    Myotoxins play a major role in the pathogenesis of the envenomations caused by snake bites in large parts of the world where this is a very relevant public health problem. We show here that two myotoxins that are major constituents of the venom of Bothrops asper, a deadly snake present in Latin America, induce the release of large amounts of K(+) and ATP from skeletal muscle. We also show that the released ATP amplifies the effect of the myotoxins, acting as a "danger signal," which spreads and causes further damage by acting on purinergic receptors. In addition, the release of ATP and K(+) well accounts for the pain reaction characteristic of these envenomations. As Bothrops asper myotoxins are representative of a large family of snake myotoxins with phospholipase A(2) structure, these findings are expected to be of general significance for snake bite envenomation. Moreover, they suggest potential therapeutic approaches for limiting the extent of muscle tissue damage based on antipurinergic drugs.

  19. Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

    Science.gov (United States)

    Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

    2013-07-15

    ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. PANX1 FORMS FUNCTIONAL CHANNELS IN T LYMPHOCYTES AND PARTICIPATES IN ATP-AND FASL-INDUCED CELL DEATH

    OpenAIRE

    SHOJI SANCHEZ, KENJI FABRICIO

    2012-01-01

    Numerosos procesos dentro del sistema inmune son mediados por la señalización estimulada por ATP extracelular. En linfocitos T, las respuestas gatilladas por ATP extracelular se dan principalmente por la activación de receptores purinérgicos P2X7. Además, se ha observado, que la activación de los receptores P2 conlleva al reclutamiento de hemicanales formados por la panexinal (HCs Panx1). Estos últimos son canales presentes en la membrana, y permiten el paso a iones y moléculas pequeñas, i...

  1. Automated propellant leak detection

    Science.gov (United States)

    Makel, D. B.; Jansa, E. D.; Bickmore, T. W.; Powers, W. T.

    1993-01-01

    An automated hydrogen leak detection system is being developed for earth-to-orbit rocket engine applications. The system consists of three elements, a sensor array, a signal processing unit, and a diagnostic processor. The sensor array consists of discrete solid state sensors which are located at specific potential leak sites and in potential leak zones. The signal processing unit provides excitations power for the sensors and provides analog to digital data conversion of the sensor signals. The diagnostic computer analyzes the sensor outputs to determine leak sources and magnitude. Leak data from the sensor network is interpreted using knowledge based software and displayed on-line through a graphical user interface including 3-D leak visualization. The system requirements have been developed assuming eventual application to the Space Shuttle Main Engine which requires approximately 72 measurement locations. A prototype system has been constructed to demonstrate the operational features of the system. This system includes both prototype electronics and data processing software algorithms. Experiments are in progress to evaluate system operation at conditions which simulate prelaunch and flight. The prototype system consists of a network using 16 sensors within a testbed which simulates engine components. Sensor response, orientation, and data analysis algorithms are being evaluated using calibrated leaks produced within the testbed. A prototype flight system is also under development consisting of 8 sensors and flight capable electronics with autonomous control and data recording.

  2. Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

    Science.gov (United States)

    Foertsch, Franziska; Ilchenko, Mykhailo; Heitkamp, Thomas; Noßmann, Silke; Hoffmann, Birgit; Starke, Ilka; Mrowka, Ralf; Biskup, Christoph; Börsch, Michael

    2017-02-01

    Cytochrome C oxidase and FoF1-ATP synthase constitute complex IV and V, respectively, of the five membrane-bound enzymes in mitochondria comprising the respiratory chain. These enzymes are located in the inner mitochondrial membrane (IMM), which exhibits large invaginations called cristae. According to recent electron cryotomography, FoF1-ATP synthases are located predominantly at the rim of the cristae, while cytochrome C oxidases are likely distributed in planar membrane areas of the cristae. Previous FLIM measurements (K. Busch and coworkers) of complex II and III unravelled differences in the local environment of the membrane enzymes in the cristae. Here, we tagged complex IV and V with mNeonGreen and investigated their mitochondrial nano-environment by FLIM and superresolution microscopy in living human cells. Different lifetimes and anisotropy values were found and will be discussed.

  3. Human antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial ATP synthesis in Leishmania

    NARCIS (Netherlands)

    Luque-Ortega, R.J.; van 't Hof, W.; Veerman, E.C.I.; Saugar, J.M.; Rivas, L.

    2008-01-01

    Histatin 5 (Hst5) is a human salivary antimicrobial peptide that targets fungal mitochondria. In the human parasitic protozoa Leishmania, the mitochondrial ATP production is essential, as it lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. On

  4. Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; Yan, Z.; Obšil, T.; Zemková, Hana

    2010-01-01

    Roč. 30, č. 8 (2010), s. 1251-1258 ISSN 0272-4340 R&D Projects: GA AV ČR(CZ) IAA500110910; GA AV ČR(CZ) IAA501110801 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATP-gated receptor channels * gating * ivermectin Subject RIV: ED - Physiology Impact factor: 2.423, year: 2010

  5. Leak detection by ultrasonics

    International Nuclear Information System (INIS)

    Murphy, R.V.

    1978-01-01

    The design of a remotely-operated ultrasonic leak detector is described. The detector is basically an ultrasonic microphone with parabolic reflector, amplifier, stepping motors to move the microphone, and a display unit. (E.C.B.)

  6. WRSS jumper leak assessment

    International Nuclear Information System (INIS)

    BAILEY, J.W.

    1999-01-01

    The purpose of this assessment is: (1) to assemble and document the facts associated with three recently installed jumpers which have leaked either during actual process operation or during post installation testing; (2) to describe the corrective actions taken and to identify any process improvements which need to be implemented in the Hanford jumper design and installation activities; and (3) to document WRSS jumper leak lessons learned for use by future projects and other jumper design, fabrication, and installation activities

  7. Pipeline Leak Detection Techniques

    Directory of Open Access Journals (Sweden)

    Timur Chis, Ph.D., Dipl.Eng.

    2007-01-01

    Full Text Available Leak detection systems range from simple, visual line walking and checking ones to complex arrangements of hard-ware and software. No one method is universally applicable and operating requirements dictate which method is the most cost effective. The aim of the paper is to review the basic techniques of leak detection that are currently in use. The advantages and disadvantages of each method are discussed and some indications of applicability are outlined.

  8. Pipeline Leak Detection Techniques

    OpenAIRE

    Timur Chis, Ph.D., Dipl.Eng.

    2007-01-01

    Leak detection systems range from simple, visual line walking and checking ones to complex arrangements of hard-ware and software. No one method is universally applicable and operating requirements dictate which method is the most cost effective. The aim of the paper is to review the basic techniques of leak detection that are currently in use. The advantages and disadvantages of each method are discussed and some indications of applicability are outlined.

  9. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    International Nuclear Information System (INIS)

    Rocafull, Miguel A.; Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H + ), have been reported. They include reticulum and plasma-membrane Ca 2+ -ATPases, Na + /K + -ATPase and H + /K + -ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K + -ATPase α1-isoform, H + /K + -ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K + -ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  10. Leaks in pipe networks

    Science.gov (United States)

    Pudar, Ranko S.; Liggett, James A.

    1992-01-01

    Leak detection in water-distribution systems can be accomplished by solving an inverse problem using measurements of pressure and/or flow. The problem is formulated with equivalent orifice areas of possible leaks as the unknowns. Minimization of the difference between measured and calculated heads produces a solution for the areas. The quality of the result depends on number and location of the measurements. A sensitivity matrix is key to deciding where to make measurements. Both location and magnitude of leaks are sensitive to the quantity and quality of pressure measurements and to how well the pipe friction parameters are known. The overdetermined problem (more measurements than suspected leaks) gives the best results, but some information can be derived from the underdetermined problem. The variance of leak areas, based on the quality of system characteristics and pressure data, indicates the likely accuracy of the results. The method will not substitute for more traditional leak surveys but can serve as a guide and supplement.

  11. Colchicine inhibits cationic dye uptake induced by ATP in P2X2 and P2X7 receptor-expressing cells: implications for its therapeutic action

    Science.gov (United States)

    Marques-da-Silva, C; Chaves, MM; Castro, NG; Coutinho-Silva, R; Guimaraes, MZP

    2011-01-01

    BACKGROUND AND PURPOSE The two longest C-termini of the purinergic P2X receptors occur in the P2X2 and P2X7 receptors and are thought to interact with multiple cytoplasmic proteins, among which are members of the cytoskeleton, including microtubules. In this work we asked whether disrupting the microtubule cytoskeleton might affect the functions of these receptors. EXPERIMENTAL APPROACH Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTS Disrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1β release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1β, interferon-γ and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONS Colchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action – inhibition of pore formation induced by activation of P2X receptors – which could explain some of the anti-inflammatory effects of colchicine. LINKED ARTICLE This article is commented on by Pelegrín, pp. 908–911 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01325.x PMID:21306580

  12. Impaired mitochondrial Ca2+ homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels

    International Nuclear Information System (INIS)

    Kleist-Retzow, Juergen-Christoph von; Hue-Tran Hornig-Do; Schauen, Matthias; Eckertz, Sabrina; Tuan Anh Duong Dinh; Stassen, Frank; Lottmann, Nadine; Bust, Maria; Galunska, Bistra; Wielckens, Klaus; Hein, Wolfgang; Beuth, Joseph; Braun, Jan-Matthias; Fischer, Juergen H.; Ganitkevich, Vladimir Y.; Maniura-Weber, Katharina; Wiesner, Rudolf J.

    2007-01-01

    Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca 2+ homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A > G and 3302A > G in tRNA Leu(UUR) , as well as Rho 0 cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP + ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases

  13. Leak testing using helium leak detector

    International Nuclear Information System (INIS)

    Aparicio, G.; Mathot, S.; Munoz, C.; Orlando, O.

    1997-01-01

    Most of the equipment used in the industry and particularly in the nuclear activity need to be, vacuum or pressure tight, for operative and safety requirements. These devices have to satisfy particular regulations in order to be qualified by means of operating licences. One of the most efficient system to ensure leaktightnes is using a helium leak detector with a mass spectrometer. In this paper we show the equipment and the devices employed in fuel rods fabrication for CAREM project, and some typical material defects. Operating system and the sensitivity of this method is also described. (author) [es

  14. Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

    Science.gov (United States)

    Lu, Y M; Miyazawa, K; Yamaguchi, K; Nowaki, K; Iwatsuki, H; Wakamatsu, Y; Ichikawa, N; Hashimoto, T

    2001-12-01

    T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.

  15. Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU.

    Directory of Open Access Journals (Sweden)

    Ronglan Zhao

    Full Text Available Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU. Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs, T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.

  16. Actin Polymerization and ATP Hydrolysis

    Science.gov (United States)

    Korn, Edward D.; Carlier, Marie-France; Pantaloni, Dominique

    1987-10-01

    F-actin is the major component of muscle thin filaments and, more generally, of the microfilaments of the dynamic, multifunctional cytoskeletal systems of nonmuscle eukaryotic cells. Polymeric F-actin is formed by reversible noncovalent self-association of monomeric G-actin. To understand the dynamics of microfilament systems in cells, the dynamics of polymerization of pure actin must be understood. The following model has emerged from recent work. During the polymerization process, adenosine 5'-triphosphate (ATP) that is bound to G-actin is hydrolyzed to adenosine 5'-diphosphate (ADP) that is bound to F-actin. The hydrolysis reaction occurs on the F-actin subsequent to the polymerization reaction in two steps: cleavage of ATP followed by the slower release of inorganic phosphate (Pi). As a result, at high rates of filament growth a transient cap of ATP-actin subunits exists at the ends of elongating filaments, and at steady state a stabilizing cap of ADP \\cdot Pi-actin subunits exists at the barbed ends of filaments. Cleavage of ATP results in a highly stable filament with bound ADP \\cdot Pi, and release of Pi destabilizes the filament. Thus these two steps of the hydrolytic reaction provide potential mechanisms for regulating the monomer-polymer transition.

  17. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells

    DEFF Research Database (Denmark)

    Robey, R W; Medina-Pérez, W Y; Nishiyama, K

    2001-01-01

    We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines...... overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100...... microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold...

  18. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells

    DEFF Research Database (Denmark)

    Robey, R W; Medina-Pérez, W Y; Nishiyama, K

    2001-01-01

    We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines...... overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100...... resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated...

  19. PFR evaporator leak

    International Nuclear Information System (INIS)

    Smedley, J.A.

    1975-01-01

    PFR has three heat removal circuits each one having an evaporator, superheater, reheater; all separate units. The status of the system was that circuit No 3 was steaming with 10 MW thermal nuclear power; No 1 circuit was filled with sodium but with the evaporator awaiting modification to cure gas entrainment problems already reported. The leak was in No 2 circuit and was located in the evaporator unit. The evaporator is rated at 120 M thermal at full power and as such is a large unit. The circuit was filled with both sodium and water for the first time three weeks before the conference so it was recent history being reported and therefore any figures quoted should be taken as indicative only. The history of the steam generator was that it was built at works to a very high standard and underwent all the usual tests of strength, inspection of welds and helium leak testing. The steam generator is of U tube design with a tube plate to which the boiler tubes are welded, with all the welds in one of two gas spaces. The inlet and outlet sides are separated by a baffle and the salient features are illustrated in the attached figure. The unit achieved a leak tightness better than the detection limit in the helium leak test at works. This limit was assessed as being less than an equivalent leak of 10 -6 g/s water under steam generator service conditions. However even though all the steam generator units passed this test at works a further test was carried out when the circuits had been completed. The test was carried out during commissioning after sodium filling and with the units hot. The method was to introduce a mixture of helium/ argon at 500 pounds/square inch into the water side of the steam generators and measure the helium concentration in the sodium side gas spaces of the circuit. The test lasted many days and under these conditions the sensitivity is such that a leak equivalent to somewhere between 10 -7 to 10 -6 g/s equivalent water leak could be detected, i

  20. Fuel rod leak detector

    International Nuclear Information System (INIS)

    Womack, R.E.

    1978-01-01

    A typical embodiment of the invention detects leaking fuel rods by means of a radiation detector that measures the concentration of xenon-133 ( 133 Xe) within each individual rod. A collimated detector that provides signals related to the energy of incident radiation is aligned with one of the ends of a fuel rod. A statistically significant sample of the gamma radiation (γ-rays) that characterize 133 Xe is accumulated through the detector. The data so accumulated indicates the presence of a concentration of 133 Xe appropriate to a sound fuel rod, or a significantly different concentration that reflects a leaking fuel rod

  1. SEALING SIMULATED LEAKS

    Energy Technology Data Exchange (ETDEWEB)

    Michael A. Romano

    2004-09-01

    This report details the testing equipment, procedures and results performed under Task 7.2 Sealing Simulated Leaks. In terms of our ability to seal leaks identified in the technical topical report, Analysis of Current Field Data, we were 100% successful. In regards to maintaining seal integrity after pigging operations we achieved varying degrees of success. Internal Corrosion defects proved to be the most resistant to the effects of pigging while External Corrosion proved to be the least resistant. Overall, with limitations, pressure activated sealant technology would be a viable option under the right circumstances.

  2. Suppression of PTEN/AKT signaling decreases the expression of TUBB3 and TOP2A with subsequent inhibition of cell growth and induction of apoptosis in human breast cancer MCF-7 cells via ATP and caspase-3 signaling pathways.

    Science.gov (United States)

    Yang, Zhenhua; Liu, Ying; Shi, Changzheng; Zhang, Yuqin; Lv, Rongzhao; Zhang, Rong; Wang, Qian; Wang, Yiming

    2017-02-01

    The aim of the present study was to evaluate the effects of PTEN/AKT signaling on TUBB3 and TOP2A expression and on the subsequent cell growth of human breast cancer MCF-7 cells. We found that the disease-free survival (DFS) and overall survival (OS) of breast cancer patients with TUBB3‑positive tumors were lower than these rates in the patients with TUBB3-negative tumors. Meanwhile, DFS and OS of breast cancer patients with TOP2A-positive tumors were also lower than these rates in patients with TOP2A-negative tumors. Suppression of PTEN reduced the protein expression of TUBB3 and TOP2A in MCF-7 cells. Suppression of PTEN also reduced cell proliferation and induced apoptosis and caspase-3 activity in MCF-7 cells. Moreover, an increase in ATP also reduced TUBB3 and TOP2A protein expression, reduced cell proliferation and induced apoptosis and caspase-3 activity in the MCF-7 cells following suppression of PTEN. Suppression of phosphorylation-AKT (p-AKT) reduced the protein expression of TUBB3 and TOP2A in the MCF-7 cells. Suppression of p-AKT also reduced cell proliferation and induced apoptosis and caspase-3 activity in the MCF-7 cells. Then, ATP also reduced TUBB3 and TOP2A protein expression, reduced cell proliferation and induced apoptosis and caspase-3 activity in MCF-7 cells following suppression of p-AKT. These results suggest that PTEN/AKT signaling affects the expression of TUBB3 and TOP2A reducing cell growth and inducing apoptosis of human breast cancer MCF-7 cells through ATP and caspase-3 signaling pathways. TUBB3 and TOP2A may be promising prognostic markers for the efficacy of adjuvant cisplatin-based chemotherapy.

  3. Utilization of calibrated leaks for leak detectors in industrial use

    International Nuclear Information System (INIS)

    Rubet, L.; Meurgues, J.P.

    1982-01-01

    After a brief introduction to the use of reference calibrated leaks in leak detection by helium spectrometry, the calibration of helium calibrated leaks is mentioned. Three European laboratories have carried out the calibration of several helium leaks. The determination results of these three laboraties were found to be in good agreement. The effect of temperature remains to be made more clear in the future; more frequent calibrated with three temperature figures would be desirable [fr

  4. Elevated levels of mitochonrial respiratory complexes activities and ATP production in 17-β-estradiol-induced prolactin-secretory tumor cells in male rats are inhibited by melatonin in vivo and in vitro.

    Science.gov (United States)

    Wang, Bao-Qiang; Yang, Quan-Hui; Xu, Rong-Kun; Xu, Jian-Ning

    2013-01-01

    Our earlier studies indicate that melatonin inhibits the proliferation of prolactinoma and induces apoptosis of pituitary prolactin-secreting tumor in rats. Melatonin has also been shown to induce apoptosis and to reduce the production of ATP in breast tumor cells. This study analyzed the levels of the four mitochondrial respiratory complexes and the production of ATP and also the effects of melatonin treatment of prolactinoma. In the in vivo study, mitochondria were harvested from control pituitaries or prolactinoma collected from the pituitaries of melatonin- and 17-β-estradiol (E2)-treated male rats. In the in vitro study, prolactinoma cells mitochondria were harvested. Activities of the four mitochondrial respiratory complexes were assayed using fluorometer. ATP production of prolactinoma cells was estimated using bioluminescent methods. Elevated levels of four mitochondrial respiratory complexes activities and ATP production were recorded in prolactinoma cells. Moreover, in both in vivo and in vitro studies, melatonin inhibited the activities of mitochondrial respiratory complexes and the production of ATP in prolactinoma cells. There is a link between mitochondrial function increase and tumorigenesis. Melatonin induces apoptosis of pituitary prolactin-secreting tumor of rats via the induction of mitochondrial dysfunction and inhibition of energy metabolism.

  5. The combination of tephrosin with 2-deoxy-D-glucose enhances the cytotoxicity via accelerating ATP depletion and blunting autophagy in human cancer cells.

    Science.gov (United States)

    Choi, Yunjin; Lee, Jeong-Hyung

    2011-12-01

    2-Deoxy-D-glucose (2-DG), a synthetic glucose analog that acts as a glycolytic inhibitor, is currently under clinical evaluation for targeting tumor cells. Tephrosin (TSN), a plant rotenoid, is known as an anticancer agent. In this study, we describe that the addition of TSN to 2-DG enhanced the cytotoxic activity of 2-DG against various types of cancer cells by accelerating ATP depletion and blunting autophagy. TSN increased the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The combination of TSN and 2-DG induced acceleration of intracellular ATP depletion and the drastic activation of AMP-activated protein kinase (AMPK), which resulted in the inactivation of the mammalian target of rapamycin (mTOR) pathway. Of particular interest, TSN suppressed 2-DG-induced autophagy, a cell survival process in response to nutrient deprivation. We also showed that TSN inhibited 2-DG-induced activation of elongation factor-2 kinase (eEF-2K), which has been known to regulate 2-DG-induced autophagy. Inhibition of eEF-2K by RNA interference blunted 2-DG-induced autophagy and increased the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The addition of TSN to 2-DG, however, did not enhance the cytotoxic activity of 2-DG by knockdown of eEF-2K, suggesting that inhibition of eEF-2K by tephrsoin could be a critical role in the potentiating effect of TSN on the cytotoxicity of 2-DG. Furthermore, we showed that the blunted autophagy and enhanced cytotoxicity of 2-DG was accompanied by the augmentation of apoptosis. These results show that TSN may be valuable for augmenting the therapeutic efficacy of 2-DG.

  6. Ultrasonic leak detection

    International Nuclear Information System (INIS)

    Murphy, R.V.

    1977-01-01

    A scanning ultrasonic microphone was used to detect the presence and locate the sources of hydraulic noises in piping systems in a reactor environment. The intensity changes of the noises correspond to changes of flow conditions within the system caused by throttled valves, flow rate changes, and leaks. (author)

  7. Overview of leak testing techniques

    International Nuclear Information System (INIS)

    Dutta, N.G.

    2002-01-01

    In the past ten years leak testing techniques have gained greater importance as a powerful non-destructive testing tool particularly in nuclear, space, petro-chemical, fertilizer and chemical industries. Leak for various reasons is an undesirable feature in a component or system, still leaks do occur in practice and can some-times cause significant loss in terms of both money and human life. Hence it is essential that leaks are effectively detected and remedial measures to plug the leaks are taken sufficiently in advance. Over the years more than twenty different leak test methods have been developed with different detection sensitivities. In this article commonly used leak-detection methods have been described with emphasis to Helium leak detection technique which is one of the most widely used techniques today

  8. Metal-Dependent Regulation of ATP7A and ATP7B in Fibroblast Cultures

    DEFF Research Database (Denmark)

    Lenartowicz, Malgorzata; Moos, Torben; Ogórek, Mateusz

    2016-01-01

    the expression level of the two genes at various concentrations of iron, copper, and insulin. Treating fibroblasts from controls or from individuals with MD or WD for 3 and 10 days with iron chelators revealed that iron deficiency led to increased transcript levels of both ATP7A and ATP7B. Copper deficiency...... obtained by treatment with the copper chelator led to a downregulation of ATP7A in the control fibroblasts, but surprisingly not in the WD fibroblasts. In contrast, the addition of copper led to an increased expression of ATP7A, but a decreased expression of ATP7B. Thus, whereas similar regulation patterns...... for the two genes were observed in response to iron deficiency, different responses were observed after changes in the access to copper. Mosaic fibroblast cultures from female carriers of MD treated with copper or copper chelator for 6-8 weeks led to clonal selection. Cells that express the normal ATP7A...

  9. Leak Detection: Technology and Implementation

    OpenAIRE

    Hamilton , Stuart; Charalambous, Bambos

    2013-01-01

    Ageing infrastructure and declining water resources are major concerns with a growing global population.  Controlling water loss has therefore become a priority for water utilities around the world. In order to improve efficiencies, water utilities need to apply good practices in leak detection. Leak Detection: Technology and Implementation assists water utilities with the development and implementation of leak detection programs. Leak detection and repair is one of the components of controll...

  10. High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity.

    Science.gov (United States)

    Tahara, Haruna; Matsuda, Shun; Yamamoto, Yusuke; Yoshizawa, Hiroe; Fujita, Masaharu; Katsuoka, Yasuhiro; Kasahara, Toshihiko

    2017-11-01

    Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator. We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice. The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r 2 cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC 50 values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98. From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Study of Internal and External Leaks in Tests of Anode-Supported SOFCs

    DEFF Research Database (Denmark)

    Rasmussen, Jens Foldager Bregnballe; Hendriksen, Peter Vang; Hagen, Anke

    2008-01-01

    Spectrometer were used to detect both internal (through electrolyte) and external (through seals) gas leaks. The internal gas leak through the electrolyte was quantified under different conditions, as was the external leak from the surroundings to the anode. The internal gas leak did not depend on the pressure...... difference between the anode and the cathode gas compartment, and can thus be described as diffusion driven. External leaks between the surroundings and the anode, but not the cathode gas compartment was observed. They were influenced by the pressure difference and are thus driven by both concentration...... and pressure gradients. The measured gas leaks deduced from the probe gas experiments and the total leak calculated from the deviation between the Emf defined by the gases and the cell OCV (which contains all gas leaks as well as effects of electronic leaks) were compared. Good agreement between the two...

  12. Synthetic peptides target ATP translocase of ‘Candidatus Liberibacter asiaticus’ to block ATP uptake

    Science.gov (United States)

    As an obligate intracellular pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) may act as an “energy parasite” by importing ATP from its host’s cells. We previously demonstrated that the Las translocase NttA (gb|ACX71867.1) is functional in Escherichia coli and enables the direct import of ATP/ADP...

  13. Diltiazem Reduces Mortality and Breakdown of ATP in Red Blood Cell Induced by Isoproterenol in a Freely Moving Rat Model in Vivo

    Directory of Open Access Journals (Sweden)

    Pollen K.F. Yeung

    2014-09-01

    Full Text Available The benefit of calcium channel blockers for cardiovascular prevention against heart attack and stroke has not been firmly supported. We investigated the possible cardiovascular protective effect of diltiazem (DTZ against injury induced by isoproterenol using a freely moving rat model in vivo. Sprague Dawley rats were injected subcutaneously (sc with either 5 or 10 mg/kg of DTZ, or saline as control, twice daily for five doses. One hour after the last injection, a single dose of isoproterenol (30 mg/kg was injected sc to each rat. Blood samples were collected serially for 6 h for measurement of adenine nucleotides (ATP, ADP and AMP in red blood cell (RBC by a validated HPLC. The study has shown isoproterenol induced 50% mortality and also increased RBC concentrations of AMP from 0.04 ± 0.02 to 0.29 ± 0.21 mM at the end of the experiment (p < 0.05. Treatment with 10 mg/kg of DTZ reduced mortality from 50% to <20% and attenuated the increase of RBC concentrations of AMP from +0.25 ± 0.22 in the control rats to +0.072 ± 0.092 mM (p < 0.05. The study concluded that 10 mg/kg of DTZ reduced mortality and breakdown of ATP induced by isoproterenol in rats.

  14. Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for Y(ATP) (cell mass produced per mole of ATP) at their physiological pHs

    NARCIS (Netherlands)

    Fiedler, T.; Bekker, M.; Jonsson, M.; Mehmeti, I.; Pritzschke, A.; Siemens, N.; Nes, I.; Hugenholtz, J.; Kreikemeyer, B.

    2011-01-01

    Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion

  15. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B

    1999-01-01

    Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m...... inactive at enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open......-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change...

  16. Leaking Fuel Impacts and Practices

    International Nuclear Information System (INIS)

    Hozer, Zoltan; Szabo, Peter; Somfai, Barbara; Cherubini, Marco; Aldworth, Robin; Waeckel, Nicolas; Delorme, Tim; Dickson, Raymond; Fujii, Hajime; Rey Gayo, Jose Maria; Grant, Wade; Gorzel, Andreas; Hellwig, Christian; Kamimura, Katsuichiro; Sugiyama, Tomoyuki; Klouzal, Jan; Miklos, Marek; Nagase, Fumihisa; Nilsson, Marcus; Petit, Marc; Richards, Stuart; Lundqvist Saleh, Tobias; Stepniewski, Marek; Sim, Ki Seob; ); Rehacek, Radomir; Kissane, Martin; )

    2014-01-01

    The impact of leaking fuel rods on the operation of nuclear power plants and the practices of handling leaking fuel has been reviewed by the CSNI Working Group on Fuel Safety in order to promote a better understanding on the handling of leaking fuel in power reactors, as well as to discuss and review the current practices in member countries to help in decisions on the specification of reactor operation conditions with leaking fuel rods and on the handling of leaking fuel after removal from reactor. Experts from 15 countries provided data on the handling of leaking fuel in PWR, BWR, VVER and PHWR reactor types. The review covered the operation of NPP reactors with leaking fuel, wet and dry storage and transport of leaking assemblies. The methods and applied instruments to identify leaking fuel assemblies and the repair of them were addressed in the review. Special attention was paid to the activity release from leaking rods in the reactor and under storage conditions. The consideration of leaking fuel in safety analyses on core behaviour during postulated accidents was also discussed in the review. The main conclusions of the review pointed out that the activity release from leaking fuel rods in the reactor can be handled by technological systems, or in case of failure of too many rods the reactor can be shutdown to minimize activity release. Under accident conditions and operational transients the leaking rods may produce coolant activity concentration peaks. The storage of spent leaking fuel is normally characterised by moderate release of radionuclides from the fuel. The power plants apply limits for activity concentration to limit the amount of leaking rods in the core. In different countries, the accident analyses take into consideration the potential release from leaking fuel rods in design basis accidents in different ways. Some power plants apply special tools for handling and repair of leaking assemblies and rods. The leaking rods are stored together with

  17. MPA Modulates Tight Junctions' Permeability via Midkine/PI3K Pathway in Caco-2 Cells: A Possible Mechanism of Leak-Flux Diarrhea in Organ Transplanted Patients

    Science.gov (United States)

    Khan, Niamat; Binder, Lutz; Pantakani, D. V. Krishna; Asif, Abdul R.

    2017-01-01

    Mycophenolic acid (MPA) is prescribed to prevent allograft rejection in organ transplanted patients. However, its use is sporadically linked to leak flux diarrhea and other gastrointestinal (GI) disturbances in around 75% of patients through yet unknown mechanisms. Recently, we identified Midkine as a modulator of tight junctions (TJs) permeability in MPA treated Caco-2 monolayer. In the present study, we investigated the possible involvement of Midkine dependent PI3K pathway in alteration of TJs under MPA treatment. Caco-2 cells were grown as monolayer to develop TJs and were treated for 72 h with DMSO (control) or MPA in presence and absence of Midkine inhibitor (iMDK) or PI3K inhibitors (LY/AMG). Caco-2 monolayer integrity was assessed by transepithelial electrical resistance (TEER) and FITC-dextran assays. Our functional assays showed that PI3K inhibitors (LY/AMG) can significantly inhibit the compromised TJs integrity of MPA-treated Caco-2 cells monolayer. Chromatin immunoprecipitation analyses showed a significant epigenetic activation of Midkine, PI3K, Cdx-2, and Cldn-2 genes and epigenetic repression of Cldn-1 gene after MPA treatment. The MPA-induced epigenetic alterations were further confirmed by mRNA and protein expression analysis. Collectively, our data shows that PI3K pathway as the downstream target of Midkine which in turn modulates p38MAPK and pAKT signaling to alter TJs permeability in Caco-2 cell monolayers treated with MPA. These results highlight the possible use of either Midkine or PI3K inhibitors as therapeutic agents to prevent MPA induced GI disturbances. PMID:28694783

  18. Hazardous fluid leak detector

    Science.gov (United States)

    Gray, Harold E.; McLaurin, Felder M.; Ortiz, Monico; Huth, William A.

    1996-01-01

    A device or system for monitoring for the presence of leaks from a hazardous fluid is disclosed which uses two electrodes immersed in deionized water. A gas is passed through an enclosed space in which a hazardous fluid is contained. Any fumes, vapors, etc. escaping from the containment of the hazardous fluid in the enclosed space are entrained in the gas passing through the enclosed space and transported to a closed vessel containing deionized water and two electrodes partially immersed in the deionized water. The electrodes are connected in series with a power source and a signal, whereby when a sufficient number of ions enter the water from the gas being bubbled through it (indicative of a leak), the water will begin to conduct, thereby allowing current to flow through the water from one electrode to the other electrode to complete the circuit and activate the signal.

  19. Aspects of leak detection

    International Nuclear Information System (INIS)

    Chivers, T.C.

    1997-01-01

    A requirement of a Leak before Break safety case is that the leakage from the through wall crack be detected prior to any growth leading to unacceptable failure. This paper sets out to review some recent developments in this field. It does not set out to be a comprehensive guide to all of the methods available. The discussion concentrates on acoustic emission and how the techniques can be qualified and deployed on operational plant

  20. Aspects of leak detection

    Energy Technology Data Exchange (ETDEWEB)

    Chivers, T.C. [Berkeley Technology Centre, Glos (United Kingdom)

    1997-04-01

    A requirement of a Leak before Break safety case is that the leakage from the through wall crack be detected prior to any growth leading to unacceptable failure. This paper sets out to review some recent developments in this field. It does not set out to be a comprehensive guide to all of the methods available. The discussion concentrates on acoustic emission and how the techniques can be qualified and deployed on operational plant.

  1. The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1 as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

    Directory of Open Access Journals (Sweden)

    Martin Jakab

    2014-10-01

    Full Text Available Background/Aims: The ATP12A gene codes for a non-gastric H+/K+ ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H+/K+ ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H+/K+ ATPase inhibitor SCH28080 in apoptosis. Methods: Real-time reverse-transcription PCR (qRT-PCR was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi after acute intracellular acid load (NH4Cl prepulsing. Mean cell volumes (MCV and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle, to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining and differentiation (CD86 expression. Results: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H+/K+ ATPase inhibitor SCH28080 (100 µM diminishes K+-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently

  2. The mitochondrial H(+)-ATP synthase and the lipogenic switch: new core components of metabolic reprogramming in induced pluripotent stem (iPS) cells.

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Cufi, Sílvia; Vellon, Luciano; Oliveras-Ferraros, Cristina; Menendez, Octavio J; Joven, Jorge; Lupu, Ruth; Menendez, Javier A

    2013-01-15

    AMPK agonist metformin, which endows somatic cells with a bioenergetic infrastructure that is protected against reprogramming, was found to drastically elongate fibroblast mitochondria, fully reverse the high IF1/β-F1-ATPase ratio and downregulate the ACACA/FASN lipogenic enzymes in iPS cells. The mitochondrial H(+)-ATP synthase and the ACACA/FASN-driven lipogenic switch are newly characterized as instrumental metabolic events that, by coupling the Warburg effect to anabolic metabolism, enable de-differentiation during the reprogramming of somatic cells to iPS cells.

  3. Electrophysiological evidence for an ATP-gated ion channel in the principal cells of the frog skin epithelium

    DEFF Research Database (Denmark)

    Brodin, Birger; Nielsen, Robert

    2000-01-01

    P2X receptor, Na+ absorption, Short circuit current, Cell potential, Microelectrodes, Frog skin, Cytosolic Ca2+......P2X receptor, Na+ absorption, Short circuit current, Cell potential, Microelectrodes, Frog skin, Cytosolic Ca2+...

  4. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

    DEFF Research Database (Denmark)

    Maynard, Scott; Hejl, Anne-Mette; Dinh, Thuan-Son T

    2015-01-01

    , and APE1 incision activity (in cell lysates) on a DNA substrate containing an AP site (to estimate DNA repair efficiency). RESULTS: In the PBMCs of AD patients, we found reduced basal mitochondrial oxygen consumption, reduced proton leak, higher dATP level, and lower AP endonuclease 1 activity, depending...

  5. Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    OpenAIRE

    Sander, Philip; Mostafa, Haouraa; Soboh, Ayman; Schneider, Julian M.; Pala, Andrej; Baron, Ann-Kathrin; Moepps, Barbara; Wirtz, C. Rainer; Georgieff, Michael; Schneider, Marion

    2017-01-01

    Glioblastomas (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutic options are urgently needed. A novel drug, Vacquinol-1 (Vac), a quinolone derivative, displays promising properties by inducing rapid cell death in GBM but not in non-transformed tissues. Features of this type of cell death are compatible with a process termed methuosis. Here we tested Vac on a highly malignant glioma cell line observed by long-term video microscopy. Human dental-...

  6. Leak test fitting

    Science.gov (United States)

    Pickett, P.T.

    A hollow fitting for use in gas spectrometry leak testing of conduit joints is divided into two generally symmetrical halves along the axis of the conduit. A clip may quickly and easily fasten and unfasten the halves around the conduit joint under test. Each end of the fitting is sealable with a yieldable material, such as a piece of foam rubber. An orifice is provided in a wall of the fitting for the insertion or detection of helium during testing. One half of the fitting also may be employed to test joints mounted against a surface.

  7. MASS SPECTROMETER LEAK

    Science.gov (United States)

    Shields, W.R.

    1960-10-18

    An improved valve is described for precisely regulating the flow of a sample fluid to be analyzed, such as in a mass spectrometer, where a gas sample is allowed to "leak" into an evacuated region at a very low, controlled rate. The flow regulating valve controls minute flow of gases by allowing the gas to diffuse between two mating surfaces. The structure of the valve is such as to prevent the corrosive feed gas from contacting the bellows which is employed in the operation of the valve, thus preventing deterioration of the bellows.

  8. The Role of ATP in Sleep Regulation

    Directory of Open Access Journals (Sweden)

    Sachiko eChikahisa

    2011-12-01

    Full Text Available One of the functions of sleep is to maintain energy balance in the brain. There are a variety of hypotheses related to how metabolic pathways interact with sleep/wake regulation. A major finding that demonstrates an interaction between sleep and metabolic homeostasis is the involvement of adenosine in sleep homeostasis. An accumulation of adenosine is supplied from ATP, which can act as an energy currency in the cell. Extracellularly, ATP can act as an activity-dependent signaling molecule, especially in regard to communication between neurons and glia, including astrocytes. Furthermore, the intracellular AMP/ATP ratio controls the activity of AMP-activated protein kinase (AMPK, which is a potent energy regulator and is recently reported to play a role in the regulation of sleep homeostasis. Brain ATP may support multiple functions in the regulation of the sleep/wake cycle and sleep homeostasis.

  9. Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters

    Directory of Open Access Journals (Sweden)

    David A. Rhodes

    2018-04-01

    Full Text Available Activation of human Vγ9/Vδ2 T cells by “phosphoantigens” (pAg, the microbial metabolite (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated

  10. Cholesterol transporter ATP-binding cassette A1 (ABCA1) is elevated in prion disease and affects PrPC and PrPSc concentrations in cultured cells.

    Science.gov (United States)

    Kumar, Rajeev; McClain, Denise; Young, Rebecca; Carlson, George A

    2008-06-01

    Prion diseases are transmissible neurodegenerative disorders of prion protein (PrP) conformation. Prion replication by conversion of benign PrPC isoforms into disease-specific PrPSc isoforms is intimately involved in prion disease pathogenesis and may be initiated in cholesterol-rich caveolae-like domains (CLD). Concentrations of the cholesterol transporter ATP-binding cassette A1 protein (ABCA1) are elevated in pre-clinical scrapie prion-infected mice and in prion-infected cells in vitro. Elevation of ABCA1 in prion-infected brain is not a direct consequence of local PrPSc accumulation, indeed levels of ABCA1 are comparable in brain regions that differ dramatically in the amount of PrPSc. Similarly, ABCA1 concentrations are identical in normal mice, transgenic mice overexpressing PrP and PrP knockout mice. In contrast, PrPC and PrPSc levels, but not Prnp mRNA, were increased by overexpression of ABCA1 in N2a neuroblastoma cells and scrapie prion-infected N2a cells (ScN2a). Conversely, RNAi-mediated knock down of Abca1 expression decreased the concentrations of PrPC in N2a cells and of PrPSc in ScN2a cells. These results suggest that ABCA1's effects on PrPC levels are post-translational and may reflect an increase in of PrPC stability, mediated either indirectly by increasing membrane cholesterol and CLD formation or by other functions of ABCA1. The increased supply of PrPC available for conversion would lead to increased PrPSc formation.

  11. ATP: The crucial component of secretory vesicles.

    Science.gov (United States)

    Estévez-Herrera, Judith; Domínguez, Natalia; Pardo, Marta R; González-Santana, Ayoze; Westhead, Edward W; Borges, Ricardo; Machado, José David

    2016-07-12

    The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

  12. The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Qian Liu

    2017-05-01

    Full Text Available Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA. The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

  13. The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

    Science.gov (United States)

    Liu, Qian; Wang, Xing; Qin, Juanxiu; Cheng, Sen; Yeo, Won-Sik; He, Lei; Ma, Xiaowei; Liu, Xiaoyun; Li, Min; Bae, Taeok

    2017-01-01

    Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection. PMID:28555174

  14. Caspase-independent apoptosis in Friend's erythroleukemia cells: role of mitochondrial ATP synthesis impairment in relocation of apoptosis-inducing factor and endonuclease G.

    Science.gov (United States)

    Comelli, Marina; Genero, Nadia; Mavelli, Irene

    2009-02-01

    Mitochondria have emerged as the central components of both caspase-dependent and independent apoptosis signalling pathways through release of different apoptogenic proteins. We previously documented that parental and differentiated Friend's erythroleukemia cells were induced to apoptosis by oligomycin and H(2)O(2) exposure, showing that the energy impairment occurring in both cases as a consequence of a severe mitochondrial F(0)F(1)ATPsynthase inactivation was a common early feature. Here we provide evidence for AIF and Endo G mitochondrio-nuclear relocation in both cases, as a component of caspase-independent apoptosis pathways. No detectable change in mitochondrial transmembrane potential and no variation in mitochondrial levels of Bcl-2 and Bax are observed. These results point to the osmotic rupture of the mitochondrial outer membrane as occurring in response to cell exposure to the two energy-impairing treatments under conditions preserving the mitochondrial inner membrane. A critical role of the mitochondrial F(0)F(1)ATP synthase inhibition in this process is also suggested.

  15. Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly.

    Directory of Open Access Journals (Sweden)

    Agnes Csanadi

    Full Text Available Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient's prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules '(i.e. nucleotides, aminoacids, fatty acids. In cancer, Malic enzyme (ME and ATP-citrate lyase (ACLY are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC.ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient's survival.Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite.In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.

  16. Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly.

    Science.gov (United States)

    Csanadi, Agnes; Kayser, Claudia; Donauer, Marcel; Gumpp, Vera; Aumann, Konrad; Rawluk, Justyna; Prasse, Antje; zur Hausen, Axel; Wiesemann, Sebastian; Werner, Martin; Kayser, Gian

    2015-01-01

    Lung cancer is the leading cause of death among malignancies worldwide. Understanding its biology is therefore of pivotal importance to improve patient's prognosis. In contrast to non-neoplastic tissues, cancer cells utilize glucose mainly for production of basic cellular modules '(i.e. nucleotides, aminoacids, fatty acids). In cancer, Malic enzyme (ME) and ATP-citrate lyase (ACLY) are key enzymes linking aerobic glycolysis and fatty acid synthesis and may therefore be of biological and prognostic significance in non-small cell lung cancer (NSCLC). ME and ACLY expression was analyzed in 258 NSCLC in correlation with clinico-pathological parameters including patient's survival. Though, overall expression of both enzymes correlated positively, ACLY was associated with local tumor stage, whereas ME correlated with occurrence of mediastinal lymph node metastases. Young patients overexpressing ACLY and/or ME had a significantly longer overall survival. This proved to be an independent prognostic factor. This contrasts older NSCLC patients, in whom overexpression of ACLY and/or ME appears to predict the opposite. In NSCLC, ME and ACLY show different enzyme expressions relating to local and mediastinal spread. Most important, we detected an inverse prognostic impact of ACLY and/or ME overexpression in young and elderly patients. It can therefore be expected, that treatment of NSCLC especially, if targeting metabolic pathways, requires different strategies in different age groups.

  17. Cellular ATP release in the lung and airway

    Directory of Open Access Journals (Sweden)

    Satoru Ito

    2016-11-01

    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  18. A bioluminescence ATP assay for estimating surface hydrophobicity and membrane damage of Escherichia coli cells treated with pulsed electric fields

    Science.gov (United States)

    Pulse Electric Field (PEF) treatments, a non-thermal process have been reported to injure and inactivate bacteria in liquid foods. However, the effect of this treatment on bacterial cell surface charge and hydrophobicity has not been investigated. Apple juice (AJ, pH 3.8) purchased from a wholesale ...

  19. Device provides controlled gas leaks

    Science.gov (United States)

    Kami, S. K.; King, H. J.

    1968-01-01

    Modified palladium leak device provides a controlled release /leak/ of very small quantities of gas at low or medium pressures. It has no moving parts, requires less than 5 watts to operate, and is capable of releasing the gas either continuously or in pulses at adjustable flow rates.

  20. Effects of the hypoglycaemic drugs repaglinide and glibenclamide on ATP-sensitive potassium-channels and cytosolic calcium levels in beta TC3 cells and rat pancreatic beta cells

    DEFF Research Database (Denmark)

    Gromada, J; Dissing, S; Kofod, Hans

    1995-01-01

    increase in intracellular free Ca2+ concentration ([Ca2+]i) with a half-maximal effect at 0.5 nmol/l for both drugs in long-term experiments (30 min). The rise in [Ca2+]i results from Ca2+ entry through voltage-dependent L-type Ca(2+)-channels since it is inhibited by verapamil (10 mumol/l). The effect......The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open...... probability of single ATP-sensitive K(+)-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (

  1. [ATP content in cryopreserved sperm of Siberian white cranes Grus leucogeranus (Aves: Gruiformes)].

    Science.gov (United States)

    Maksudov, G Iu; Erokhin, A S; Nesterenko, O N; Panchenko, V G

    2002-01-01

    ATP contents were studied in the native and cryoconserved sperm of Siberian white cranes Grus leucogeranus using bioluminescence analysis. The ATP content in freshly obtained spermatozoa was 12.7 nmol/10(8) cells. No ATP was found in the seminal plasma. In the process of freezing-thawing, the ATP concentration in the spermatozoa decreased by 30%. The differences in the dynamics of ATP content during cryoconservation of sperm of white cranes and other birds and mammals are discussed.

  2. Hermetic Seal Leak Detection Apparatus

    Science.gov (United States)

    Kelley, Anthony R. (Inventor)

    2013-01-01

    The present invention is a hermetic seal leak detection apparatus, which can be used to test for hermetic seal leaks in instruments and containers. A vacuum tight chamber is created around the unit being tested to minimize gas space outside of the hermetic seal. A vacuum inducing device is then used to increase the gas chamber volume inside the device, so that a slight vacuum is pulled on the unit being tested. The pressure in the unit being tested will stabilize. If the stabilized pressure reads close to a known good seal calibration, there is not a leak in the seal. If the stabilized pressure reads closer to a known bad seal calibration value, there is a leak in the seal. The speed of the plunger can be varied and by evaluating the resulting pressure change rates and final values, the leak rate/size can be accurately calculated.

  3. Ultrasonic Leak Detection System

    Science.gov (United States)

    Youngquist, Robert C. (Inventor); Moerk, J. Steven (Inventor)

    1998-01-01

    A system for detecting ultrasonic vibrations. such as those generated by a small leak in a pressurized container. vessel. pipe. or the like. comprises an ultrasonic transducer assembly and a processing circuit for converting transducer signals into an audio frequency range signal. The audio frequency range signal can be used to drive a pair of headphones worn by an operator. A diode rectifier based mixing circuit provides a simple, inexpensive way to mix the transducer signal with a square wave signal generated by an oscillator, and thereby generate the audio frequency signal. The sensitivity of the system is greatly increased through proper selection and matching of the system components. and the use of noise rejection filters and elements. In addition, a parabolic collecting horn is preferably employed which is mounted on the transducer assembly housing. The collecting horn increases sensitivity of the system by amplifying the received signals. and provides directionality which facilitates easier location of an ultrasonic vibration source.

  4. Tank car leaks gasoline

    International Nuclear Information System (INIS)

    Anon.

    1997-01-01

    On January 27, 1994, a Canadian National (CN) tank car loaded with gasoline began to leak from a crack in the tank shell on the end of the car near the stub sill. The tank car had been damaged from impact switching. A part of the tank car was sent for laboratory analysis which concluded that: (1) the fracture originated in two locations in welds, (2) the cracks propagated in a symmetrical manner and progressed into the tank plate, (3) the fracture surface revealed inadequate weld fusion. A stress analysis of the tank car was conducted to determine the coupling force necessary to cause the crack. It was noted that over the last decade several problems have occurred pertaining to stub sill areas of tank cars that have resulted in hazardous material spills. An advisory was sent to Transport Canada outlining many examples where tank cars containing serious defects had passed CN inspections that were specifically designed to identify such defects. 4 figs

  5. Expandable coating cocoon leak detection system

    Science.gov (United States)

    Hauser, R. L.; Kochansky, M. C.

    1972-01-01

    Development of system and materials for detecting leaks in cocoon protective coatings are discussed. Method of applying materials for leak determination is presented. Pressurization of system following application of materials will cause formation of bubble if leak exists.

  6. Mg(2+,ATP-dependent plasma membrane calcium pump of smooth muscle cells. ІІ. Regulation of activity

    Directory of Open Access Journals (Sweden)

    T. О. Veklich

    2015-04-01

    Full Text Available Plasma membrane Ca2+-pump is one of key proteins, which takes part in Ca2+ exchange in smooth muscle cells. It has a lot of diverse functions from control of basal cytoplasmal Ca2+ concentration to regulation of proteins involved in Ca2+-dependent signal pathway. Ca2+ pump function is often depen­dent on the isoform or even form of alternative splicing. Allowing for a variety of Ca2+-pump functions and properties, which were reviewed in detail in the first part of our review article cycle (Ukr. Biochem. J., 2015; 87(1, the precise control of the mentioned pump activity is very important for cell functioning­. The other part of this article is dedicated to different regulation factors of smooth muscle plasma membrane Ca2+-pump activity: endogenous and exo­genous, biotic and abiotic factors. Special attention is given to literature data and own results about design and the search of selective plasma membrane Ca2+-pump inhibitor which would allow examining its functioning in smooth muscle cells more meticulously.

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

    Science.gov (United States)

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

    2002-01-01

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

  8. SSME propellant path leak detection

    Science.gov (United States)

    Crawford, Roger; Shohadaee, Ahmad Ali; Powers, W. T.

    1995-01-01

    The primary objective of this phase of the investigation is the experimental validation of techniques for detecting and analyzing propellant path external leaks which have a high probability of occurring on the SSME. The selection of candidate detection methods requires a good analytic model for leak plumes which would develop from external leaks and an understanding of radiation transfer through the leak plume. One advanced propellant path leak detection technique is obtained by using state-of-art technology of infrared (IR) thermal imaging systems combined with computer, digital image processing and expert systems for the engine protection. The feasibility of the IR leak plume detection will be evaluated on subscale simulated laboratory plumes to determine sensitivity, signal to noise, and general suitability for the application. The theoretical analysis was undertaken with the objective of developing and testing simple, easy-to-use models to predict the amount of radiation coming from a radiation source, background plate (BP), which can be absorbed, emitted and scattered by the gas leaks.

  9. α-Lipoic acid ameliorates foam cell formation via liver X receptor α-dependent upregulation of ATP-binding cassette transporters A1 and G1.

    Science.gov (United States)

    Cheng, Li-Ching; Su, Kuo-Hui; Kou, Yu Ru; Shyue, Song-Kun; Ching, Li-Chieh; Yu, Yuan-Bin; Wu, Yuh-Lin; Pan, Ching-Chian; Lee, Tzong-Shyuan

    2011-01-01

    α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal

    Science.gov (United States)

    Quazi, Faraz; Molday, Robert S.

    2014-01-01

    The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration. PMID:24707049

  11. Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs.

    Science.gov (United States)

    Fiedler, Tomas; Bekker, Martijn; Jonsson, Maria; Mehmeti, Ibrahim; Pritzschke, Anja; Siemens, Nikolai; Nes, Ingolf; Hugenholtz, Jeroen; Kreikemeyer, Bernd

    2011-01-01

    Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.

  12. Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Gruenhagen, Jason Alan [Iowa State Univ., Ames, IA (United States)

    2003-01-01

    The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activated a G{sub q}-type protein to initiate ATP release from HUVECs. Ca2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K+ and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol

  13. Basis UST leak detection systems

    International Nuclear Information System (INIS)

    Silveria, V.

    1992-01-01

    This paper reports that gasoline and other petroleum products are leaking from underground storage tanks (USTs) at an alarming rate, seeping into soil and groundwater. Buried pipes are an even greater culprit, accounting for most suspected and detected leaks according to Environmental Protection Agency (EPA) estimates. In response to this problem, the EPA issued regulations setting standards for preventing, detecting, reporting, and cleaning up leaks, as well as fiscal responsibility. However, federal regulations are only a minimum; some states have cracked down even harder Plant managers and engineers have a big job ahead of them. The EPA estimates that there are more than 75,000 fuel USTs at US industrial facilities. When considering leak detection systems, the person responsible for making the decision has five primary choices: inventory reconciliation combined with regular precision tightness tests; automatic tank gauging; groundwater monitoring; interstitial monitoring of double containment systems; and vapor monitoring

  14. Leak detection with expandable coatings

    Science.gov (United States)

    1971-01-01

    Developed and evaluated is a system for leak detection that can be easily applied over separable connectors and that expands into a bubble or balloon if a leak is present. This objective is accomplished by using thin films of Parafilm tape wrapped over connectors, which are then overcoated with a special formulation. The low yield strength and the high elongation of the envelope permit bubble formation if leakage occurs. This system is appropriate for welds and other hardware besides separable connectors. The practical limit of this system appears to be for leaks exceeding 0.000001 cc/sec. If this envelope is used to trap gases for mass spectrometer inspection, leaks in the range of ten to the minus 8th power cc/sec. may be detectable.

  15. Bioluminometric assay of ATP in mouse brain

    Indian Academy of Sciences (India)

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the ...

  16. Sensors for Fluid Leak Detection

    OpenAIRE

    Martinsanz, Gonzalo Pajares

    2015-01-01

    Fluid leak detection represents a problem that has attracted the interest of researchers, but not exclusively because in industries and services leaks are frequently common. Indeed, in water or gas supplies, chemical or thermal plants, sea-lines or cooling/heating systems leakage rates can cause important economic losses and sometimes, what it is more relevant, environmental pollution with human, animal or plant lives at risk. This last issue has led to increased national and international re...

  17. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B

    1999-01-01

    -AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change......-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance....... demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost...

  18. ATP release and purinergic signaling in NLRP3 inflammasome activation

    Directory of Open Access Journals (Sweden)

    Isabelle eCOUILLIN

    2013-01-01

    Full Text Available The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns. One step- or two step- models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers ATP release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP (eATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key proinflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

  19. Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels.

    NARCIS (Netherlands)

    Graaf, A.O. de; Meijerink, J.P.P.; Heuvel, L.P.W.J. van den; Abreu, R.A. de; Witte, T.J.M. de; Jansen, J.H.; Smeitink, J.A.M.

    2002-01-01

    Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production

  20. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    NARCIS (Netherlands)

    Tjaden, J.; Haferkamp, I.; Boxma, B.; Tielens, A.G.; Huynen, M.A.; Hackstein, J.H.P.

    2004-01-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and

  1. Mitigated Transfer Line Leaks that Result in Surface Pools and Spray Leaks into Pits

    Energy Technology Data Exchange (ETDEWEB)

    HEY, B.E.

    1999-12-07

    This analysis provides radiological and toxicological consequence calculations for postulated mitigated leaks during transfers of six waste compositions. Leaks in Cleanout Boxes equipped with supplemental covers and leaks in pits are analyzed.

  2. Cell ATP level of Saccharomyces cerevisiae sensitively responds to culture growth and drug-inflicted variations in membrane integrity and PDR pump activity

    Czech Academy of Sciences Publication Activity Database

    Krasowska, A.; Lukaszewicz, M.; Bartosiewicz, D.; Sigler, Karel

    2010-01-01

    Roč. 395, č. 1 (2010), s. 51-55 ISSN 0006-291X R&D Projects: GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z50200510 Keywords : S. cerevisiae * ABC transporters * ATP level Subject RIV: EE - Microbiology, Virology Impact factor: 2.595, year: 2010

  3. Endocrine Disruptors Differentially Target ATP-Binding Cassette Transporters in the Blood-Testis Barrier and Affect Leydig Cell Testosterone Secretion In Vitro

    NARCIS (Netherlands)

    Dankers, A.C.A.; Roelofs, M.J.; Piersma, A.H.; Sweep, F.C.; Russel, F.G.M.; Berg, M. van den; Duursen, M.B. van; Masereeuw, R.

    2013-01-01

    Endocrine-disrupting chemicals (EDCs) are considered to cause testicular toxicity primarily via interference with steroid hormone function. Alternatively, EDCs could possibly exert their effects by interaction with ATP-binding cassette (ABC) transporters that are expressed in the blood-testis

  4. Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

    Science.gov (United States)

    Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

  5. Review of leak detection methods

    International Nuclear Information System (INIS)

    Choumoff, S.; Le Pavec, M.

    The development of the leak-tightness concept and the industrial sectors mainly concerned are reviewed, after which the various methods of leak detection are outlined briefly. The extent to which gases such as ammonia, freon, sulfur hexafluoride and helium are used, generally involving the use of specific detectors, leads to considerations on the principles of leak detection by tracer gas. Practical applications are discussed with regard to criteria of choice, sensitivity required and cost price. Finally the possibilities of using detection methods for industrial purposes are pointed out. They imply that equipment called on to fulfil tightness criteria can be designed to account for certain factors necessary for successful application of the detection methods chosen [fr

  6. Sensors for Fluid Leak Detection

    Directory of Open Access Journals (Sweden)

    Gonzalo Pajares Martinsanz

    2015-02-01

    Full Text Available Fluid leak detection represents a problem that has attracted the interest of researchers, but not exclusively because in industries and services leaks are frequently common. Indeed, in water or gas supplies, chemical or thermal plants, sea-lines or cooling/heating systems leakage rates can cause important economic losses and sometimes, what it is more relevant, environmental pollution with human, animal or plant lives at risk. This last issue has led to increased national and international regulations with different degrees of severity regarding environmental conservation.[...

  7. Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

    Science.gov (United States)

    Tripathi, Diwaker; Zhang, Tong; Koo, Abraham J; Stacey, Gary; Tanaka, Kiwamu

    2018-01-01

    Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis ( Arabidopsis thaliana ) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1 - 3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1 , but not in the JA biosynthesis mutant, aos , or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses. © 2018

  8. ATP and potassium ions: a deadly combination for astrocytes

    Science.gov (United States)

    Jackson, David G.; Wang, Junjie; Keane, Robert W.; Scemes, Eliana; Dahl, Gerhard

    2014-04-01

    The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

  9. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Amstrup, Jan; Henriksen, Katrine Lütken

    2003-01-01

    concentrations were higher, about 10µM, around acinar cells after cholinergic stimulation. Fluorescence of quinacrine and MANT-ATP indicated that some ATP is stored in secretory granules. Pancreatic acini have transcripts for P2X1, P2X4, P2Y2, and P2Y4 receptors, but measurements of Ca2+ signals in isolated...... acini using Fura-2 and CLSM revealed that only about 15% of acini respond to extracellular ATP or UTP. Hence, in acini only a few P2 receptors are functional and the distribution seems heterogenous. In contrast, pancreatic ducts have transcripts for P2Y2, P2Y4, P2X4, and P2X7 receptors that consistently...... increase intracellular Ca2+. Patch-clamp studies show that P2Y2/P2Y4 receptors inhibit K+ channels, and thus downregulate secretion. P2X4/P2X7 channels stimulate Na+/Ca2+ influx and may upregulate secretion by yet unknown mechanisms. Taken together, our studies show that pancreatic acini release ATP...

  10. Pipe Leak Detection Technology Development

    Science.gov (United States)

    The U. S. Environmental Protection Agency (EPA) has determined that one of the nation’s biggest infrastructural needs is the replacement or rehabilitation of the water distribution and transmission systems. The institution of more effective pipe leak detection technology will im...

  11. Detecting leaks in vacuum bags

    Science.gov (United States)

    Carlstrom, E. E.

    1980-01-01

    Small leaks in vacuum bag can be readily detected by eye, using simple chemical reaction: combination of ammonia and acetic acid vapors to produce cloudy white smoke. Technique has been successfully used to test seam integrity and to identify minute pinholes in vacuum bag used in assembly of ceramic-tile heat shield for Space Shuttle Orbiter.

  12. Stochastic Consequence Analysis for Waste Leaks

    Energy Technology Data Exchange (ETDEWEB)

    HEY, B.E.

    2000-05-31

    This analysis evaluates the radiological consequences of potential Hanford Tank Farm waste transfer leaks. These include ex-tank leaks into structures, underneath the soil, and exposed to the atmosphere. It also includes potential misroutes, tank overflow

  13. Rapid leak detection with liquid crystals

    Science.gov (United States)

    Heisman, R. M.; Iceland, W. F.; Ruppe, E. P.

    1978-01-01

    Small leaks in vacuum lines are detected by applying liquid-crystal coating, warming suspected area, and observing color change due to differential cooling by leak jet. Technique is used on inside or outside walls of vacuum-jacketed lines.

  14. Reliability of leak detection systems in LWRs

    International Nuclear Information System (INIS)

    Kupperman, D.S.

    1986-10-01

    In this paper, NRC guidelines for leak detection will be reviewed, current practices described, potential safety-related problems discussed, and potential improvements in leak detection technology (with emphasis on acoustic methods) evaluated

  15. Stochastic Consequence Analysis for Waste Leaks

    International Nuclear Information System (INIS)

    HEY, B.E.

    2000-01-01

    This analysis evaluates the radiological consequences of potential Hanford Tank Farm waste transfer leaks. These include ex-tank leaks into structures, underneath the soil, and exposed to the atmosphere. It also includes potential misroutes, tank overflow

  16. Presidential Leaks: Rhetoric and Mediated Political Knowledge.

    Science.gov (United States)

    Erickson, Keith V.

    1989-01-01

    Argues that presidential leaks constitute rhetorical acts, enabling administrations to exercise a variety of rhetorically potent options not afforded by the public forum. Proposes a typology of presidential leaks and analyzes their rhetorical functions, benefits, and liabilities. (MM)

  17. Helium leak testing methods in nuclear applications

    International Nuclear Information System (INIS)

    Ahmad, Anis

    2004-01-01

    Helium mass-spectrometer leak test is the most sensitive leak test method. It gives very reliable and sensitive test results. In last few years application of helium leak testing has gained more importance due to increased public awareness of safety and environment pollution caused by number of growing chemical and other such industries. Helium leak testing is carried out and specified in most of the critical area applications like nuclear, space, chemical and petrochemical industries

  18. Simulation of leaking fuel rods

    International Nuclear Information System (INIS)

    Hozer, Z.

    2006-01-01

    The behaviour of failed fuel rods includes several complex phenomena. The cladding failure initiates the release of fission product from the fuel and in case of large defect even urania grains can be released into the coolant. In steady state conditions an equilibrium - diffusion type - release is expected. During transients the release is driven by a convective type leaching mechanism. There are very few experimental data on leaking WWER fuel rods. For this reason the activity measurements at the nuclear power plants provide very important information. The evaluation of measured data can help in the estimation of failed fuel rod characteristics and the prediction of transient release dynamics in power plant transients. The paper deals with the simulation of leaking fuel rods under steady state and transient conditions and describes the following new results: 1) A new algorithm has been developed for the simulation of leaking fuel rods under steady state conditions and the specific parameters of the model for the Paks NPP has been determined; 2) The steady state model has been applied to calculation of leaking fuel characteristics using iodine and noble gas activity measurement data; 3) A new computational method has been developed for the simulation of leaking fuel rods under transient conditions and the specific parameters for the Paks NPP has been determined; 4) The transient model has been applied to the simulation of shutdown process at the Paks NPP and for the prediction of the time and magnitude of 123 I activity peak; 5) Using Paks NPP data a conservative value has been determined for the upper limit of the 123 I release from failed fuel rods during transients

  19. Structure of ATP-Bound Human ATP:Cobalamin Adenosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Schubert,H.; Hill, C.

    2006-01-01

    Mutations in the gene encoding human ATP:cobalamin adenosyltransferase (hATR) can result in the metabolic disorder known as methylmalonic aciduria (MMA). This enzyme catalyzes the final step in the conversion of cyanocobalamin (vitamin B{sub 12}) to the essential human cofactor adenosylcobalamin. Here we present the 2.5 {angstrom} crystal structure of ATP bound to hATR refined to an R{sub free} value of 25.2%. The enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces. Only two of the three active sites within the trimer contain the bound ATP substrate, thereby providing examples of apo- and substrate-bound-active sites within the same crystal structure. Comparison of the empty and occupied sites indicates that twenty residues at the enzyme's N-terminus become ordered upon binding of ATP to form a novel ATP-binding site and an extended cleft that likely binds cobalamin. The structure explains the role of 20 invariant residues; six are involved in ATP binding, including Arg190, which hydrogen bonds to ATP atoms on both sides of the scissile bond. Ten of the hydrogen bonds are required for structural stability, and four are in positions to interact with cobalamin. The structure also reveals how the point mutations that cause MMA are deficient in these functions.

  20. Sensitivities of Soap Solutions in Leak Detection

    Science.gov (United States)

    Stuck, D.; Lam, D. Q.; Daniels, C.

    1985-01-01

    Document describes method for determining minimum leak rate to which soap-solution leak detectors sensitive. Bubbles formed at smaller leak rates than previously assumed. In addition to presenting test results, document discusses effects of joint-flange configurations, properties of soap solutions, and correlation of test results with earlier data.

  1. Endoscopic management of bile leaks after laparoscopic ...

    African Journals Online (AJOL)

    Of 84 patients managed endoscopically, 44 had a cystic duct (CD) leak, 26 a CD leak and common bile duct (CBD) stones, and 14 a CBD injury amenable to endoscopic stenting. Of the 70 patients with CD leaks (group A), 24 underwent sphincterotomy only (including 8 stone extractions), 43 had a sphincterotomy with stent ...

  2. Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

    Science.gov (United States)

    Tang, Wenqiang; Brady, Shari R.; Sun, Yu; Muday, Gloria K.; Roux, Stanley J.

    2003-01-01

    Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.

  3. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  4. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.

    Science.gov (United States)

    Arslan, Fatih; Lai, Ruenn Chai; Smeets, Mirjam B; Akeroyd, Lars; Choo, Andre; Aguor, Eissa N E; Timmers, Leo; van Rijen, Harold V; Doevendans, Pieter A; Pasterkamp, Gerard; Lim, Sai Kiang; de Kleijn, Dominique P

    2013-05-01

    We have previously identified exosomes as the paracrine factor secreted by mesenchymal stem cells. Recently, we found that the key features of reperfusion injury, namely loss of ATP/NADH, increased oxidative stress and cell death were underpinned by proteomic deficiencies in ischemic/reperfused myocardium, and could be ameliorated by proteins in exosomes. To test this hypothesis in vivo, mice (C57Bl6/J) underwent 30 min ischemia, followed by reperfusion (I/R injury). Purified exosomes or saline was administered 5 min before reperfusion. Exosomes reduced infarct size by 45% compared to saline treatment. Langendorff experiments revealed that intact but not lysed exosomes enhanced viability of the ischemic/reperfused myocardium. Exosome treated animals exhibited significant preservation of left ventricular geometry and contractile performance during 28 days follow-up. Within an hour after reperfusion, exosome treatment increased levels of ATP and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated-GSK-3β, and reduced phosphorylated-c-JNK in ischemic/reperfused hearts. Subsequently, both local and systemic inflammation were significantly reduced 24h after reperfusion. In conclusion, our study shows that intact exosomes restore bioenergetics, reduce oxidative stress and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury. Hence, mesenchymal stem cell-derived exosomes are a potential adjuvant to reperfusion therapy for myocardial infarction. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

    Science.gov (United States)

    He, Jiuya; Ford, Holly C; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-03-28

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1 , ATP5G2 , and ATP5G3 , encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1 , ATP5G2 , and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F 1 -catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ 0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

  6. CEGB research on boiler leaks and their detection in service

    International Nuclear Information System (INIS)

    Hayes, D.J.

    1978-01-01

    The penalty in loss of output to an electricity generation organisation as a consequence of failure to deal effectively with small LMFBR boiler leaks would be large. There is therefore a considerable incentive for these organisations to satisfy themselves that proper provisions are made to ensure that both the incidence and the severity of boiler leaks are minimised. In the UK, responsibility for the research, development and design work for this and indeed for most aspects of future nuclear power plant rests with the UKAEA and NPC; nevertheless as a consequence of its 'informed operator' policy the Central Electricity Generating Board has devoted some research effort to this field in recent years. o date, research work has been put in hand with the objective of achieving an understanding of the basic behaviour of boiler leaks. In addition, attention has been given to leak detection by monitoring the sodium for increases in oxygen and hydrogen levels. In both cases leaks into liquid sodium rather than into the gas space have been considered. In the course of the work hydrogen and oxygen meters based on the galvanic cell principle have been constructed and evaluated. The former is a new device which is comparable in performance with hydrogen meters based on the ion pump. The present state of the work is briefly described in this paper

  7. Leak signature space: an original representation for robust leak location in water distribution networks

    OpenAIRE

    Casillas, Myrna V.; Garza Castañón, Luís Eduardo; Puig Cayuela, Vicenç; Vargas Martinez, Adriana

    2015-01-01

    In this paper, an original model-based scheme for leak location using pressure sensors in water distribution networks is introduced. The proposed approach is based on a new representation called the Leak Signature Space (LSS) that associates a specific signature to each leak location being minimally affected by leak magnitude. The LSS considers a linear model approximation of the relation between pressure residuals and leaks that is projected onto a selected hyperplane. This new approach allo...

  8. Materials and methods for preventing and treating anastomotic leaks

    Energy Technology Data Exchange (ETDEWEB)

    Alverdy, John C.

    2018-04-10

    Materials and methods for preventing and treating anastomotic leaks are disclosed. Data establishes that pathogenic microbes interfere with establishing epithelial cell barriers in anastomoses and, more generally, with the reconnection of any two portions of like or different tissues comprising epithelia. Suitable prophylactic and therapeutic composition comprise, e.g., a phosphorylated high molecular weight polyethylene glycol compound.

  9. Leak rate measurements and detection systems

    International Nuclear Information System (INIS)

    Kupperman, D.; Shack, W.J.; Claytor, T.

    1984-01-01

    A research program is under way at the Argonne National Laboratory under the sponsorship of the USNRC Office of Nuclear Regulatory Research to evaluate and develop improved leak detection systems. The primary focus of the work has been on acoustic emission detection of leaks. Leaks from artificial flaws, laboratory-generated IGSCCs and thermal fatigue cracks, and field-induced intergranular stress corrosion cracks (IGSCCs) from reactor piping have been examined. The effects of pressure, temperature, and leak rate and geometry on the acoustic signature are under study. The use of cross-correlation techniques for leak location and pattern recognition and autocorrelation for source discrimination is also being considered

  10. Leak testing and repair of fusion devices

    International Nuclear Information System (INIS)

    Kozman, T.A.

    1983-01-01

    The leak testing, reporting and vacuum leak repair techniques of the MFTF yin-yang number one magnet system, the world's largest superconducting magnet system, are discussed. Based on this experience, techniques will be developed for testing and repairing leaks on the 42 MFTF-B magnets. The leak-hunting techniques for the yin-yang magnet systems were applied to two helium circuits (the coil bundle and guard vacuum; both require helium flow for magnet cooldown), their associated piping, liquid nitrogen radiation shields, and piping. Additionally, during MFTF-B operation there will be warm water plasma shields and piping that require leak checking

  11. Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes.

    Science.gov (United States)

    Burkeen, Jeff F; Womac, Alisa D; Earnest, David J; Zoran, Mark J

    2011-06-08

    The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN, and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of ATP. Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Because ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca(2+) and ATP accumulation and have demonstrated that intracellular Ca(2+) levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca(2+) levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca(2+). In contrast, our finding that peak mitochondrial Ca(2+) occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca(2+) uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium, and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Together, these results demonstrate that mitochondrial Ca(2+) mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

  12. Leak detector for radioactive materials

    International Nuclear Information System (INIS)

    Masuda, Masataka; Yamaga, Satoru.

    1982-01-01

    Purpose: To improve the detection sensitivity for radioactive substances leaking from pipeways or the likes in nuclear reactors. Constitution: A monitoring center s disposed apart from a chamber for which the leakage of radioactive substances is to be detected and the chamber to be detected and the monitoring center are connected by way of pipeways through which airs from the chamber to be detected are introduced into the monitoring center by way of a suction pump. The airs thus introduced are passed through air filters to remove dusts and the radioactivity of which is measured in a first radioactivity monitoring instrument. Then, the radioactivity of the airs removed with the dusts is measured by a second instrument. Since the detection is carried out by measuring the radioactive substances released into airs not by direct detection for the leaked fluid, the detection sensitivity can be improved. (Ikeda, J.)

  13. HDMCP uncouples yeast mitochondrial respiration and alleviates steatosis in L02 and hepG2 cells by decreasing ATP and H2O2 levels: a novel mechanism for NAFLD.

    Science.gov (United States)

    Jin, Xi; Yang, Yi-da; Chen, Kun; Lv, Zhi-yuan; Zheng, Lin; Liu, Ya-ping; Chen, Shao-hua; Yu, Chao-hui; Jiang, Xue-yuan; Zhang, Cheng-yu; Li, You-ming

    2009-05-01

    To explore the uncoupling activity of hepatocelluar downregulated mitochondrial carrier protein (HDMCP) in a yeast expression system and its function in non-alcoholic fatty liver disease (NAFLD). Molecular cloning and RT-PCR were used for yeast protein expression and uncoupling activity was assessed. Western blot analysis was used to determine HDMCP level in rat NAFLD and steatotic L02 and hepG2 cell models where their presence was confirmed by pathologic (Nile red and H-E staining) and biochemical changes. RNA interference was used to knock down HDMCP level and mitochondrial ATP and hydroperoxide levels were measured for potential mechanism exploration. We found a significant GDP insensitive uncoupling activity of HDMCP in yeast mitochondria and its increased expression in animal and cell models. HDMCP was significantly increased with culture time and steatosis was aggravated when HDMCP level was knocked down. Furthermore, we found that HDMCP might function through promoting ATP depletion and decreasing H(2)O(2) production. This study adds supportive data to the hypothesis that HDMCP might be a long postulated liver-specific uncoupling protein and broadens our understanding of the pathogenesis of NAFLD. More importantly, HDMCP might become a novel drug target for its ability in alleviating hepatic steatosis.

  14. Hydrogen Leak Detection Sensor Database

    Science.gov (United States)

    Baker, Barton D.

    2010-01-01

    This slide presentation reviews the characteristics of the Hydrogen Sensor database. The database is the result of NASA's continuing interest in and improvement of its ability to detect and assess gas leaks in space applications. The database specifics and a snapshot of an entry in the database are reviewed. Attempts were made to determine the applicability of each of the 65 sensors for ground and/or vehicle use.

  15. Schlieren optics for leak detection

    Science.gov (United States)

    Peale, Robert E.; Ruffin, Alranzo B.

    1995-01-01

    The purpose of this research was to develop an optical method of leak detection. Various modifications of schlieren optics were explored with initial emphasis on leak detection of the plumbing within the orbital maneuvering system of the space shuttle (OMS pod). The schlieren scheme envisioned for OMS pod leak detection was that of a high contrast pattern on flexible reflecting material imaged onto a negative of the same pattern. We find that the OMS pod geometry constrains the characteristic length scale of the pattern to the order of 0.001 inch. Our experiments suggest that optical modulation transfer efficiency will be very low for such patterns, which will limit the sensitivity of the technique. Optical elements which allow a negative of the scene to be reversibly recorded using light from the scene itself were explored for their potential in adaptive single-ended schlieren systems. Elements studied include photochromic glass, bacteriorhodopsin, and a transmissive liquid crystal display. The dynamics of writing and reading patterns were studied using intensity profiles from recorded images. Schlieren detection of index gradients in air was demonstrated.

  16. Development of sodium leak detectors for PFBR

    International Nuclear Information System (INIS)

    Sylvia, J.I.; Rao, P. Vijayamohana; Babu, B.; Madhusoodanan, K.; Rajan, K.K.

    2012-01-01

    Highlights: ► Sodium leak detection system developed for PFBR using diverse principle. ► Miniature, remotely locatable diverse leak detector developed for Main Vessel. ► Mutual inductance type leak detectors designed and adapted for different locations. ► Sodium Ionisation detectors used for area monitoring. ► Crosswire type leak detector designed, developed and tested. - Abstract: The 500 MWe Prototype Fast Breeder Reactor (PFBR) is under advanced stage of construction at Kalpakkam near Chennai in India. The wide and high operating temperature, highly chemically active nature of sodium and its reaction with air make the sodium instrumentation complex over the conventional instrumentation. Over the years, traditional instruments such as wire type leak detectors, spark plug type leak detectors were developed and used in different sodium systems. The redundant and diverse leak detection method calls for development of special instrumentation for sodium systems which include sodium ionization (leak) detector for detecting minute sodium leak in addition to those systems based on mutual inductance principle. For detection of sodium leak from reactor Main Vessel (MV), diverse methods are used such as miniature, remotely locatable, Mutual Inductance type Leak Detector(MILD) and specially modified spark plug type leak detector. The design of MILD is suitably modified for detecting leak in double wall pipes and Diverse Safety Rod drive Mechanism (DSRDM). Steam/water leak in steam generator produces hydrogen and leads to high pressure and temperature in the system. Rupture disc is used as a safety device which punctures itself due to sudden pressure rise. To detect the discharge of sodium and its reaction products at the downstream of the rupture disc due to bursting of the rupture disc, cross wire type leak detector has been designed, developed and tested. The selection of the leak detection system depends on the location where leak has to be detected. This paper

  17. Glutamate and ATP at the Interface Between Signaling and Metabolism in Astroglia

    DEFF Research Database (Denmark)

    Parpura, Vladimir; Fisher, Elizabeth S; Lechleiter, James D

    2017-01-01

    Glutamate is the main excitatory transmitter in the brain, while ATP represents the most important energy currency in any living cell. Yet, these chemicals play an important role in both processes, enabling them with dual-acting functions in metabolic and intercellular signaling pathways. Glutamate...... can fuel ATP production, while ATP can act as a transmitter in intercellular signaling. We discuss the interface between glutamate and ATP in signaling and metabolism of astrocytes. Not only do glutamate and ATP cross each other's paths in physiology of the brain, but they also do so in its pathology...

  18. [ATP-P2X7R signalling pathway and its effects in parasitic diseases].

    Science.gov (United States)

    Wang-Fang, J; Chun-Jie, J; Fei, G; Jia-Hui, L; Ming, L

    2017-07-24

    ATP (Adenosine triphosphate) is an important endogenous damage - associated molecular pattern (DAMP). P2X 7 R is an ATP-gated cation channel. ATP-P2X 7 R plays a vital role in the pathophysiology of many diseases because P2X 7 R is distributed on various immune cells. ATP-P2X 7 R signal transduction pathway has been implicated to participate in the body's immune defense against pathogens. This paper reviews the recent progress regarding ATP-P2X 7 R and its effects on parasitic diseases.

  19. A cytotoxic Petiveria alliacea dry extract induces ATP depletion and decreases β-F1-ATPase expression in breast cancer cells and promotes survival in tumor-bearing mice

    Directory of Open Access Journals (Sweden)

    John F. Hernández

    Full Text Available Abstract Metabolic plasticity in cancer cells assures cell survival and cell proliferation under variable levels of oxygen and nutrients. Therefore, new anticancer treatments endeavor to target such plasticity by modifying main metabolic pathways as glycolysis or oxidative phosphorylation. In American traditional medicine Petiveria alliacea L., Phytolaccacea, leaf extracts have been used for leukemia and breast cancer treatments. Herein, we study cytotoxicity and antitumoral effects of P. alliacea extract in tumor/non-tumorigenic cell lines and murine breast cancer model. Breast cancer cells treated with P. alliacea dry extract showed reduction in β-F1-ATPase expression, glycolytic flux triggering diminished intracellular ATP levels, mitochondrial basal respiration and oxygen consumption. Consequently, a decline in cell proliferation was observed in conventional and three-dimension spheres breast cancer cells culture. Additionally, in vivo treatment of BALB/c mice transplanted with the murine breast cancer TS/A tumor showed that P. alliacea extract via i.p. decreases the primary tumor growth and increases survival in the TS/A model.

  20. Paeoniflorin down-regulates ATP-induced inflammatory cytokine production and P2X7R expression on peripheral blood mononuclear cells from patients with primary Sjögren's syndrome.

    Science.gov (United States)

    Yu, Jingya; Chen, Yong; Li, Mingcai; Gao, Qiaoyan; Peng, Yong; Gong, Qiongyao; Zhang, Zhen; Wu, Xiudi

    2015-09-01

    This study determined the effects of paeoniflorin (PF) on the expression of purinergic receptor P2X ligand-gated ion channel 7 (P2X7R) expressed on peripheral blood mononuclear cells (PBMCs) and production of ATP-induced pro-inflammatory cytokines released by PBMCs in patients with primary Sjögren's syndrome (pSS). The pharmacological functions and cytotoxic effects of PF were dose dependent in PBMCs from 20 newly diagnosed pSS patients and 20 normal individuals. The optimum dose of PF was 100μM. PF significantly down-regulated the production of interleukin (IL)-1β and IL-6 from pSS PBMCs, and significantly inhibited ATP-induced expression of P2X7R, that might contribute to reduced IL-1β and IL-6. mRNA and protein levels of P2X7R on pSS PBMCs were significantly higher than in normal individuals (p=0.03, pP2X7R mRNA and protein levels were decreased significantly (pP2X7R on pSS PBMCs, indicating that PF might be useful for the management of pSS via down-regulating P2X7R expression. Thus, PF may provide a new therapeutic approach to regulate P2X7R-mediated pathologic responses of pSS. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Atomic model for the dimeric FOregion of mitochondrial ATP synthase.

    Science.gov (United States)

    Guo, Hui; Bueler, Stephanie A; Rubinstein, John L

    2017-11-17

    Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded F O region turns the rotor that drives ATP synthesis in the soluble F 1 region. Although crystal structures of the F 1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the F O region. Using cryo-electron microscopy, we determined the structure of the dimeric F O complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae. Copyright © 2017, American Association for the Advancement of Science.

  2. Mesurements of intracellular ATP provide new insight into the regulation of glycolysis in the yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Ytting, Cecilie Karkov; Fuglsang, Anja Thoe; Hiltunen, J. Kalervo

    2012-01-01

    Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F0F1 ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating...... of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal...... activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose...

  3. Direct ATP photolabeling of Escherichia coli recA proteins: identification of regions required for ATP binding

    International Nuclear Information System (INIS)

    Banks, G.R.; Sedgwick, S.G.

    1986-01-01

    When the Escherichia coli RecA protein is UV irradiated in the presence of [alpha- 32 P]ATP, a labeled protein--ATP adduct is formed. All the experimental evidence indicates that, in forming such an adduct, the ATP becomes specifically immobilized in the catalytically relevant ATP binding site. The adduct can also be identified after irradiation of E. coli cell lysates in a similar manner. This direct ATP photolabeling of RecA proteins has been used to identify regions of the polypeptide chain involved in the binding of ATP. The photolabeling of a RecA protein that lacks wild-type carboxy-terminal amino acids is not detectable. A RecA protein in which the amino-terminal sequence NH2-Ala-Ile-Asp-Glu-Asn- is replaced by NH2-Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser- is only about 5% as efficiently photolabeled as the wild-type protein. Both of these RecA protein constructions, however, contain all the elements previously implicated, directly or indirectly, in the binding of ATP. ATP-photolabeled RecA protein has also been chemically cleaved at specific amino acids in order to identify regions of the polypeptide chain to which the nucleotide becomes covalently photolinked. The evidence is consistent with a region comprising amino acids 116-170. Thus, this work and that of others suggest that several disparate regions of the unfolded polypeptide chain may combine to form the ATP binding site upon protein folding or may influence binding through long-range effects

  4. Leak detection capability in CANDU reactors

    International Nuclear Information System (INIS)

    Azer, N.; Barber, D.H.; Boucher, P.J.

    1997-01-01

    This paper addresses the moisture leak detection capability of Ontario Hydro CANDU reactors which has been demonstrated by performing tests on the reactor. The tests confirmed the response of the annulus gas system (AGS) to the presence of moisture injected to simulate a pressure tube leak and also confirmed the dew point response assumed in leak before break assessments. The tests were performed on Bruce A Unit 4 by injecting known and controlled rates of heavy water vapor. To avoid condensation during test conditions, the amount of moisture which could be injected was small (2-3.5 g/hr). The test response demonstrated that the AGS is capable of detecting and annunciating small leaks. Thus confidence is provided that it would alarm for a growing pressure tube leak where the leak rate is expected to increase to kg/hr rapidly. The measured dew point response was close to that predicted by analysis

  5. Acoustic leak detection of LMFBR steam generator

    International Nuclear Information System (INIS)

    Kumagai, Hiromichi; Yoshida, Kazuo

    1993-01-01

    The development of a water leak detector with short response time for LMFBR steam generators is required to prevent the failure propagation caused by the sodium-water reaction and to maintain structural safety in steam generators. The development of an acoustic leak detector assuring short response time has attracted. The purpose of this paper is to confirm the basic detection feasibility of the active acoustic leak detector, and to investigate the leak detection method by erasing the background noise by spectrum analysis of the passive acoustic leak detector. From a comparison of the leak detection sensitivity of the active and the passive method, the active method is not influenced remarkably by the background noise, and it has possibility to detect microleakage with short response time. We anticipate a practical application of the active method in the future. (author)

  6. Leak detection capability in CANDU reactors

    Energy Technology Data Exchange (ETDEWEB)

    Azer, N.; Barber, D.H.; Boucher, P.J. [and others

    1997-04-01

    This paper addresses the moisture leak detection capability of Ontario Hydro CANDU reactors which has been demonstrated by performing tests on the reactor. The tests confirmed the response of the annulus gas system (AGS) to the presence of moisture injected to simulate a pressure tube leak and also confirmed the dew point response assumed in leak before break assessments. The tests were performed on Bruce A Unit 4 by injecting known and controlled rates of heavy water vapor. To avoid condensation during test conditions, the amount of moisture which could be injected was small (2-3.5 g/hr). The test response demonstrated that the AGS is capable of detecting and annunciating small leaks. Thus confidence is provided that it would alarm for a growing pressure tube leak where the leak rate is expected to increase to kg/hr rapidly. The measured dew point response was close to that predicted by analysis.

  7. Hermetic compartments leak-tightness enhancement

    International Nuclear Information System (INIS)

    Murani, J.

    2000-01-01

    In connection with the enhancement of the nuclear safety of the Jaslovske Bohunice V-1 NPP actions for the increase of the leak tightness are performed. The reconstruction has been done in the following directions: hermetic compartments leak tightness enhancement; air lock installation; installation of air lock in SP 4 vent system; integrated leakage rate test to hermetic compartments with leak detection. After 'major' leaks on the hermetic boundary components had been eliminated, since 1994 works on a higher qualitative level began. The essence of the works consists in the detection and identification of leaks in the structural component of the hermetic boundary during the planned refueling outages. The results of the Small Reconstruction and gradual enhancement of leak tightness are presented

  8. ATP Modifies the Proteome of Extracellular Vesicles Released by Microglia and Influences Their Action on Astrocytes

    Directory of Open Access Journals (Sweden)

    Francesco Drago

    2017-12-01

    Full Text Available Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs, which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012. However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.

  9. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Vacuum leak detector and method

    Science.gov (United States)

    Edwards, Jr., David

    1983-01-01

    Apparatus and method for detecting leakage in a vacuum system involves a moisture trap chamber connected to the vacuum system and to a pressure gauge. Moisture in the trap chamber is captured by freezing or by a moisture adsorbent to reduce the residual water vapor pressure therein to a negligible amount. The pressure gauge is then read to determine whether the vacuum system is leaky. By directing a stream of carbon dioxide or helium at potentially leaky parts of the vacuum system, the apparatus can be used with supplemental means to locate leaks.

  11. Leak detection of KNI seals

    International Nuclear Information System (INIS)

    Baranyai, G.; Peter, A.; Windberg, P.

    1990-03-01

    In Unit 3 and 4 of the Paks Nuclear Power Plant, Hungary, KNI type seals are used as lead-throughs with conical nickel sealing rings. Their failure can be critical for the operation of the reactor. An Acoustical Leak Detection System (ALDS) was constructed and tested for the operational testing of the seals. Some individual papers are presented in this collection on the calibration and testing of the ALDS intended to be placed on the top of the reactor vessels. The papers include simulation measurements of Unit 3 of NPP, laboratory experiments, evaluation of measurements, and further development needs with the ALDS. (R.P.) 50 figs.; 19 tabs

  12. Leaking mycotic abdominal aortic aneurysm

    International Nuclear Information System (INIS)

    Sing, T.M.Y.; Young, N.; O'Rourke, I.C.; Tomlinson, P.

    1994-01-01

    A case of leaking mycotic abdominal aortic aneurysm is reported, with a brief review of the literature. A 58 year old female presented with shoulder and abdominal pain associated with diarrhea, vomiting and fever with leucocytosis. Computed tomography of the abdomen showed pooling of contrast in the retroperitoneum anterior to a non-dilated abdominal aorta. There was considerable retroperitoneal blood accumulating in a mass-like lesion in the right lower abdomen and pelvis obstructing the right renal collecting system. Laparotomy revealed a 4 cm diameter saccular aneurysm of the abdominal aorta, with a 1 cm diameter neck. Culture of the thrombus grew Streptococcus pyogens. 11 refs., 2 figs

  13. Intermediate-Scale Laboratory Experiments of Subsurface Flow and Transport Resulting from Tank Leaks

    Energy Technology Data Exchange (ETDEWEB)

    Oostrom, Martinus; Wietsma, Thomas W.

    2014-09-30

    Washington River Protection Solutions contracted with Pacific Northwest National Laboratory to conduct laboratory experiments and supporting numerical simulations to improve the understanding of water flow and contaminant transport in the subsurface between waste tanks and ancillary facilities at Waste Management Area C. The work scope included two separate sets of experiments: •Small flow cell experiments to investigate the occurrence of potential unstable fingering resulting from leaks and the limitations of the STOMP (Subsurface Transport Over Multiple Phases) simulator to predict flow patterns and solute transport behavior under these conditions. Unstable infiltration may, under certain conditions, create vertically elongated fingers potentially transporting contaminants rapidly through the unsaturated zone to groundwater. The types of leak that may create deeply penetrating fingers include slow release, long duration leaks in relatively permeable porous media. Such leaks may have occurred below waste tanks at the Hanford Site. •Large flow experiments to investigate the behavior of two types of tank leaks in a simple layered system mimicking the Waste Management Area C. The investigated leaks include a relatively large leak with a short duration from a tank and a long duration leak with a relatively small leakage rate from a cascade line.

  14. Leak detection using structure-borne noise

    Science.gov (United States)

    Holland, Stephen D. (Inventor); Chimenti, Dale E. (Inventor); Roberts, Ronald A. (Inventor)

    2010-01-01

    A method for detection and location of air leaks in a pressure vessel, such as a spacecraft, includes sensing structure-borne ultrasound waveforms associated with turbulence caused by a leak from a plurality of sensors and cross correlating the waveforms to determine existence and location of the leak. Different configurations of sensors and corresponding methods can be used. An apparatus for performing the methods is also provided.

  15. Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry

    2007-01-01

    Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2......)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO...... activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell...

  16. Acoustic surveillance techniques for SGU leak monitoring

    International Nuclear Information System (INIS)

    McKnight, J.A.; Rowley, R.; Beesley, M.J.

    1990-01-01

    The paper presents a brief review of the acoustic techniques applicable to the detection of steam generator unit leaks that have been studied in the UK. Before discussion of the acoustic detection methods a reference representation of the required performance as developed in the UK is given. The conclusion is made that preliminary specification for the acoustic leak detection of sodium/water leaks in steam generating units suggests that it will be necessary to detect better than a leak rate of 3 g/s within a few seconds. 10 refs, 12 figs

  17. The pump and leak steady-state concept with a variety of regulated leak pathways

    DEFF Research Database (Denmark)

    Hoffmann, E K

    2001-01-01

    physiological significance in most cells. We found that Cl- transport in Ehrlich ascites tumor cells (EATC) was completely dominated by an exchange diffusion process, as defined by Ussing, and, thus, the Cl- conductance was much lower than previously estimated from measurements of unidirectional tracer fluxes...... found that the passive ion leaks play an active role in cell volume control. (iii) The use of isotopes and Ussing's famous flux ratio equation provided an ingenious instrument for distinguishing the various transport routes. We used this to identify the Na,K,2Cl cotransport system as accounting...... for maintaining a [Cl-]i in the EATC far above thermodynamic equilibrium, as well as accounting for the ion uptake during a regulatory volume increase (RVI) in EATC, similar to what Ussing had found in frog skin. (iv) Short-circuit current setup in the Ussing chamber is still used in laboratories around the world...

  18. Be(2+) complexing with ATP

    International Nuclear Information System (INIS)

    Dobrynina, N.A.; Dmitrieva, S.V.; Reshetova, L.N.; Smirnova, N.S.

    1997-01-01

    The equilibria in the BeSO 4 -CON and BeSO 4 -ATP-CON water solutions are studied through the method of the pH-metric titration with application of mathematical modeling. It is shown that the BeOH, Be(OH) 2 , Be(OH) 3 , BeATP, BeHATP and BeOHATP complexes with the (lgβ) formation constants equal to 9.05±0.2, 16.4±0.4, 18.54±5.2, 8.49±0.3, 7.51±0.32 correspondingly, are formed

  19. Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension

    DEFF Research Database (Denmark)

    Beuschlein, Felix; Boulkroun, Sheerazed; Osswald, Andrea

    2013-01-01

    Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na+/K+ ATPase α...... subunit) and ATP2B3 (encoding a Ca2+ ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced...... affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation...

  20. Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B

    NARCIS (Netherlands)

    Materia, Stephanie; Cater, Michael A.; Klomp, Leo W. J.; Mercer, Julian F. B.; La Fontaine, Sharon

    2011-01-01

    The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper.

  1. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    Science.gov (United States)

    Haagsma, Anna C; Driessen, Nicole N; Hahn, Marc-Manuel; Lill, Holger; Bald, Dirk

    2010-12-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhaojing [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Xu, Yonghong [Institute of Ophthalmological Research, Department of Ophthalmology, Renmin Hospital of Wuhan University, 430060 Wuhan (China); Meng, Xiangning [School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Watari, Fumio [Department of Biomedical, Dental Materials and Engineering, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586 (Japan); Liu, Hudan, E-mail: hudanliu@hust.edu.cn [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Chen, Xiao, E-mail: mornsmile@yahoo.com [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China)

    2015-01-01

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

  3. Leak testing requirements at a research facility

    International Nuclear Information System (INIS)

    Conner, J.B.

    1979-01-01

    Since September, 1952, Lawrence Livermore Laboratory has conducted pioneering research in applied science. A vital part of this activity has been the development of a variety of high vacuum and ultrahigh vacuum systems. Leaks occur in everything, including vacuum systems. The mass spectrometer leak detection equipment is described

  4. Capacitive system detects and locates fluid leaks

    Science.gov (United States)

    1966-01-01

    Electronic monitoring system automatically detects and locates minute leaks in seams of large fluid storage tanks and pipelines covered with thermal insulation. The system uses a capacitive tape-sensing element that is adhesively bonded over seams where fluid leaks are likely to occur.

  5. ISOLDE Off-line Gas Leak Upgrade

    CERN Document Server

    Nielsen, Kristoffer Bested

    2017-01-01

    This study investigates gas injection system of the ISOLDE Off-line separator. A quadrupole mass spectrometer is used to analysis the composition of the gas. Based on these measurements a contamination of the injected gas is found and a system upgrade is purposed. Furthermore a calibration of the leak rate of the leak valve is made.

  6. Leak detection for underground storage tanks

    International Nuclear Information System (INIS)

    Durgin, P.B.; Young, T.M.

    1993-01-01

    This symposium was held in New Orleans, Louisiana on January 29, 1992. The purpose of this conference was to provide a forum for exchange of state-of-the-art information on leak detection for underground storage tanks that leaked fuel. A widespread concern was protection of groundwater supplies from these leaking tanks. In some cases, the papers report on research that was conducted two or three years ago but has never been adequately directed to the underground storage tank leak-detection audience. In other cases, the papers report on the latest leak-detection research. The symposium was divided into four sessions that were entitled: Internal Monitoring; External Monitoring; Regulations and Standards; and Site and Risk Evaluation. Individual papers have been cataloged separately for inclusion in the appropriate data bases

  7. Leak Tightness of LHC Cold Vacuum Systems

    CERN Document Server

    Cruikshank, P; Maan, M; Mourier, L; Perrier-Cornet, A; Provot, N

    2011-01-01

    The cold vacuum systems of the LHC machine have been in operation since 2008. While a number of acceptable helium leaks were known to exist prior to cool down and have not significantly evolved over the last years, several new leaks have occurred which required immediate repair activities or mitigating solutions to permit operation of the LHC. The LHC vacuum system is described together with a summary and timetable of known air and helium leaks and their impact on the functioning of the cryogenic and vacuum systems. Where leaks have been investigated and repaired, the cause and failure mechanism is described. We elaborate the mitigating solutions that have been implemented to avoid degradation of known leaks and minimize their impact on cryogenic operation and LHC availability, and finally a recall of the consolidation program to be implemented in the next LHC shutdown.

  8. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants

    OpenAIRE

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded) plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs). ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling role in animals, includ...

  9. Small sodium-to-gas leak behavior in relation to LMFBR leak detection system design

    International Nuclear Information System (INIS)

    Hopenfeld, J.; Taylor, G.R.; James, L.A.

    1976-01-01

    Various aspects of sodium-to-gas leaks which must be considered in the design of leak detection systems for LMFBR's are discussed. Attention is focused primarily on small, weeping type leaks. Corrosion rates of steels in fused sodium hydroxide and corrosion damage observed at the site of small leaks lead to the conclusion that the sodium-gas reaction products could attack the primary hot leg piping at rates up to 0.08 mils per hour. Based on theoretical considerations of the corrosion mechanism and on visual observations of pipe topography following small sodium leak tests, it is concluded that pipe damage will be manifested by the formation of small detectable leaks prior to the appearance of larger leaks. The case for uniform pipe corrosion along the pipe circumference or along a vertical section of the pipe is also examined. Using a theoretical model for the gravity flow of sodium and reaction products along the pipe surface and a mass transport controlled corrosion process, it is shown that below sodium leak rates of about 30 g/hr for the primary piping corrosion damage will not extend beyond one radius distance from the leak site. A method of estimating the time delay between the initiation of such leaks and the development of a larger leak due to increased pipe stresses resulting from corrosion is presented

  10. A mathematical model for leak location and leak area determination in pipeline networks

    Directory of Open Access Journals (Sweden)

    Oyedokun O.I.

    2013-01-01

    Full Text Available Prompt leak location and leak area determination in oil and gas pipeline installations is an indispensable approach to controlling petroleum products wastages in pipes. However, there is an evident lack of literature information on this subject. In this paper, we modelled leak location detection and leak area determination in pipes by applying two methodologies and gave an illustrative example using simulated data with the aid of Matlab. A comparison of these two approaches resulted in an error of 6.24%, suggesting that the closer the leak is to the measurement station, the lower will be the time interval between two successive waves that will pass through the leak and get to the measurement station. The relationship between the pipe area and coefficient of reflection is parabolic. This contribution is valuable to pipeline engineers in the economic control of leaks.

  11. Leak Signature Space: An Original Representation for Robust Leak Location in Water Distribution Networks

    Directory of Open Access Journals (Sweden)

    Myrna V. Casillas

    2015-03-01

    Full Text Available In this paper, an original model-based scheme for leak location using pressure sensors in water distribution networks is introduced. The proposed approach is based on a new representation called the Leak Signature Space (LSS that associates a specific signature to each leak location being minimally affected by leak magnitude. The LSS considers a linear model approximation of the relation between pressure residuals and leaks that is projected onto a selected hyperplane. This new approach allows to infer the location of a given leak by comparing the position of its signature with other leak signatures. Moreover, two ways of improving the method’s robustness are proposed. First, by associating a domain of influence to each signature and second, through a time horizon analysis. The efficiency of the method is highlighted by means of a real network using several scenarios involving different number of sensors and considering the presence of noise in the measurements.

  12. Development of an ATP assay for rapid onboard testing to detect living microorganisms in ballast water

    Science.gov (United States)

    Hyun, Bonggil; Cha, Hyung-Gon; Lee, Nayoung; Yum, Seungshic; Baek, Seung Ho; Shin, Kyoungsoon

    2018-03-01

    Ballast water is a principal pathway for the introduction of pathogens and non-indigenous species to ports worldwide. The International Maritime Organization (IMO) and the United States Coast Guard (USCG) have adopted ballast water management regulations that require, e.g., the installation of shipboard ballast water management systems (BWMS). Rapid and simple analytical methods are needed to monitor whether ballast water disinfection ensures compliance with the discharge standards. In this study laboratory and full scale land-based testing was used to investigate the suitability of an adenosine triphosphate (ATP) assay for quantifying living organisms (≥ 10 and < 50 μm minimum dimension) in ballast water. In laboratory experiments the ATP assay was highly sensitive, with a detection limit of < 5 cells 0.1 mL- 1. Diatom species (Chaetoceros simplex and Skeletonema costatum) had low ATP concentrations compared with dinoflagellate, Raphidophyceae, and Chrysophyceae species. This was because of differences in cell volume, as the ATP concentration increased exponentially with increasing cell volume. Using a regression model between ATP concentration and cell volume, an estimated the pass and fail ATP concentration in this study (788-98,610 pg mL- 1) was developed for the discharge of ballast water. In land-based testing the ATP assay also showed a good correlation with the presence of living natural plankton cells in control samples, but the ATP concentration (137 pg mL- 1) was much lower than the ATP guideline. The low ATP concentration in natural plankton cells may reflect a decline in their biological activity because of extended exposure to dark conditions. Although our results need further validation, the ATP assay is a suitable tool for monitoring compliance of ballast water treatment.

  13. Rosuvastatin activates ATP-binding cassette transporter A1-dependent efflux ex vivo and promotes reverse cholesterol transport in macrophage cells in mice fed a high-fat diet.

    Science.gov (United States)

    Shimizu, Tomohiko; Miura, Shin-ichiro; Tanigawa, Hiroyuki; Kuwano, Takashi; Zhang, Bo; Uehara, Yoshinari; Saku, Keijiro

    2014-10-01

    It is controversial whether statins improve high-density lipoprotein (HDL) function, which plays an important role in reverse cholesterol transport in vivo. The aim of the present study was to clarify the effects of rosuvastatin and atorvastatin on reverse cholesterol transport in macrophage cells in vivo and their underlying mechanisms. Male C57BL mice were divided into 3 groups (rosuvastatin, atorvastatin, and control groups) and orally administered rosuvastatin, atorvastatin, or placebo for 6 weeks under feeding with a 0.5% cholesterol+10% coconut oil diet. After administration, although there were no changes in plasma HDL cholesterol levels among the groups, plasma from the rosuvastatin group showed an increased ability to promote ATP-binding cassette transporter A1-mediated cholesterol efflux ex vivo. In addition, capillary electrophoresis revealed a shift in HDL toward the pre-β HDL fraction only in the rosuvastatin group. Mice in all 3 groups were intraperitoneally injected with (3)H-cholesterol-labeled and cholesterol-loaded macrophages and then were monitored for the appearance of (3)H-tracer in plasma and feces. The amount of (3)H-tracer excreted into feces during 48 hours in the rosuvastatin group was greater than that in the control group. Finally, (3)H-cholesteryl oleate-HDL was intravenously injected into all groups, blood samples were taken, and the count of (3)H-cholesterol was analyzed. Plasma (3)H-cholesteryl oleate-HDL changed similarly, and no differences in fractional catabolic rates were observed. Rosuvastatin enhanced the ATP-binding cassette transporter A1-dependent HDL efflux function of reverse cholesterol transport, and this finding highlights the potential of rosuvastatin for the regression of atherosclerosis. © 2014 American Heart Association, Inc.

  14. ATP synthase from slow and fast growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  15. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    NARCIS (Netherlands)

    Haagsma, A.C.; Driessen, N.N.; Hahn, M.M.; Lill, H.; Bald, D.

    2010-01-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme

  16. 40 CFR Table 6 to Subpart IIIii of... - Examples of Techniques for Equipment Problem Identification, Leak Detection and Mercury Vapor

    Science.gov (United States)

    2010-07-01

    ... Problem Identification, Leak Detection and Mercury Vapor 6 Table 6 to Subpart IIIII of Part 63 Protection... Hazardous Air Pollutants: Mercury Emissions From Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII..., Leak Detection and Mercury Vapor As stated in Tables 1 and 2 of Subpart IIIII, examples of techniques...

  17. Blind Leak Detection for Closed Systems

    Science.gov (United States)

    Oelgoetz, Peter; Johnson, Ricky; Todd, Douglas; Russell, Samuel; Walker, James

    2003-01-01

    The current inspection technique for locating interstitial leaking in the Space Shuttle Main Engine nozzles is the application of a liquid leak check solution in the openings where the interstitials space between the tubing and the structural jacket vent out the aft end of the nozzle, while its cooling tubes are pressurized to 25 psig with Helium. When a leak is found, it is classified, and if the leak is severe enough the suspect tube is cut open so that a boroscope can be inserted to find the leak point. Since the boroscope can only cover a finite tube length and since it is impossible to identify which tube (to the right or left of the identified interstitial) is leaking, many extra and undesired repairs have been made to fix just one leak. In certain instances when the interstitials are interlinked by poor braze bonding, many interstitials will show indications of leaking from a single source. What is desired is a technique that can identify the leak source so that a single repair can be performed. Dr, Samuel Russell and James Walker, both with NASA/MSFC have developed a thermographic inspection system that addresses a single repair approach. They have teamed with Boeing/Rocketdyne to repackage the inspection processes to be suitable to address full scale Shuttle development and flight hardware and implement the process at NASA centers. The methods and results presented address the thermographic identification of interstitial leaks in the Space Shuttle Main Engine nozzles. A highly sensitive digital infrared camera (capable of detecting a delta temperature difference of 0.025 C) is used to record the cooling effects associated with a leak source, such as a crack or pinhole, hidden within the nozzle wall by observing the inner hot wall surface as the nozzle is pressurized, These images are enhanced by digitally subtracting a thermal reference image taken before pressurization. The method provides a non-intrusive way of locating the tube that is leaking and the

  18. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells

    NARCIS (Netherlands)

    Hinrichs, JWJ; Klappe, K; Hummel, [No Value; Kok, JW

    2004-01-01

    In this study we show that P-glycoprotein in multi-drug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multi-drug-resistant HT29(col) human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched

  19. Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

    Science.gov (United States)

    Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

    2013-01-01

    Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

  20. Imaging review of cerebrospinal fluid leaks

    Directory of Open Access Journals (Sweden)

    Naga V Vemuri

    2017-01-01

    Full Text Available Cerebrospinal fluid (CSF leak occurs due to a defect in the dura and skull base. Trauma remains the most common cause of CSF leak; however, a significant number of cases are iatrogenic, and result from a complication of functional endoscopic sinus surgery (FESS. Early diagnosis of CSF leak is of paramount importance to prevent life-threatening complications such as brain abscess and meningitis. Imaging plays a crucial role in the detection and characterization of CSF leaks. Three-dimensional, isotropic, high resolution computed tomography (HRCT accurately detects the site and size of the bony defect. CT cisternography, though invasive, helps accurately identify the site of CSF leak, especially in the presence of multiple bony defects. Magnetic resonance imaging (MRI accurately detects CSF leaks and associated complications such as the encephaloceles and meningoceles. In this review, we emphasize the importance and usefulness of 3D T2 DRIVE MR cisternography in localizing CSF leaks. This sequence has the advantages of effective bone and fat suppression, decreased artefacts, faster acquisition times, three-dimensional capability, y and high spatial resolution in addition to providing very bright signal from the CSF.

  1. Pipeline leak detection using volatile tracers

    International Nuclear Information System (INIS)

    Thompson, G.M.; Golding, R.D.

    1993-01-01

    A method of leak detection for underground storage tanks and pipelines adds volatile tracers to the products in the tanks and analyzes the surrounding shallow soil gases for tracer vapors. This method has several advantages: the success of the test is not limited by the size and structural design of the vessels, tanks can be tested at any fill level without taking the tank out of service, the location of a leak along a pipeline is clearly marked by the location of the tracer, and liquid leaks as small as 0.2 liters per hour (lph) can be detected. A limitation is: the backfill material must have some degree of air permeability in the zone above the water table. Several field tests document the success achieved using this method. A tracer leak detection system was installed at Homestead AFB after several other testing methods failed to locate a leak at a valve pit location along approximately 4 kilometers of fuel transfer piping. The leak was detected to the side of the valve pit at a depth of approximately 2.5 meters below the ground surface. Another installation of Edwards AFB involved the collection of 415 soil gas samples along approximately 3,050 meters of 15.25-centimeter fiberglass pipeline. Fourteen separate leaks were detected

  2. Successful Endoscopic Therapy of Traumatic Bile Leaks

    Directory of Open Access Journals (Sweden)

    Matthew P. Spinn

    2013-02-01

    Full Text Available Traumatic bile leaks often result in high morbidity and prolonged hospital stay that requires multimodality management. Data on endoscopic management of traumatic bile leaks are scarce. Our study objective was to evaluate the efficacy of the endoscopic management of a traumatic bile leak. We performed a retrospective case review of patients who were referred for endoscopic retrograde cholangiopancreatography (ERCP after traumatic bile duct injury secondary to blunt (motor vehicle accident or penetrating (gunshot trauma for management of bile leaks at our tertiary academic referral center. Fourteen patients underwent ERCP for the management of a traumatic bile leak over a 5-year period. The etiology included blunt trauma from motor vehicle accident in 8 patients, motorcycle accident in 3 patients and penetrating injury from a gunshot wound in 3 patients. Liver injuries were grade III in 1 patient, grade IV in 10 patients, and grade V in 3 patients. All patients were treated by biliary stent placement, and the outcome was successful in 14 of 14 cases (100%. The mean duration of follow-up was 85.6 days (range 54-175 days. There were no ERCP-related complications. In our case review, endoscopic management with endobiliary stent placement was found to be successful and resulted in resolution of the bile leak in all 14 patients. Based on our study results, ERCP should be considered as first-line therapy in the management of traumatic bile leaks.

  3. Extracellular ATP induces albuminuria in pregnant rats

    NARCIS (Netherlands)

    Faas, M.M.; van der Schaaf, G.; Borghuis, T.; Jongman, R.M.; van Pampus, Maria; de Vos, P.; van Goor, Harry; Bakker, W.W.

    BACKGROUND: As circulating plasma ATP concentrations are increased in pre-eclampsia, we tested whether increased plasma ATP is able to induce albuminuria during pregnancy. METHODS: Pregnant (day 14) and non-pregnant rats were infused with ATP (3000 microg/kg bw) via a permanent jugular vein cannula.

  4. Hazard Analysis for In Tank Spray Leaks

    International Nuclear Information System (INIS)

    GRAMS, W.H.

    2000-01-01

    The River Protection Project (RPP) Authorization Basis (AB) contains controls that address spray leaks in tanks. However, there are no hazardous conditions in the Hazards Database that specifically identify in-tank spray leak scenarios. The purpose of this Hazards Evaluation is to develop hazardous conditions related to in-tank spray leaks for the Hazards Database and to provide more complete coverage of Tank Farm facilities. Currently, the in-tank spray leak is part of the ''Spray Leak in Structures or From Waste Transfer Lines'' accidents in Section 3.4.2.9 of the Final Safety Analysis Report (FSAR) (CHG, 2000a). The accident analysis for the ''Spray Leak in Structure or From Waste Transfer Lines'' states the following regarding the location of a possible spray leak: Inside ventilated waste storage tanks (DSTs, DCRTs, and some SSTs). Aerosols could be generated inside a storage tank during a transfer because of a leak from the portion of the transfer pipe inside the tank. The tank ventilation system could help disperse the aerosols to the atmosphere should the vent system HEPA filters fail. This Hazards Evaluation also evaluates the controls currently assigned to the spray leak in structure accident and determines the applicability of the controls to the new hazardous conditions. This comparison reviews both the analysis in the FSAR and the controls found in the Technical Safety Requirements (TSRs) (CHG, 2000h). If the new hazardous conditions do not match the analyzed accident conditions and controls, then additional analysis may be required. This document is not intended to authorize the activity or determine the adequacy of controls; it is only intended to provide information about the hazardous conditions associated with this activity. The Control decision process as defined in the AB will be used to determine the adequacy of controls and whether the proposed activity is within the AB. This hazard evaluation does not constitute an accident analysis

  5. Acoustic leak detector in Monju steam generator

    International Nuclear Information System (INIS)

    Wachi, E.; Inoue, T.

    1990-01-01

    Acoustic leak detectors are equipped with the Monju steam generators for one of the R and D activities, which are the same type of the detectors developed in the PNC 50MW Steam Generator Test Facility. Although they are an additional leak detection system to the regular one in Monju SG, they would also detect the intermediate or large leaks of the SG tube failures. The extrapolation method of a background noise analysis is expected to be verified by Monju SG data. (author). 4 figs

  6. Leak detection system for RBMK coolant circuit

    International Nuclear Information System (INIS)

    Cherkashov, Ju.M.; Strelkov, B.P.; Korolev, Yu.V.; Eperin, A.P.; Kozlov, E.P.; Belyanin, L.A.; Vanukov, V.N.

    1996-01-01

    In report the description of an object of the control is submitted, requests to control of leak-tightness and functioning of system are formulated, analysis of a current status on NPP with RBMK is submitted, review of methods of the leak-tightness monitoring, their advantage and defects with reference to conditions and features of a design RBMK is indicated, some results of tests and operation of various monitoring methods are submitted, requests on interaction of operative staff, leak-tightness monitoring system and protection system of reactor are submitted. (author). 11 figs, 1 tab

  7. Diverse Functional Properties of Wilson Disease ATP7B Variants

    Science.gov (United States)

    Huster, Dominik; Kühne, Angelika; Bhattacharjee, Ashima; Raines, Lily; Jantsch, Vanessa; Noe, Johannes; Schirrmeister, Wiebke; Sommerer, Ines; Sabri, Osama; Berr, Frieder; Mössner, Joachim; Stieger, Bruno; Caca, Karel; Lutsenko, Svetlana

    2012-01-01

    BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis. PMID:22240481

  8. Excessive Extracellular ATP Desensitizes P2Y2 and P2X4 ATP Receptors Provoking Surfactant Impairment Ending in Ventilation-Induced Lung Injury

    Directory of Open Access Journals (Sweden)

    Djo Hasan

    2018-04-01

    Full Text Available Stretching the alveolar epithelial type I (AT I cells controls the intercellular signaling for the exocytosis of surfactant by the AT II cells through the extracellular release of adenosine triphosphate (ATP (purinergic signaling. Extracellular ATP is cleared by extracellular ATPases, maintaining its homeostasis and enabling the lung to adapt the exocytosis of surfactant to the demand. Vigorous deformation of the AT I cells by high mechanical power ventilation causes a massive release of extracellular ATP beyond the clearance capacity of the extracellular ATPases. When extracellular ATP reaches levels >100 μM, the ATP receptors of the AT II cells become desensitized and surfactant impairment is initiated. The resulting alteration in viscoelastic properties and in alveolar opening and collapse time-constants leads to alveolar collapse and the redistribution of inspired air from the alveoli to the alveolar ducts, which become pathologically dilated. The collapsed alveoli connected to these dilated alveolar ducts are subject to a massive strain, exacerbating the ATP release. After reaching concentrations >300 μM extracellular ATP acts as a danger-associated molecular pattern, causing capillary leakage, alveolar space edema, and further deactivation of surfactant by serum proteins. Decreasing the tidal volume to 6 mL/kg or less at this stage cannot prevent further lung injury.

  9. Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants.

    Science.gov (United States)

    Tanaka, Kiwamu; Choi, Jeongmin; Cao, Yangrong; Stacey, Gary

    2014-01-01

    As sessile organisms, plants have evolved effective mechanisms to protect themselves from environmental stresses. Damaged (i.e., wounded) plants recognize a variety of endogenous molecules as danger signals, referred to as damage-associated molecular patterns (DAMPs). ATP is among the molecules that are released by cell damage, and recent evidence suggests that ATP can serve as a DAMP. Although little studied in plants, extracellular ATP is well known for its signaling roles in animals, including acting as a DAMP during the inflammatory response and wound healing. If ATP acts outside the cell, then it is reasonable to expect that it is recognized by a plasma membrane-localized receptor. Recently, DORN1, a lectin receptor kinase, was shown to recognize extracellular ATP in Arabidopsis. DORN1 is the founding member of a new purinoceptor subfamily, P2K (P2 receptor kinase), which is plant-specific. P2K1 (DORN1) is required for ATP-induced cellular responses (e.g., cytosolic Ca(2+) elevation, MAPK phosphorylation, and gene expression). Genetic analysis of loss-of-function mutants and overexpression lines showed that P2K1 participates in the plant wound response, consistent with the role of ATP as a DAMP. In this review, we summarize past research on the roles and mechanisms of extracellular ATP signaling in plants, and discuss the direction of future research on extracellular ATP as a DAMP signal.

  10. Gamma-radiation effect of the ATP-ASE-activity in various parts of cotton sprouts

    International Nuclear Information System (INIS)

    Kazimov, A.K.

    1975-01-01

    ATP-ase is a thiol enzyme whose sulfhydryl group plays an important role. The transport of substances through biological membranes is the result of the action of the sodium-potassium pump of the cell, which functions with ATP energy. The action of this transport mechanism depends on the activity of ATP-ase. It may be postulated, therefore, that the suppression of the active transport of Na + and K + ions in cells under irradiation is partially the result of a disturbance of the activity of the ATP enzyme system. The author studied the effect of gamma radiation on ATP-ase activity in various parts of seven-day-old seedlings of type 108-F cotton, which were irradiated using Co 60 gamma radiation. The results of the experiment showed that the ATP-ase activity of the cotton seedling rootlets depends on the dose and the time elapsed after irradiation (a table is given). Small radiation doses (0.2 and 0.5 krad) significantly increased ATP-ase activity in the rootlets, while heavy doses inhibited it significantly. Similar results were obtained for the stems and leaves (tables are given). It was estblished that the ATP-ase of cotton seedlings has varying sensitivity to irradiation. The most sensitive ATP-ases were those of the rootlets. The activity of background ATP-ase is less subject to change than Na + and K + activated ATP-ases. For example, while the activity of ATP-ase (without ions) was inhibited by 25% when a 25 krad irradiation dose was administered, the retardation of Na + and K + activated ATP-ases reached 41%. The author suggests that the inhibition of ATP-ase activity under irradiation is mainly the result of a disturbance of the structure of the membrane functions. It is also possible that ATP-ase activity decreases because of a lack of the enzyme substrate - ATP, which is formed during the process of oxydative phosphorylization. A table is also provided showing the effect of irradiation on the activity of ATP-ase activated by various ions in the roots of

  11. Hair bundles are specialized for ATP delivery via creatine kinase.

    NARCIS (Netherlands)

    Shin, J.B.; Streijger, F.; Beynon, A.J.; Peters, T.; Gadzala, L.; McMillen, D.; Bystrom, C.; Zee, C.E.E.M. van der; Wallimann, T.; Gillespie, P.G.

    2007-01-01

    When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to

  12. Bioluminometric assay of ATP in mouse brain: Determinant factors ...

    Indian Academy of Sciences (India)

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the ...

  13. The Role of ATP in the Regulation of NCAM Function

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    Extracellular ATP is an abundant signaling molecule that has a number of functions in the nervous system. It is released by both neurons and glial cells, activates purinergic receptors and acts as a trophic factor as well as a neurotransmitter. In this review, we summarize the evidence for a dire...

  14. Cloning and characterization of ATP synthase CF1 α gene from ...

    African Journals Online (AJOL)

    ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

  15. Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B.

    Science.gov (United States)

    Materia, Stephanie; Cater, Michael A; Klomp, Leo W J; Mercer, Julian F B; La Fontaine, Sharon

    2011-03-25

    The copper-transporting P(1B)-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.

  16. Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase.

    Directory of Open Access Journals (Sweden)

    Carole H Sellem

    2016-07-01

    Full Text Available Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO of the ATP synthase drive the rotation of a ring of 8-15 c-subunits, which induces conformational changes in the catalytic part (F1 of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary cells while Atp9-5 is expressed in the cells at the extremity (apex of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before.

  17. ATP measurements for monitoring microbial drinking water quality

    DEFF Research Database (Denmark)

    Vang, Óluva Karin

    Current standard methods for surveillance of microbial drinking water quality are culture based, which are laborious and time-consuming, where results not are available before one to three days after sampling. This means that the water may have been consumed before results on deteriorated water....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP...... in drinking water. For on-line continuous real-time monitoring it is essential to choose an adequate enzyme reagent in terms of limit of detection, stability in catalytic activity and an efficient extraction of microbial ATP from cells. Experiments with different types of commercial and R&D reagents...

  18. The leak location package for assessment of the time-frequency correlation method for leak location

    OpenAIRE

    Faerman, Vladimir Andreevich; Cheremnov, A. G.; Avramchuk, Valery Stepanovich; Shepetovsky, Denis Vladimirovich

    2017-01-01

    The paper describes the simplest implementation of a software and hardware package for acoustic correlation leak location and results of its performance assessment for location of water leaks from a metallic pipe in laboratory conditions. A distinctive feature of this leak locator is the use of the software based on the time-frequency correlation analysis of signals, which was proposed in our previous papers. Comparative analysis results are given for the information content of classical and ...

  19. Acoustic leak detection in piping systems, 4

    International Nuclear Information System (INIS)

    Kitajima, Akira; Naohara, Nobuyuki; Aihara, Akihiko

    1983-01-01

    To monitor a high-pressure piping of nuclear power plants, a possibility of acoustic leak detection method has been experimentally studied in practical field tests and laboratory tests. Characteristics of background noise in field test and the results of experiment are summarized as follows: (1) The level of background noise in primary loop (PWR) was almost constant under actual plant operation. But it is possible that it rises at the condition of the pressure in primary loop. (2) Based on many experience of laboratory tests and practical field tests. The leak monitoring system for practical field was designed and developed. To improve the reliability, a judgment of leak on this system is used three factors of noise level, duration time of phenomena and frequency spectrum of noise signal emitted from the leak point. (author)

  20. Evaluation of leak rate by EPRI code

    International Nuclear Information System (INIS)

    Isozaki, Toshikuni; Hashiguchi, Issei; Kato, Kiyoshi; Miyazono, Shohachiro

    1987-08-01

    From 1987, a research on the leak rate from a cracked pipe under BWR or PWR operating condition is going to be carried out at the authors' laboratory. This report describes the computed results by EPRI's leak rate code which was mounted on JAERI FACOM-M380 machine. Henry's critical flow model is used in this program. For the planning of an experimental research, the leak rate from a crack under BWR or PWR operating condition is computed, varying a crack length 2c, crack opening diameter COD and pipe diameter. The COD value under which the minimum detectable leak rate of 5 gpm is given is 0.22 mm or 0.21 mm under the BWR or PWR condition with 2c = 100 mm and 16B pipe geometry. The entire lists are shown in the appendix. (author)

  1. Leak Detectives Saving Money, Water in Virginia

    Science.gov (United States)

    “Circuit riders” from the Virginia Rural Water Association (VRWA) are traveling to small communities across the Commonwealth using special equipment financed by EPA to locate expensive and wasteful leaks in drinking water distribution systems.

  2. Multiple-Unit MRE Leak Detector

    National Research Council Canada - National Science Library

    Litman, Neal

    2000-01-01

    ... testing configuration, materials handling and data collection, and cost benefits. This STP uses the baseline leak detection system, ATC-3, and determine the impact of such variables as pouch temperature and cartoning...

  3. Leaking Underground Storage Tank Sites in Iowa

    Data.gov (United States)

    Iowa State University GIS Support and Research Facility — Leaking Underground Storage Tank (LUST) sites where petroleum contamination has been found. There may be more than one LUST site per UST site.

  4. The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway

    Directory of Open Access Journals (Sweden)

    Weijie Liang

    2017-02-01

    Full Text Available Background/Aims: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS, toll-like receptor 4 (TLR4, receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis, which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP channel opening against high glucose-induced cardiac injury and inflammation. Methods: H9c2 cardiac cells were treated with 35 mM glucose (HG to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP and secretion of inflammatory cytokines were measured as injury indexes. Results: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis or TAK-242 (an inhibitor of TLR4 co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener or pinacidil (Pin, a non-selective KATP channel opener or N-acetyl-L-cysteine (NAC, a ROS scavenger pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker or glibenclamide (Gli, a non-selective KATP channel blocker pre-treatment did not aggravate HG-induced injury and inflammation. Conclusion: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

  5. Detection of primary coolant leaks in NPP

    International Nuclear Information System (INIS)

    Slavov, S.; Bakalov, I.; Vassilev, H.

    2001-01-01

    The thermo-hydraulic analyses of the SG box behaviour of Kozloduy NPP units 3 and 4 in case of small primary circuit leaks and during normal operation of the existing ventilation systems in order to determine the detectable leakages from the primary circuit by analysing different parameters used for the purposes of 'Leak before break' concept, performed by ENPRO Consult Ltd. are presented. The following methods for leak detection: measurement of relative air humidity in SG box (can be used for detection of leaks with flow rate 3.78 l/min within one hour at ambient parameters - temperature 40 0 - 60 0 C and relative humidity form 30% to 60%); measurement of water level in SG box sumps (can not be used for reliable detection of small primary circuit leakages with flow rate about 3.78 l/min); measurement of gaseous radioactivity in SG box( can be used as a general global indication for detection of small leakages from the primary circuit); measurement of condensate flow after the air coolers of P-1 venting system (can be used for primary circuit leak detection) are considered. For determination of the confinement behaviour, a model used with computer code MELCOR has been developed by ENPRO Consult Ltd. A brief summary based on the capabilities of the different methods of leak detection, from the point of view of the applicability of a particular method is given. For both Units 3 and 4 of Kozloduy NPP a qualified complex system for small leak detection is planned to be constructed. Such a system has to unite the following systems: acoustic system for leak detection 'ALUS'; system for control of the tightness of the main primary circuit pipelines by monitoring the local humidity; system for primary circuit leakage detection by measuring condensate run-off in collecting tank after ventilation system P-1 air coolers

  6. Cable-Based Water Leak Detection Technology

    OpenAIRE

    ECT Team, Purdue

    2007-01-01

    Water leaks can be considered as a serious problem from many sources such as water supply and return chains, air conditioning units, cold-water chillers, clogged drains, damaged skylights or windows, or even construction errors. The new water leak detection technologies can provide significant advantages in cost, reliability, and easy adoption have continued since the traditional technology mainly focusing on a spot detector revealed several limitations.

  7. Leak of draft report on radioactive waste

    International Nuclear Information System (INIS)

    1986-01-01

    The Environment Committee's second report discusses the leak of the draft report on radioactive waste. The circumstances of the leak are discussed and ''The Times'' and ''The Guardian'' articles of December 1985 are printed in full, as are the letter from the Chairman of the environmental committee to the members and their replies. The letters from Mr John Large of Large and Associates to the Chairman of the Environment Committee, and Mr Large's correspondence with Greenpeace are also printed. (UK)

  8. Leak detection systems for VVER units based on leak before break concept. PowerPoint presentation

    International Nuclear Information System (INIS)

    Matal, Oldrich

    2010-01-01

    To comply with international standards, independent leak monitoring systems should be installed based on the monitoring of different physical parameters capable of detecting any small leak within one hour from the start of the leak. Such leak detection systems are based mainly on acoustic emission monitoring, humidity monitoring and/or radiation monitoring. Advanced systems integrate the monitoring of different physical parameters into one integrated leak detection system. The Integrated Leak Detection System (ILDS) for NPP Metsamor is described. This system consists of three independent leak detection subsystems, viz. LEMOP (LEak MOnitoring of Pipelines) based on acoustic emission monitoring, HUMOS (HUmidity MOnitoring System) based on humidity monitoring, and RAMOS (RAdiation MOnitoring System) based on radiation monitoring). The Integrated Leak Detection System (ILDS) collects data from the three systems, performs data evaluation, data storage, generates alarms and provides a user interface for the whole system including all subsystems. An example of DiagAssist user interface in the ILDS system in the pictorial form. (P.A.)

  9. Leak detector for reactor pressure vessel

    International Nuclear Information System (INIS)

    Morimoto, Mikio.

    1991-01-01

    A branched pipe is disposed to a leak off pipeline led from a flange surface which connects the main body and the upper lid of a reactor pressure vessel. An exhaust pump is disposed to the branched pipe and a moisture gage is disposed on the side of the exhaustion and a dry air supplier is connected to the branched pipe. Upon conducting a pressure-proof leak test for the reactor pressure vessel, the exhaust pump is operated and an electromagnet valve disposed at the upstream of the dry air supplier is opened and closed repeatedly. The humidity of air sucked by the exhaust pump is detected by the moisture gage. If leaks should be caused in the joining surface of the flange, leaked water is diffused as steams. Accordingly, occurrence of leak can be detected instantly based on the comparison with the moisture level of the dry air as a standard. In this way, a leak test can be conducted reliably in a short period of time with no change of for the reactor pressure container itself. (I.N.)

  10. Bile leak detection by radionuclide scintigraphy.

    Science.gov (United States)

    Gupta, V

    2006-01-01

    Bile leak after liver injury has been reported to be a complication associated with significant mortality. Tc99m-IDA (Tc: Technetium) nuclear scan allows rapid and noninvasive diagnosis of bile leak. An accidental case was referred for the detection of suspicious bile leak. The ultrasound could not detect any abnormality. Dynamic Hepatobiliary Scintigraphy (DHBS) using radiolabeled tracer was performed. DHBS promptly detected the site of leak. This technique should be opted because it has been found to be quite sensitive, specific and accurate for detecting the bile leak, whereas, Ultrasound and CT imaging are less sensitive and nonspecific. Though, they are initially helpful in determining the presence of abdominal fluid collections, they are unable to differentiate between a seroma, lymphocele, hematoma or bile leak. Hepatobiliary scintigraphy is more accurate because it can demonstrate continuity of these fluid collections with the biliary tree. Further, hepatobiliary scintigraphy can provide real-time assessment of hepatocytes function and bile progression from the liver to the intestine.

  11. Automated Hydrogen Gas Leak Detection System

    Science.gov (United States)

    1995-01-01

    The Gencorp Aerojet Automated Hydrogen Gas Leak Detection System was developed through the cooperation of industry, academia, and the Government. Although the original purpose of the system was to detect leaks in the main engine of the space shuttle while on the launch pad, it also has significant commercial potential in applications for which there are no existing commercial systems. With high sensitivity, the system can detect hydrogen leaks at low concentrations in inert environments. The sensors are integrated with hardware and software to form a complete system. Several of these systems have already been purchased for use on the Ford Motor Company assembly line for natural gas vehicles. This system to detect trace hydrogen gas leaks from pressurized systems consists of a microprocessor-based control unit that operates a network of sensors. The sensors can be deployed around pipes, connectors, flanges, and tanks of pressurized systems where leaks may occur. The control unit monitors the sensors and provides the operator with a visual representation of the magnitude and locations of the leak as a function of time. The system can be customized to fit the user's needs; for example, it can monitor and display the condition of the flanges and fittings associated with the tank of a natural gas vehicle.

  12. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

    2016-05-16

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

  13. Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction

    Science.gov (United States)

    Giniatullin, AR; Grishin, SN; Sharifullina, ER; Petrov, AM; Zefirov, AL; Giniatullin, RA

    2005-01-01

    During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2′,7′-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings. PMID:15774519

  14. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

    Directory of Open Access Journals (Sweden)

    Kimberly L. James

    2016-08-01

    Full Text Available Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1 for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4 in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase.

  15. Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation

    Science.gov (United States)

    James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; Mouttaki, Housna; Sieber, Jessica R.; Sheik, Cody S.; Nguyen, Hong H.; Yang, Yanan; Xie, Yongming; Erde, Jonathan; Rohlin, Lars; Karr, Elizabeth A.; Loo, Joseph A.; Ogorzalek Loo, Rachel R.; Hurst, Gregory B.; Gunsalus, Robert P.; Szweda, Luke I.

    2016-01-01

    ABSTRACT Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. PMID:27531911

  16. Liquid metal-to-gas leak-detection instruments

    International Nuclear Information System (INIS)

    Matlin, E.; Witherspoon, J.E.; Johnson, J.L.

    1982-01-01

    It is desirable for liquid-metal-cooled reactors that small liquid metal-to-gas leaks be reliably detected. Testing has been performed on a number of detection systems to evaluate their sensitivity, response time, and performance characteristics. This testing has been scheduled in three phases. The first phase was aimed at screening out the least suitable detectors and optimizing the performance of the most promising. In the second phase, candidates were tested in a 1500 ft 3 walk-in type enclosure in which leaks were simulated on 24-in. and 3-in. piping. In the third phase of testing, selected type detectors were tested in the 1500-ft 3 enclosure with Clinch River Breeder Reactor Plant (CRBRP) pipe insulation configurations and detector tubing configuration with cell gas recirculation simulated. Endurance testing of detection equipment was also performed as part of this effort. Test results have been shown that aerosol-type detectors will reliably detect leaks as small as a few grams per hour when sampling pipe insulation annuli

  17. Remote leak localization approach for fusion machines

    International Nuclear Information System (INIS)

    Durocher, Au.; Bruno, V.; Chantant, M.; Gargiulo, L.; Gherman, T.; Hatchressian, J.-C.; Houry, M.; Le, R.; Mouyon, D.

    2013-01-01

    Highlights: ► Description of leaks issue. ► Selection of leak localization concepts. ► Qualification of leak localization concepts. -- Abstract: Fusion machine operation requires high-vacuum conditions and does not tolerate water or gas leak in the vacuum vessels, even if they are micrometric. Tore Supra, as a fully actively cooled tokamak, has got a large leak management experience; 34 water leaks occurred since the beginning of its operation in 1988. To handle this issue, after preliminary machine protection phases, the current process for leak localization is based on water or helium pressurization network by network. It generally allows the identification of a set of components where the leakage element is located. However, the unique background of CEA-IRFM laboratory points needs of accuracy and promptness out in the leak localization process. Moreover, in-vessel interventions have to be performed trying to minimize time and risks for the persons. They are linked to access conditions, radioactivity, tracer gas high pressure and vessel conditioning. Remote operation will be one of the ways to improve these points on future fusion machines. In this case, leak sensors would have to be light weight devices in order to be integrated on a carrier or to be located outside with a sniffing process set up. A leak localization program is on-going at CEA-IRFM Laboratory with the first goal of identifying and characterizing relevant concepts to localize helium or water leaks on ITER. In the same time, CEA has developed robotic carrier for effective in-vessel intervention in a hostile environment. Three major tests campaigns with the goal to identify leak sensors have been achieved on several CEA test-beds since 2010. Very promising results have been obtained: relevant scenario of leak localization performed, concepts tested in a high volume test-bed called TITAN, and, in several conditions of pressure and temperature (ultrahigh vacuum to atmospheric pressure and 20

  18. Remote leak localization approach for fusion machines

    Energy Technology Data Exchange (ETDEWEB)

    Durocher, Au., E-mail: aurelien.durocher@cea.fr [CEA-IRFM, F-13108 Saint Paul-Lez-Durance (France); Bruno, V.; Chantant, M.; Gargiulo, L. [CEA-IRFM, F-13108 Saint Paul-Lez-Durance (France); Gherman, T. [Floralis UJF Filiale, F-38610 Gières (France); Hatchressian, J.-C.; Houry, M.; Le, R.; Mouyon, D. [CEA-IRFM, F-13108 Saint Paul-Lez-Durance (France)

    2013-10-15

    Highlights: ► Description of leaks issue. ► Selection of leak localization concepts. ► Qualification of leak localization concepts. -- Abstract: Fusion machine operation requires high-vacuum conditions and does not tolerate water or gas leak in the vacuum vessels, even if they are micrometric. Tore Supra, as a fully actively cooled tokamak, has got a large leak management experience; 34 water leaks occurred since the beginning of its operation in 1988. To handle this issue, after preliminary machine protection phases, the current process for leak localization is based on water or helium pressurization network by network. It generally allows the identification of a set of components where the leakage element is located. However, the unique background of CEA-IRFM laboratory points needs of accuracy and promptness out in the leak localization process. Moreover, in-vessel interventions have to be performed trying to minimize time and risks for the persons. They are linked to access conditions, radioactivity, tracer gas high pressure and vessel conditioning. Remote operation will be one of the ways to improve these points on future fusion machines. In this case, leak sensors would have to be light weight devices in order to be integrated on a carrier or to be located outside with a sniffing process set up. A leak localization program is on-going at CEA-IRFM Laboratory with the first goal of identifying and characterizing relevant concepts to localize helium or water leaks on ITER. In the same time, CEA has developed robotic carrier for effective in-vessel intervention in a hostile environment. Three major tests campaigns with the goal to identify leak sensors have been achieved on several CEA test-beds since 2010. Very promising results have been obtained: relevant scenario of leak localization performed, concepts tested in a high volume test-bed called TITAN, and, in several conditions of pressure and temperature (ultrahigh vacuum to atmospheric pressure and 20

  19. SINGLE-SHELL TANKS LEAK INTEGRITY ELEMENTS/SX FARM LEAK CAUSES AND LOCATIONS - 12127

    Energy Technology Data Exchange (ETDEWEB)

    VENETZ TJ; WASHENFELDER D; JOHNSON J; GIRARDOT C

    2012-01-25

    Washington River Protection Solutions, LLC (WRPS) developed an enhanced single-shell tank (SST) integrity project in 2009. An expert panel on SST integrity was created to provide recommendations supporting the development of the project. One primary recommendation was to expand the leak assessment reports (substitute report or LD-1) to include leak causes and locations. The recommendation has been included in the M-045-9IF Hanford Federal Facility Agreement and Consent Order (Tri-Party Agreement) as one of four targets relating to SST leak integrity. The 241-SX Farm (SX Farm) tanks with leak losses were addressed on an individual tank basis as part of LD-1. Currently, 8 out of 23 SSTs that have been reported to having a liner leak are located in SX Farm. This percentage was the highest compared to other tank farms which is why SX Farm was analyzed first. The SX Farm is comprised of fifteen SSTs built 1953-1954. The tanks are arranged in rows of three tanks each, forming a cascade. Each of the SX Farm tanks has a nominal I-million-gal storage capacity. Of the fifteen tanks in SX Farm, an assessment reported leak losses for the following tanks: 241-SX-107, 241-SX-108, 241-SX-109, 241-SX-111, 241-SX-112, 241-SX-113, 241-SX-114 and 241-SX-115. The method used to identify leak location consisted of reviewing in-tank and ex-tank leak detection information. This provided the basic data identifying where and when the first leaks were detected. In-tank leak detection consisted of liquid level measurement that can be augmented with photographs which can provide an indication of the vertical leak location on the sidewall. Ex-tank leak detection for the leaking tanks consisted of soil radiation data from laterals and drywells near the tank. The in-tank and ex-tank leak detection can provide an indication of the possible leak location radially around and under the tank. Potential leak causes were determined using in-tank and ex-tank information that is not directly related to

  20. The danger signal extracellular ATP is an inducer of Fusobacterium nucleatum biofilm dispersal

    Directory of Open Access Journals (Sweden)

    Qinfeng Ding

    2016-11-01

    Full Text Available Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP is an example of a danger associated molecular pattern (DAMP employed by mammalian cells to elicit inflammatory and damage healing responses. Although the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were significantly more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also exhibited significantly higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a

  1. The Danger Signal Extracellular ATP Is an Inducer ofFusobacterium nucleatumBiofilm Dispersal.

    Science.gov (United States)

    Ding, Qinfeng; Tan, Kai Soo

    2016-01-01

    Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum , an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also showed higher expression of fadA , a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to

  2. The Danger Signal Extracellular ATP Is an Inducer of Fusobacterium nucleatum Biofilm Dispersal

    Science.gov (United States)

    Ding, Qinfeng; Tan, Kai Soo

    2016-01-01

    Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also showed higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to detach

  3. Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by oestradiol and tamoxifen.

    Science.gov (United States)

    Radde, Brandie N; Ivanova, Margarita M; Mai, Huy Xuan; Salabei, Joshua K; Hill, Bradford G; Klinge, Carolyn M

    2015-01-01

    Oestrogen receptor α (ERα+) breast tumours rely on mitochondria (mt) to generate ATP. The goal of the present study was to determine how oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete compared with dextran-coated charcoal (DCC)-stripped foetal bovine serum (FBS)-containing medium ('serum-starved'). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respiratory stateapparent was unchanged by serum deprivation. 4-OHT reduced OCR independent of serum status. Despite having a higher mt DNA/nuclear DNA ratio than MCF-7 cells, T47D cells have a lower OCR and ATP levels and higher proton leak. T47D express higher nuclear respiratory factor-1 (NRF-1) and NRF-1-regulated, nuclear-encoded mitochondrial transcription factor TFAM and cytochrome c, but lower levels of cytochrome c oxidase, subunit IV, isoform 1 (COX4, COX4I1). Mitochondrial reserve capacity, reflecting tolerance to cellular stress, was higher in serum-starved T47D cells and was increased by 4-OHT, but was decreased by 4-OHT in MCF-7 cells. These data demonstrate critical differences in cellular energetics and responses to 4-OHT in these two ERα+ cell lines, likely reflecting cancer cell avoidance of apoptosis.

  4. Persister formation in Staphylococcus aureus is associated with ATP depletion

    Energy Technology Data Exchange (ETDEWEB)

    Conlon, Brian P.; Rowe, Sarah E.; Gandt, Autumn Brown; Nuxoll, Austin S.; Donegan, Niles P.; Zalis, Eliza A.; Clair, Geremy; Adkins, Joshua N.; Cheung, Ambrose L.; Lewis, Kim

    2016-04-18

    Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic bacterial infection and antibiotic treatment failure. In Escherichia coli, toxin/antitoxin (TA) modules are responsible for persister formation. The mechanism of persister formation in Gram positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance to stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers was associated with a 100-1000 fold increased likelihood of survival to antibiotic challenge. We find that the antibiotic tolerance of these cells is due to a drop in intracellular ATP. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotic treatment.

  5. 'Domino' systems biology and the 'A' of ATP.

    Science.gov (United States)

    Verma, Malkhey; Zakhartsev, Maksim; Reuss, Matthias; Westerhoff, Hans V

    2013-01-01

    We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  6. Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique

    Science.gov (United States)

    Geer, M. Ariel; Fitzgerald, Michael C.

    2016-02-01

    The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins.

  7. MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

    Science.gov (United States)

    Zhao, Yumei; Migita, Keisuke; Sun, Jing; Katsuragi, Takeshi

    2010-04-01

    Adenosine triphosphate (ATP) plays the role of an autocrine/paracrine signal molecule in a variety of cells. So far, however, the membrane machinery in the export of intracellular ATP remains poorly understood. Activation of B2-receptor with bradykinin-induced massive release of ATP from cultured taenia coli smooth muscle cells. The evoked release of ATP was unaffected by gap junction hemichannel blockers, such as 18alpha-glycyrrhetinic acid and Gap 26. Furthermore, the cystic fibrosis transmembrane regulator (CFTR) coupled Cl(-) channel blockers, CFTR(inh)172, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, Gd3(+) and glibenclamide, failed to suppress the export of ATP by bradykinin. On the other, the evoked release of ATP was greatly reduced by multidrug resistance protein (MRP) transporter inhibitors, MK-571, indomethacin, and benzbromarone. From western blotting analysis, blots of MRP 1 protein only, but not MRP 2 and MRP 3 protein, appeared at 190 kD. However, the MRP 1 protein expression was not enhanced after loading with 1 muM bradykinin for 5 min. Likewise, niflumic acid and fulfenamic acid, Ca2(+)-activated Cl(-) channel blockers, largely abated the evoked release of ATP. The possibility that the MRP transporter system couples with Ca2(+)-activated Cl(-) channel activities is discussed here. These findings suggest that MRP transporters, probably MRP 1, unlike CFTR-Cl(-) channels and gap junction hemichannels, may contribute as membrane machinery to the export of ATP induced by G-protein-coupled receptor stimulation.

  8. Extracellular ATP induces graded reactive response of astrocytes and strengthens their antioxidative defense in vitro.

    Science.gov (United States)

    Adzic, Marija; Stevanovic, Ivana; Josipovic, Natasa; Laketa, Danijela; Lavrnja, Irena; Bjelobaba, Ivana M; Bozic, Iva; Jovanovic, Marija; Milosevic, Milena; Nedeljkovic, Nadezda

    2017-04-01

    It is widely accepted that adenosine triphosphate (ATP) acts as a universal danger-associated molecular pattern with several known mechanisms for immune cell activation. In the central nervous system, ATP activates microglia and astrocytes and induces a neuroinflammatory response. The aim of the present study was to describe responses of isolated astrocytes to increasing concentrations of ATP (5 µM to 1 mM), which were intended to mimic graded intensity of the extracellular stimulus. The results show that ATP induces graded activation response of astrocytes in terms of the cell proliferation, stellation, shape remodeling, and underlying actin and GFAP filament rearrangement, although the changes occurred without an apparent increase in GFAP and actin protein expression. On the other hand, ATP in the range of applied concentrations did not evoke IL-1β release from cultured astrocytes, nor did it modify the release from LPS and LPS+IFN-γ-primed astrocytes. ATP did not promote astrocyte migration in the wound-healing assay, nor did it increase production of reactive oxygen and nitrogen species and lipid peroxidation. Instead, ATP strengthened the antioxidative defense of astrocytes by inducing Cu/ZnSOD and MnSOD activities and by increasing their glutathione content. Our current results suggest that although ATP triggers several attributes of activated astrocytic phenotype with a magnitude that increases with the concentration, it is not sufficient to induce full-blown reactive phenotype of astrocytes in vitro. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. CT scan-based modelling of anastomotic leak risk after colorectal surgery.

    Science.gov (United States)

    Gervaz, P; Platon, A; Buchs, N C; Rocher, T; Perneger, T; Poletti, P-A

    2013-01-01

    Prolonged ileus, low-grade fever and abdominal discomfort are common during the first week after colonic resection. Undiagnosed anastomotic leak carries a poor outcome and computed tomography (CT) scan is the best imaging tool for assessing postoperative abdominal complications. We used a CT scan-based model to quantify the risk of anastomotic leak after colorectal surgery. A case-control analysis of 74 patients who underwent clinico-radiological evaluation after colorectal surgery for suspicion of anastomotic leak was undertaken and a multivariable analysis of risk factors for leak was performed. A logistic regression model was used to identify determinant variables and construct a predictive score. Out of 74 patients with a clinical suspicion of anastomotic leak, 17 (23%) had this complication confirmed following repeat laparotomy. In multivariate analysis, three variables were associated with anastomotic leak: (1) white blood cells count > 9 × 10(9) /l (OR = 14.8); (2) presence of ≥ 500 cm(3) of intra- abdominal fluid (OR = 13.4); and (3) pneumoperitoneum at the site of anastomosis (OR = 9.9). Each of these three parameters contributed one point to the risk score. The observed risk of leak was 0, 6, 31 and 100%, respectively, for patients with scores of 0, 1, 2 and 3. The area under the receiver operating characteristic curve for the score was 0.83 (0.72-0.94). This CT scan-based model seems clinically promising for objective quantification of the risk of a leak after colorectal surgery. Colorectal Disease © 2013 The Association of Coloproctology of Great Britain and Ireland.

  10. Leaks in fixed-ring banded sleeve gastrectomies: a management approach.

    Science.gov (United States)

    Foo, Jonathan W; Balshaw, James; Tan, Michael H L; Tan, Jeremy T H

    2017-08-01

    The use of a Fobi ring to prevent pouch dilation is sometimes used in Roux-en-Y gastric bypass (RYGB). Recently, it has been extrapolated to laparoscopic sleeve gastrectomy (LSG) procedures by placing a fixed-ring band a few centimeters below the gastroesophageal junction (GEJ). What is the consequence if a patient develops a leak? Tertiary metropolitan referral center, Australia. Over 18 months, all patients with either a conventional LSG or a fixed-ring banded sleeve gastrectomy (BLSG) who presented with a proven leak complication were included. The management approaches along with the surgical, endoscopic, and percutaneous procedures used were examined. Six patients had a BLSG leak and 6 had a LSG leak. All patients had leak resolution. There was no significant difference in body mass index (BMI), time to leak, initial white cell count (WCC), and C-reactive protein (CRP) levels between the 2 groups. LSG patients required a median of 2 endoscopic procedures (range: 1-3). Stents were deployed in 3 patients. All BLSG patients required a single surgical intervention with laparoscopic washout, drainage, removal of band±feeding jejunostomy. One stent was deployed in 1 BLSG patient. BLSG leak resolution was found at 34±12 days versus 85±12 days in the LSG group (P< .05). The BLSG is a new modification of the sleeve gastrectomy procedure. This study presents a management strategy for leak resolution employed in BLSG patients. The presence of a foreign body as a persistent nidus of infection mandates band removal. Copyright © 2017 American Society for Bariatric Surgery. All rights reserved.

  11. Risk factors for postoperative cerebrospinal fluid leak and meningitis after expanded endoscopic endonasal surgery.

    Science.gov (United States)

    Ivan, Michael E; Iorgulescu, J Bryan; El-Sayed, Ivan; McDermott, Michael W; Parsa, Andrew T; Pletcher, Steven D; Jahangiri, Arman; Wagner, Jeffrey; Aghi, Manish K

    2015-01-01

    Postoperative cerebrospinal fluid (CSF) leak is a serious complication of transsphenoidal surgery, which can lead to meningitis and often requires reparative surgery. We sought to identify preoperative risk factors for CSF leaks and meningitis. We reviewed 98 consecutive expanded endoscopic endonasal surgeries performed from 2008-2012 and analyzed preoperative comorbidities, intraoperative techniques, and postoperative care. Univariate and multivariate analyses were performed. The most common pathologies addressed included pituitary adenoma, Rathke cyst, chordoma, esthesioneuroblastoma, meningioma, nasopharyngeal carcinoma, and squamous cell carcinoma. There were 11 CSF leaks (11%) and 10 central nervous system (CNS) infections (10%). Univariate and multivariate analysis of preoperative risk factors showed that patients with non-ideal body mass index (BMI) were associated with higher rate of postoperative CSF leak and meningitis (both p<0.01). Also, patients with increasing age were associated with increased CSF leak (p = 0.03) and the length of time a lumbar drain was used postoperatively was associated with infection in a univariate analysis. In addition, three of three endoscopic transsphenoidal surgeries combined with open cranial surgery had a postoperative CSF leak and CNS infection rate which was a considerably higher rate than for transsphenoidal surgeries alone or surgeries staged with open cases (p<0.01 and p=0.04, respectively) In this series of expanded endoscopic transsphenoidal surgeries, preoperative BMI remains the most important preoperative predictor for CSF leak and infection. Other risk factors include age, intraoperative CSF leak, lumbar drain duration, and cranial combined cases. Risks associated with complex surgical resections when combining open and endoscopic approaches could be minimized by staging these procedures. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

    Directory of Open Access Journals (Sweden)

    Avery Vicky M

    2009-11-01

    Full Text Available Abstract Background Human African Trypanosomiasis (HAT is caused by two trypanosome species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS of large compound library collections. Results We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of T.b.brucei. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC50 data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for T.b.brucei. Conclusion Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening.

  13. Operational Philosophy Concerning Manned Spacecraft Cabin Leaks

    Science.gov (United States)

    DeSimpelaere, Edward

    2011-01-01

    The last thirty years have seen the Space Shuttle as the prime United States spacecraft for manned spaceflight missions. Many lessons have been learned about spacecraft design and operation throughout these years. Over the next few decades, a large increase of manned spaceflight in the commercial sector is expected. This will result in the exposure of commercial crews and passengers to many of the same risks crews of the Space Shuttle have encountered. One of the more dire situations that can be encountered is the loss of pressure in the habitable volume of the spacecraft during on orbit operations. This is referred to as a cabin leak. This paper seeks to establish a general cabin leak response philosophy with the intent of educating future spacecraft designers and operators. After establishing a relative definition for a cabin leak, the paper covers general descriptions of detection equipment, detection methods, and general operational methods for management of a cabin leak. Subsequently, all these items are addressed from the perspective of the Space Shuttle Program, as this will be of the most value to future spacecraft due to similar operating profiles. Emphasis here is placed upon why and how these methods and philosophies have evolved to meet the Space Shuttle s needs. This includes the core ideas of: considerations of maintaining higher cabin pressures vs. lower cabin pressures, the pros and cons of a system designed to feed the leak with gas from pressurized tanks vs. using pressure suits to protect against lower cabin pressures, timeline and consumables constraints, re-entry considerations with leaks of unknown origin, and the impact the International Space Station (ISS) has had to the standard Space Shuttle cabin leak response philosophy. This last item in itself includes: procedural management differences, hardware considerations, additional capabilities due to the presence of the ISS and its resource, and ISS docking/undocking considerations with a

  14. Standard practice for leaks using ultrasonics

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2011-01-01

    1.1 Practice A, Pressurization—This practice covers procedures for calibration of ultrasonic instruments, location, and estimated measurements of gas leakage to atmosphere by the airborne ultrasonic technique. 1.2 In general practice this should be limited to leaks detected by two classifications of instruments, Class I and Class II. Class I instruments should have a minimum detectable leak rate of 6.7 × 10−7 mol/s (1.5 × 10−2 std. cm3/s at 0°C) or more for the pressure method of gas leakage to atmosphere. Class II instruments should have a minimal detectable leak rate of 6.7 × 10−6 mol/s (1.5 × 10−1 std. cm3/s at 0°C) or more for the pressure method of gas leakage to atmosphere. Refer to Guide E432 for additional information. 1.3 Practice B, Ultrasonic Transmitter—For object under test not capable of being pressurized but capable of having ultrasonic tone placed/injected into the test area to act as an ultrasonic leak trace source. 1.3.1 This practice is limited to leaks producing leakage o...

  15. Leak processing system for valve gland portion

    International Nuclear Information System (INIS)

    Nishino, Masami

    1990-01-01

    When a process fluid for a valve to be checked is at such a normal temperature as during reactor operation, leaked fluid can be detected depending on the temperature increase accompanying the leakage. However, detection is difficult if the temperature of the process fluid for the valve to be checked is low and, if leakage is detected after the reactor start-up, repair has to be applied after the shutdown of the plant. Then, gland leak is detected by detecting the pressure instead of the temperature in the pipeline system and the leak flow rate is calculated based on the pressure. As a result, leakage is detected irrespective of the temperature of the leaked fluid and, for instance, leakage can be detected even in a case where the temperature is not high as in the case of pressure proof test for the pressure vessel before start-up. It can contribute much to the improvement of the plant operation efficiency and can determine the leak flow rate at a high accuracy. (N.H.)

  16. Mitochondrial ROMK channel is a molecular component of mitoK(ATP).

    Science.gov (United States)

    Foster, D Brian; Ho, Alice S; Rucker, Jasma; Garlid, Anders O; Chen, Ling; Sidor, Agnieszka; Garlid, Keith D; O'Rourke, Brian

    2012-08-03

    Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP). Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase β. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death. The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.

  17. Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase).

    Science.gov (United States)

    Pilankatta, Rajendra; Lewis, David; Inesi, Giuseppe

    2011-03-04

    ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

  18. Adenosine 5 '-triphosphate (ATP) supplements are not orally bioavailable: a randomized, placebo-controlled cross-over trial in healthy humans

    NARCIS (Netherlands)

    Arts, I.C.W.; Coolen, E.J.C.M.; Bours, M.J.L.; Huyghebaert, N.; Cohen Stuart, M.A.; Bast, A.; Dagnelie, P.C.

    2012-01-01

    Background: Nutritional supplements designed to increase adenosine 5'-triphosphate (ATP) concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity

  19. Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation

    Science.gov (United States)

    Bojovschi, A.; Liu, Ming S.; Sadus, Richard J.

    2012-08-01

    The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F1-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg2+ leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

  20. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    Directory of Open Access Journals (Sweden)

    Jonathon eTelianidis

    2013-08-01

    Full Text Available Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-Type ATPases (copper-ATPases, ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  1. Remote Leak Detection: Indirect Thermal Technique

    Science.gov (United States)

    Clements, Sandra

    2002-01-01

    Remote sensing technologies are being considered for efficient, low cost gas leak detection. Eleven specific techniques have been identified for further study and evaluation of several of these is underway. The Indirect Thermal Technique is one of the techniques that is being explored. For this technique, an infrared camera is used to detect the temperature change of a pipe or fitting at the site of a gas leak. This temperature change is caused by the change in temperature of the gas expanding from the leak site. During the 10-week NFFP program, the theory behind the technique was further developed, experiments were performed to determine the conditions for which the technique might be viable, and a proof-of-concept system was developed and tested in the laboratory.

  2. Remote leak detection for the TFTR

    International Nuclear Information System (INIS)

    Walthers, C.R.

    1977-01-01

    The planned design for the TFTR (TOKAMAK Fusion Test Reactor) remote leak detection system consists of a central console which controls the application of tracer gas to possible leak areas. Seals are tested by admitting tracer gas to machined cavities on the atmospheric side of the seal. The tracer gas is brought to the seal cavity by 1 / 8 -inch diameter tubes which connect to local tracer gas/vacuum manifolds located outside the protective radiation shielding. Vacuum shell walls and welds are checked by flowing tracer gas through annular heating/cooling passages. The detector will be either an MSLD (mass spectrometer leak detector) or an RGA (residual gas analyzer), the location of which is not finalized. Feasibility tests performed and planned include response and sensitivity measurements of possible tubing/detector configurations with several tracer gases

  3. Recent Progress in Technology of Leak detection

    Energy Technology Data Exchange (ETDEWEB)

    Jung, H. K.; Kim, S. H.; Cho, J. W.; Joo, Y. S.; Yang, D. J

    2005-07-15

    It is very important to check for leakage points of fluids and gases on primary pressure boundary of nuclear power plants in order to maintain and manage various structures safely. Even though much investigation has been performed by a number of researchers, there are a lot of problems to detect the leakage under some areas to which people can not approach. In particular, it is certainly necessary to find the leakage point in order to repair and replace the pressure boundaries. In this report, the basic principle and application situations for the development of the leak detection system which can detect micro-leaks are introduced. As the technologies and performances of recent sensors have been improving, the application range of leak detection has been increasing steadily. Therefore the sensor technologies written in this report will be able to contribute to nuclear safety to detect the leakage rate and the leakage point with an on-line monitoring system in the near future.

  4. Combustion suppressing device for leaked sodium

    International Nuclear Information System (INIS)

    Ooto, Akihiro.

    1985-01-01

    Purpose: To suppress the atmospheric temperature to secure the building safety and shorten the recovery time after the leakage in a chamber for containing sodium leaked from coolant circuit equipments or pipeways of LMFBR type rector by suppressing the combustion of sodium contained in the chamber. Constitution: To the inner wall of a chamber for containing sodium handling equipments, are vertically disposed a panel having a coolant supply port at the upper portion and a coolant discharge port at the lower portion thereof and defined with a coolant flowing channel and a panel for sucking the coolant discharged from the abovementioned panel and exhausting the same externally. Further, a corrugated combustion suppressing plate having apertures for draining the condensated leaked sodium is disposed near the sodium handling equipments. If ruptures are resulted to the sodium handling equipments or pipeway, leaked sodium is passed through the drain apertures in the suppressing plate and stored at the bottom of the containing chamber. (Horiuchi, T.)

  5. Urinary ATP may be a dynamic biomarker of detrusor overactivity in women with overactive bladder syndrome.

    Directory of Open Access Journals (Sweden)

    Miguel Silva-Ramos

    Full Text Available BACKGROUND: Nowadays, there is a considerable bulk of evidence showing that ATP has a prominent role in the regulation of human urinary bladder function and in the pathophysiology of detrusor overactivity. ATP mediates nonadrenergic-noncholinergic detrusor contractions in overactive bladders. In vitro studies have demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladder samples (OAB release more ATP than controls. Here, we compared the urinary ATP concentration in samples collected non-invasively from OAB women with detrusor overactivity and age-matched controls. METHODS: Patients with neurologic diseases, history of malignancy, urinary tract infections or renal impairment (creatinine clearance <70 ml/min were excluded. All patients completed a 3-day voiding diary, a 24 h urine collection and blood sampling to evaluate creatinine clearance. Urine samples collected during voluntary voids were immediately freeze-preserved for ATP determination by the luciferin-luciferase bioluminescence assay; for comparison purposes, samples were also tested for urinary nerve growth factor (NGF by ELISA. RESULTS: The urinary content of ATP, but not of NGF, normalized to patients' urine creatinine levels (ATP/Cr or urinary volume (ATP.Vol were significantly (P<0.05 higher in OAB women with detrusor overactivity (n = 34 than in healthy controls (n = 30. Significant differences between the two groups were still observed by boosting urinary ATP/Cr content after water intake, but these were not detected for NGF/Cr. In OAB patients, urinary ATP/Cr levels correlated inversely with mean voided volumes determined in a 3-day voiding diary. CONCLUSION: A high area under the receiver operator characteristics (ROC curve (0.741; 95% CI 0.62-0.86; P<0.001 is consistent with urinary ATP/Cr being a highly sensitive dynamic biomarker for assessing detrusor overactivity in women with OAB syndrome.

  6. CT assessment of anastomotic bowel leak

    International Nuclear Information System (INIS)

    Power, N.; Atri, M.; Ryan, S.; Haddad, R.; Smith, A.

    2007-01-01

    Aim: To evaluate the predictors of clinically important gastrointestinal anastomotic leaks using multidetector computed tomography (CT). Subjects and methods: Ninety-nine patients, 73 with clinical suspicion of anastomotic bowel leak and 26 non-bowel surgery controls underwent CT to investigate postoperative sepsis. Fifty patients had undergone large bowel and 23 small bowel anastomoses. The time interval from surgery was 3-30 days (mean 10 ± 5.9 SD) for the anastomotic group and 3-40 days (mean 14 ± 11 SD) for the control group (p = 0.3). Two radiologists blinded to the final results reviewed the CT examinations in consensus and recorded the presence of peri-anastomotic air, fluid or combination of the two; distant loculated fluid or combination of fluid and air; free air or fluid; and intestinal contrast leak. Final diagnosis of clinically important anastomotic leak (CIAL) was confirmed at surgery or by chart review of predetermined clinical and laboratory criteria. Results: The prevalence of CIAL in the group undergoing CT was 31.5% (23/73). The CT examinations with documented leak were performed 5-28 (mean; 11.4 ± 6 SD) days after surgery. Nine patients required repeat operation, 10 percutaneous abscess drainage, two percutaneous drainage followed by surgery, and two prolonged antibiotic treatment and total parenteral nutrition (TPN). Of the CT features examined, only peri-anastomotic loculated fluid containing air was more frequently seen in the CIAL group as opposed to the no leak group (p = 0.04). There was no intestinal contrast leakage in this cohort. Free air was present up to 9 days and loculated air up to 26 days without CIAL. Conclusion: Most postoperative CT features overlap between patients with and without CIAL. The only feature seen statistically more frequently with CIAL is peri-anastomotic loculated fluid containing air

  7. LMFBR steam generator leak detection development in the United States

    International Nuclear Information System (INIS)

    Magee, P.M.; Gerrels, E.E.; Greene, D.A.; McKee, J.

    1978-01-01

    Leak detection for Liquid Metal Fast Breeder Reactor steam generators is an important economic factor in the shutdown, repair and restart of a plant. Development of leak detection systems in the U.S. has concentrated on four areas: (1) chemical (H 2 and O 2 ) leak detection meters; (2) acoustic leak detection/location techniques; (3) investigation of leak behavior (enlargement, damage effects, plugging and unplugging); and (4) data management for plant operations. This paper discusses the status, design aspects, and applications of leak detection technology for LMFBR plants. (author)

  8. Technique for detecting liquid metal leaks

    International Nuclear Information System (INIS)

    Bauerle, J.E.

    1979-01-01

    In a system employing flowing liquid metal as a heat transfer medium in contact with tubular members containing a working fluid, i.e., steam, liquid metal leaks through the wall of the tubular member are detected by dislodging the liquid metal compounds forming in the tubular member at the leak locations and subsequently transporting the dislodged compound in the form of an aerosol to a detector responsive to the liquid metal compound. In the application to a sodium cooled tubular member, the detector would consist of a sodium responsive device, such as a sodium ion detector

  9. Hydrogen detection systems leak response codes

    International Nuclear Information System (INIS)

    Desmas, T.; Kong, N.; Maupre, J.P.; Schindler, P.; Blanc, D.

    1990-01-01

    A loss in tightness of a water tube inside a Steam Generator Unit of a Fast Reactor is usually monitored by hydrogen detection systems. Such systems have demonstrated in the past their ability to detect a leak in a SGU. However, the increase in size of the SGU or the choice of ferritic material entails improvement of these systems in order to avoid secondary leak or to limit damages to the tube bundle. The R and D undertaken in France on this subject is presented. (author). 11 refs, 10 figs

  10. Acoustic leak detection development in the USA

    International Nuclear Information System (INIS)

    Greene, D.A.; Malovrh, J.W.; Magee, P.M.

    1984-01-01

    Acoustic monitoring systems that detect and locate a leak of water/steam from a defective tube in an LMFBR steam generator have been developed in the United States. A low frequency (approx. 10 KHz) system was developed by General Electric, and a high frequency (200 to 300 KHz) system by Rockwell International with support from Argonne National Laboratory. A comprehensive base technology program provided absolute signal amplitudes, background noise amplitudes, and signal source-to-detector transfer functions. Field tests of these systems demonstrated an ability to detect and locate simulated leaks under operating and quiescent conditions in an LMFBR steam generator. (author)

  11. Localized leak detection using moisture sensitive tape

    International Nuclear Information System (INIS)

    Riddle, P.

    1984-01-01

    In the United States the concept of localized leak detection has become an accepted monitoring method for existing conditions such as intergranular stress corrosion cracking and for conditions which may be present in pipes which are inaccessible to currently used monitoring systems. Among the options which exist for augmented localized leak detection systems are moisture sensitive tape and acoustic emission. Choice of system depends upon the number of locations to be monitored, the environment, total installed cost and operating cost. The Techmark multiplex moisture sensitive tape system is described. (U.K.)

  12. Single-Shell Tank Leak Integrity Summary

    Energy Technology Data Exchange (ETDEWEB)

    Harlow, D. G. [Washington River Protection Solutions LLC, Richland, WA (United States); Girardot, C. L. [Washington River Protection Solutions LLC, Richland, WA (United States); Venetz, T. J. [Washington River Protection Solutions LLC, Richland, WA (United States)

    2015-03-26

    This document summarizes and evaluates the information in the Hanford Tri-Party Agreement Interim Milestone M-045-91F Targets completed between 2010 and 2015. 1) Common factors of SST liner failures (M-045-91F-T02), 2) the feasibility of testing for ionic conductivity between the inside and outside of SSTs (M-045-91F-T03, and 3) the causes, locations, and rates of leaks from leaking SSTs (M-045-91F-T04).

  13. A Correlated Active Acoustic Leak Detection in a SFR Steam Generator

    International Nuclear Information System (INIS)

    Kim, Tae Joon; Jeong, Ji Young; Kim, Jong Man; Kim, Byung Ho; Kim, Yong Il

    2009-01-01

    The methods of acoustic leak detection are active acoustic leak detection and passive acoustic leak detection. The methods for passive acoustic leak detection are already established, but because our goal is development of passive acoustic leak detection for detecting a leakage range of small and micro leak rates, it is difficult detecting a leak in steam generator using this developed passive acoustic leak detection. Thus the acoustic leak detection system is required to be able to detect wide range of water leaks. From this view point we need to develop an active acoustic leak detection technology to be able to detect intermediate leak rates

  14. ATP stimulates calcium influx in primary astrocyte cultures

    International Nuclear Information System (INIS)

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-01-01

    The effect of ATP and other purines on 45 Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular 45 Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45 Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of 45 Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced 45 Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

  15. Detection of CSF leak in spinal CSF leak syndrome using MR myelography: correlation with radioisotope cisternography.

    Science.gov (United States)

    Yoo, H-M; Kim, S J; Choi, C G; Lee, D H; Lee, J H; Suh, D C; Choi, J W; Jeong, K S; Chung, S J; Kim, J S; Yun, S-C

    2008-04-01

    Spinal CSF leak syndrome is a unique disorder caused by spinal CSF leak. In this study, we attempted to determine whether MR myelography (MRM) can detect the leakage site in the spine. We performed both MRM and radioisotope cisternography (RIC) in 15 patients with spinal CSF leak syndrome. Patients were included in this study if they had at least 2 of the following criteria: 1) orthostatic headache, 2) low CSF opening pressure, and 3) diffuse pachymeningeal enhancement on brain MR imaging. For comparison, we performed MRM in 15 subjects without symptoms of spinal CSF leak syndrome. MRM was performed with the 2D turbo spin-echo technique in the entire spine by using a 1.5T scanner. Two blinded radiologists evaluated the MRM findings in a total of 30 cases, composed of patient and control groups, with regard to the presence of leakage and the level of leakage if present. RIC was performed only in the patient group and was assessed by consensus among 3 physicians experienced in nuclear medicine. The diagnostic performance of MRM and RIC was evaluated on the basis of the clinical diagnosis of spinal CSF leak syndrome. The sensitivity, specificity, and accuracy of MR myelography for detecting CSF leak was 86.7%, 86.7%, and 86.7% for reader 1, respectively, and 80.0%, 93.3%, and 86.7% for reader 2, respectively. The sensitivity of RIC was 93.3%. Agreement between the 2 techniques for the detection of CSF leak was substantial in reader 1 and moderate in reader 2 (kappa = 0.634 and 0.444, respectively). MRM is an effective tool for detecting CSF leak in the spine in patients with spinal CSF leak syndrome.

  16. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  17. Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan; Sengupta, Rituparna; Jiang, Li; Satyshur, Kenneth A.; Xing, Yongna

    2013-10-08

    Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.

  18. Inhibition of chemokine expression in rat inflamed paws by systemic use of the antihyperalgesic oxidized ATP

    Directory of Open Access Journals (Sweden)

    Ticozzi Paolo

    2005-07-01

    Full Text Available Abstract Background We previously showed that local use of periodate oxidized ATP (oATP, a selective inhibitor of P2X7 receptors for ATP in rat paw treated with Freund's adjuvant induced a significant reduction of hyperalgesia Herein we investigate the role of oATP, in the rat paws inflamed by carrageenan, which mimics acute inflammation in humans. Results Local, oral or intravenous administration of a single dose of oATP significantly reduced thermal hyperalgesia in hind paws of rats for 24 hours, and such effect was greater than that induced by diclofenac or indomethacin. Following oATP treatment, the expression of the pro-inflammatory chemokines interferon-gamma-inducible protein-10 (IP-10, mon ocyte chemoattractant protein-1 (MCP-1 and interleukin-8 (IL-8 within the inflamed tissues markedly decreased on vessels and infiltrated cells. In parallel, the immunohistochemical findings showed an impairment, with respect to the untreated rats, in P2X7 expression, mainly on nerves and vessels close to the site of inflammation. Finally, oATP treatment significantly reduced the presence of infiltrating inflammatory macrophages in the paw tissue. Conclusion Taken together these results clearly show that oATP reduces carrageenan-induced inflammation in rats.

  19. Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

    Directory of Open Access Journals (Sweden)

    Askew Christopher D

    2009-12-01

    Full Text Available Abstract Background It has been proposed that adenosine triphosphate (ATP released from red blood cells (RBCs may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC. Results Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P Conclusions Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

  20. Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.

    Science.gov (United States)

    Wilhelm, Konrad; Ganesan, Jayanthi; Müller, Tobias; Dürr, Christoph; Grimm, Melanie; Beilhack, Andreas; Krempl, Christine D; Sorichter, Stephan; Gerlach, Ulrike V; Jüttner, Eva; Zerweck, Alf; Gärtner, Frank; Pellegatti, Patrizia; Di Virgilio, Francesco; Ferrari, Davide; Kambham, Neeraja; Fisch, Paul; Finke, Jürgen; Idzko, Marco; Zeiser, Robert

    2010-12-01

    Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

  1. Diagnosis of anastomotic leak: electrolyte detection versus barium fluoroscopy.

    Science.gov (United States)

    DeArmond, Daniel T; Carswell, Aimee; Louden, Christopher L; Simmons, Jeremy D; Bayer, Johanna; Das, Nitin A; Johnson, Scott B

    2013-06-15

    We recently described a new method of diagnosing anastomotic leak using the detection of electrical changes induced by electrolyte extravasation from a surgically created gastric leak site in experimental rats. We sought to compare the sensitivity and specificity of anastomotic leak detection for this method to that of upper gastrointestinal (GI) barium fluoroscopy. Experimental rats with a surgically created gastric leak site and controls were interrogated as to the presence of leak using either the electrolyte-gated leak detection method or upper GI barium fluoroscopy. The sensitivity and specificity of leak detection for the two methods were compared. The sensitivity and specificity of electrolyte-gated leak detection were both 100% (95% confidence interval 69-100%). Barium upper GI fluoroscopy misidentified one leak as a control and one control as a leak, for a sensitivity and specificity of 80% each (95% confidence interval 37-97%). No statistically significant difference was seen between electrolyte-gated leak detection and barium upper GI fluoroscopy in terms of the sensitivity and specificity of anastomotic leak detection. Electrolyte-gated leak detection was similarly sensitive and specific for anastomotic leak detection as upper GI barium fluoroscopy, the current standard. The electrolyte-gated method has the advantages of an inert contrast agent (normal saline) and the possibility of performing leak interrogation at the bedside. Electrolyte-gated leak detection might represent a plausible alternative to upper GI barium fluoroscopy for routine postoperative anastomotic leak surveillance after esophagectomy or other foregut surgery. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Humos monitoring system of leaks in to the containment atmosphere

    International Nuclear Information System (INIS)

    Matal, O.; Zaloudek, J.; Matal, O. Jr.; Klinga, J.; Brom, J.

    1997-01-01

    HUmidity MOnitoring System (HUMOS) has been developed and designed to detect the presence of leak in selected primary circuit high energy pipelines and components that are evaluated from the point of view of Leak Before Break (LBB) requirements. It also requires to apply technical tools for detection and identification of coolant leaks from primary circuit and components of PWRs reactors. Safety significant of leaks depend on: leak source (location); leak rate, and leak duration. Therefore to detect and monitor coolant leaks in to the containment atmosphere during reactor operation is one of important safety measures. As potential leak sources flange connection in the upper head region of WWER reactors can be considered. HUMOS does not rely on the release of radioactivity to detect leaks but rather the relies on detection of moisture in the air resulting from a primary boundary leak. Because HUMOS relies on moisture and temperature detection, leaks can be detected without requiring the reactor to be critical. Therefore leaks can be detected during integrity pressure tests and any other mode of operation provided the reactor ventilation system is operating and primary circuit and components are pressurized. 3 figs

  3. Detection of unapproachable leaks in VECC K-500 superconducting cyclotron

    International Nuclear Information System (INIS)

    Dutta, M.; Polley, A.

    2013-01-01

    K-500 super conducting cyclotron of VECC is very compact. Many areas of it are unapproachable. At the time of commissioning process of Magnet, RF cavity and other systems of VECC K-500 superconducting cyclotron, many vacuum related leaks were developed. These leaks were located by using different methods of leak detection. Special methods were also used to detect some leaks. Periodic vacuum failure was observed in cryostat of SCC at the time of commission of superconducting magnet. As cryostat was not easily approachable, thorough study was made. The study confirmed us that periodic change in pressure was due to air leak at cryostat outer vacuum chamber. This critical leak was identified using special technique and the leak was repaired after taking out the cryostat. Several unapproachable magnet dependent leaks were developed from C-seal in between liner and beam chamber and trimmer driving unit. Leaks were also formed in the ceramic part of dee stems during building up of R.F. voltage. Causes of these leaks were identified and rectified. Another unapproachable critical leak still exists in the bobbin of cryostat. This unapproachable leak also has relation with magnetic field. Leak rate increases with increase of magnetic field. For this reason upper magnetic field of SCC is limited to maintain thermal insulation of cryostat. (author)

  4. Targeted inactivation of copper transporter Atp7b in hepatocytes causes liver steatosis and obesity in mice.

    Science.gov (United States)

    Muchenditsi, Abigael; Yang, Haojun; Hamilton, James P; Koganti, Lahari; Housseau, Franck; Aronov, Lisa; Fan, Hongni; Pierson, Hannah; Bhattacharjee, Ashima; Murphy, Robert; Sears, Cynthia; Potter, James; Wooton-Kee, Clavia R; Lutsenko, Svetlana

    2017-07-01

    Copper-transporting ATPase 2 (ATP7B) is essential for mammalian copper homeostasis. Mutations in ATP7B result in copper accumulation, especially in the liver, and cause Wilson disease (WD). The major role of hepatocytes in WD pathology is firmly established. It is less certain whether the excess Cu in hepatocytes is solely responsible for development of WD. To address this issue, we generated a mouse strain for Cre-mediated deletion of Atp7b and inactivated Atp7b selectively in hepatocytes. Atp7b ΔHep mice accumulate copper in the liver, have elevated urinary copper, and lack holoceruloplasmin but show no liver disease for up to 30 wk. Liver inflammation is muted and markedly delayed compared with the age-matched Atp7b -/- null mice, which show a strong type1 inflammatory response. Expression of metallothioneins is higher in Atp7b ΔHep livers than in Atp7b -/- mice, suggesting better sequestration of excess copper. Characterization of purified cell populations also revealed that nonparenchymal cells in Atp7b ΔHep liver maintain Atp7b expression, have normal copper balance, and remain largely quiescent. The lack of inflammation unmasked metabolic consequences of copper misbalance in hepatocytes. Atp7b ΔHep animals weigh more than controls and have higher levels of liver triglycerides and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase. By 45 wk, all animals develop liver steatosis on a regular diet. Thus copper misbalance in hepatocytes dysregulates lipid metabolism, whereas development of inflammatory response in WD may depend on copper status of nonparenchymal cells. The implications of these findings for the cell-targeting WD therapies are discussed. NEW & NOTEWORTHY Targeted inactivation of copper-transporting ATPase 2 (Atp7b) in hepatocytes causes steatosis in the absence of inflammation. Copyright © 2017 the American Physiological Society.

  5. Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

    DEFF Research Database (Denmark)

    Mortensen, Stefan P.; Gonzalez-Alonso, Jose; Nielsen, Jens Jung

    2009-01-01

    ATP and NE concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and norepinephrine (NE) concentrations were measured during: 1) femoral arterial ATP infusion (0......, respectively (Pcontracting muscle (Pmuscle, whereas interstitial NE concentrations increased similarly in both active...... and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial prinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its...

  6. Thermodynamics of proton transport coupled ATP synthesis.

    Science.gov (United States)

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°(ref)=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pH(in) or pH(out)) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F(0) to the catalytic nucleotide binding sites on the β-subunits in F(1), is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Localized leak detection utilizing moisture sensitive tape

    International Nuclear Information System (INIS)

    Riddle, P.

    1984-01-01

    Moisture sensitive tape (MST) has been used in various nuclear power plants to detect leaks in reactor piping systems. The sensor assembly consists of MST, transponder, and sensor carrier, and is installed on the exterior of thermal insulation. The components, applications, installation, and purchasing information are discussed in the paper

  8. Steam generator leak detection using acoustic method

    International Nuclear Information System (INIS)

    Goluchko, V.V.; Sokolov, B.M.; Bulanov, A.N.

    1982-05-01

    The main requirements to meet by a device for leak detection in sodium - water steam generators are determined. The potentialities of instrumentation designed based on the developed requirements have been tested using a model of a 550 kw steam generator [fr

  9. Detecting gas leaks by ultrasonic emission

    DEFF Research Database (Denmark)

    Rasmussen, Karsten Bo; Henriksen, Eigil

    1997-01-01

    The emission of noise in the frequency range 10 kHz to 25.6 kHz from an experimental gas leak in a flanged joint has been experimentally investigated. The overall conclusion is that the emitted noise is almost frequency independent in level within the considered frequency range.A small PC program...

  10. Gas leak detector is simple and inexpensive

    Science.gov (United States)

    Mitchell, D. K.

    1966-01-01

    Pressure sensor monitors small gas leaks in piping and pressure vessels. A combination of a paper ribbon and adhesive plastic tape is used to cover the area to be monitored and the pressure sensor is placed over a hole in the tape and paper.

  11. Endoscopic management of bile leaks after laparoscopic ...

    African Journals Online (AJOL)

    2013-11-04

    Nov 4, 2013 ... 1 required mesh repair of a large incisional hernia. Discussion. In this study, three-quarters of postoperative bile leaks were suitable for endoscopic management and were treated with combinations of ES and bile duct ... is more rapid in dogs with stents compared with sphincterotomy alone.[8,10,18,19] The.

  12. [Ryanodine receptor, calcium leak and arrhythmias].

    Science.gov (United States)

    Rueda, Angélica; de Alba-Aguayo, David R; Valdivia, Héctor H

    2014-01-01

    The participation of the ionic Ca(2+) release channel/ryanodine receptor in cardiac excitation-contraction coupling is well known since the late '80s, when various seminal papers communicated its purification for the first time and its identity with the "foot" structures located at the terminal cisternae of the sarcoplasmic reticulum. In addition to its main role as the Ca(2+) channel responsible for the transient Ca(2+) increase that activates the contractile machinery of the cardiomyocytes, the ryanodine receptor releases Ca(2+) during the relaxation phase of the cardiac cycle, giving rise to a diastolic Ca(2+) leak. In normal physiological conditions, diastolic Ca(2+) leak regulates the proper level of luminal Ca(2+), but in pathological conditions it participates in the generation of both, acquired and hereditary arrhythmias. Very recently, several groups have focused their efforts into the development of pharmacological tools to control the altered diastolic Ca(2+) leak via ryanodine receptors. In this review, we focus our interest on describing the participation of cardiac ryanodine receptor in the diastolic Ca(2+) leak under physiological or pathological conditions and also on the therapeutic approaches to control its undesired exacerbated activity during diastole. Copyright © 2013 Instituto Nacional de Cardiología Ignacio Chávez. Published by Masson Doyma México S.A. All rights reserved.

  13. Detection of tube leaks in piles

    Energy Technology Data Exchange (ETDEWEB)

    Upson, U.L.

    1960-01-20

    This report discusses the use of liquid ammonia as a tracer for the detection of cooling water leaks into the piles. It is both safe and cheap and can be detected by methods adaptable to direct-reading instrumentation on a continuous-flow sample. Moderate capital costs and materials costs of less than $50 per pile test are anticipated.

  14. Developments in steam generator leak detection at Ontario Hydro

    International Nuclear Information System (INIS)

    Maynard, K.J.; Singh, V.P.

    1998-01-01

    A method for locating small tube leaks in steam generators has been developed and implemented at Ontario Hydro. The technique utilizes both helium leak detection and moisture leak detection. The combination of these two methods allows tube leaks to be detected in any part of the tube bundle, including those submerged below water near the tubesheet. The estimated detection limits for the helium and moisture leak detection systems are 0.001 kg/hr and 0.05 kg/hr respectively, expressed as leak rates measured at typical boiler operating conditions. This technology is best utilized in situations where the leak rate under operating conditions is smaller than the practical limit for fluorescein dye techniques (∼2 kg/hour). Other novel techniques have been utilized to increase the reliability and speed of the boiler leak search process. These include the use of argon carrier gas to stabilize the buoyant helium gas in the boiler secondary. (author)

  15. Study of Residual Gas Analyser (RGA) Response towards Known Leaks

    International Nuclear Information System (INIS)

    Pathan, Firozkhan S; Khan, Ziauddin; Semwal, Pratibha; George, Siju; Raval, Dilip C; Thankey, Prashant L; Manthena, Himabindu; Yuvakiran, Paravastu; Dhanani, Kalpesh R

    2012-01-01

    Helium leak testing is the most versatile form of weld qualification test for any vacuum application. Almost every ultra-high vacuum (UHV) system utilizes this technique for insuring leak tightness for the weld joints as well as demountable joints. During UHV system under operational condition with many other integrated components, in-situ developed leaks identification becomes one of the prime aspect for maintaining the health of such system and for continuing the experiments onwards. Since online utilization of leak detector (LD) has many practical limitations, residual gas analyser (RGA) can be used as a potential instrument for online leak detection. For this purpose, a co-relation for a given leak rate between Leak Detector and RGA is experimentally established. This paper describes the experimental aspect and the relationship between leak detector and RGA.

  16. REFLEAK: NIST Leak/Recharge Simulation Program for Refrigerant Mixtures

    Science.gov (United States)

    SRD 73 NIST REFLEAK: NIST Leak/Recharge Simulation Program for Refrigerant Mixtures (PC database for purchase)   REFLEAK estimates composition changes of zeotropic mixtures in leak and recharge processes.

  17. Development of pressure boundaries leak detection technology for nuclear reactor

    International Nuclear Information System (INIS)

    Zhang Yao; Zhang Dafa; Chen Dengke; Zhang Liming

    2008-01-01

    The leak detection for the pressure boundaries is an important safeguard in nuclear reactor operation. In the paper, the status and the characters on the development of the pressure boundaries leak detection technology for the nuclear reactor were reviewed, especially, and the advance of the radiation leak detection technology and the acoustic emission leak detection technology were analyzed. The new advance trend of the leak detection technology was primarily explored. According to the analysis results, it is point out that the advancing target of the leak detection technology is to enhance its response speed, sensitivity, and reliability, and to provide effective information for operator and decision-maker. The realization of the global leak detection and the whole life cycle health monitoring for the nuclear boundaries is a significant advancing tendency of the leak detection technology. (authors)

  18. Comparison of leak opening and leak rate calculations to HDR experimental results

    International Nuclear Information System (INIS)

    Grebner, H.; Hoefler, A.; Hunger, H.

    1993-01-01

    During the last years a number of calculations of leak opening and leak rate for through cracks in piping components have been performed. Analyses are pre- or mostly post-calculations to experiments performed at the HDR facility under PWR operating conditions. Piping components under consideration were small diameter straight pipes with circumferential cracks, pipe bends with longitudinal or circumferential cracks and pipe branches with weldment cracks. The components were loaded by internal pressure and opening as well as closing bending moment. The finite element method and two-phase flow leak rate programs were used for the calculations. Results of the analyses are presented as J-integral values, crack opening displacements and areas and leak rates as well as comparisons to the experimental results

  19. Experiences with leak rate calculations methods for LBB application

    International Nuclear Information System (INIS)

    Grebner, H.; Kastner, W.; Hoefler, A.; Maussner, G.

    1997-01-01

    In this paper, three leak rate computer programs for the application of leak before break analysis are described and compared. The programs are compared to each other and to results of an HDR Reactor experiment and two real crack cases. The programs analyzed are PIPELEAK, FLORA, and PICEP. Generally, the different leak rate models are in agreement. To obtain reasonable agreement between measured and calculated leak rates, it was necessary to also use data from detailed crack investigations

  20. Experiences with leak rate calculations methods for LBB application

    Energy Technology Data Exchange (ETDEWEB)

    Grebner, H.; Kastner, W.; Hoefler, A.; Maussner, G. [and others

    1997-04-01

    In this paper, three leak rate computer programs for the application of leak before break analysis are described and compared. The programs are compared to each other and to results of an HDR Reactor experiment and two real crack cases. The programs analyzed are PIPELEAK, FLORA, and PICEP. Generally, the different leak rate models are in agreement. To obtain reasonable agreement between measured and calculated leak rates, it was necessary to also use data from detailed crack investigations.

  1. Investigation for the sodium leak in Monju. Sodium leak and fire test-1

    International Nuclear Information System (INIS)

    Kawata, Koji; Ohno, Shuji; Miyahara, Shinya; Miyake, Osamu; Tanabe, Hiromi

    2000-08-01

    As a part of the work for investigating the sodium leak accident which occurred in the Monju reactor (hereinafter referred to as Monju) on December 8, 1995, three tests, (1) a sodium leak test, (2) a sodium leak and fire test-1, and (3) a sodium leak and fire test-II, were carried out at OEC/PEC. The main objectives of these tests were to confirm the leak and burning behavior of sodium from the damaged thermometer, and the effects of the sodium fire on the integrity of the surrounding structure. This report describes the results of the sodium fire test-I carried out as a preliminary test. The test was performed using the SOLFA-2 (Sodium Leak, Fire and Aerosol) facility on April 8, 1996. In this test, sodium heated to 480degC was leaked for approximately 1.5 hours from a leak simulating apparatus and caused to drop onto a ventilation duct and a grating with the same dimensions and layout as those in Monju. The main conclusions obtained from the test are shown below: 1) Observation from video cameras in the test revealed that in the early stages of the sodium leak, sodium dripped out of the flexible tube of the thermometer. This dripping and burning expanded in range as the sodium splashed on the duct. 2) No damage to the duct itself was detected. However, the aluminum louver frame of the ventilation duct's lower inlet was damaged. Its machine screws came off, leaving half of the grill (on the grating side) detached. 3) No large hole, like the one seen at Monju, was found when the grating was removed from the testing system for inspection, although the area centered on the point were the sodium dripped was damaged in a way indicating the first stages of grating failure. The 5mm square lattice was corroded through in some parts, and numerous blades (originally 3.2 mm thick) had become sharpened like the blade of a knife. 4) The burning pan underside thermocouple near the leak point measured 700degC in within approximately 10 minutes, and for the next hour remained

  2. Endoscopic management of biliary injuries and leaks

    Directory of Open Access Journals (Sweden)

    T S Chandrasekar

    2012-01-01

    Full Text Available Bile duct injuries and subsequent leaks can occur following laparoscopic and open cholecystectomies and also during other hepatobiliary surgeries. Various patient related and technical factors are implicated in the causation of biliary injuries. Over a period of twenty five years managing such patients of biliary injuries our team has found a practical approach to assess the cause of biliary injuries based on the symptoms, clinical examination and imaging. Bismuth classification is helpful in most of the cases. Immediate referral to a centre experienced in the management of bile duct injury and timely intervention is associated with improved outcomes. Resuscitation, correcting dyselectrolytemia, aspiration of undrained biloma and antibiotics take the priority in the management. The goal is to restore the bile conduit, and to prevent short and longterm complications such as biliary fistula, intra-abdominal abscess, biliary stricture, recurrent cholangitis and secondary biliary cirrhosis. Endoscopic therapy by reducing the transpapillary pressure gradient helps in reducing the leak. Endoscopic therapy with biliary sphincterotomy alone or with additional placement of a biliary stent/ nasobiliary drainage is advocated. In our tertiary care referral unit, we found endoscopic interventions are useful in situations where there is leak with associated CBD calculus or a foreign body, peripheral bile duct injury, cystic duct stump leak and partial bile duct injury with leak/ narrowing of the lumen. Endotherapy is not useful in case of complete transection (total cut off and complete stricture involving common hepatic or common bile ducts. In conclusion, endoscopic treatment can be considered a highly effective therapy and should be the first-line therapy in such patients. Though less successful, an endoscopic attempt is warranted in patients suffering from central bile duct leakages failing which surgical management is recommended.

  3. Report on Boeing pipeline leak detection system

    International Nuclear Information System (INIS)

    Aichele, W.T.

    1978-08-01

    Testing was performed on both simulated (test) and existing (water) pipelines to evaluate the Boeing leak detection technique. This technique uses a transformer mounted around the pipe to induce a voltage level onto the pipeline. The induced ground potential is measured from a distant ground probe, inserted into the surrounding soil, with respect to the excited pipeline. The induced voltage level will depend on the soil characteristics, the distance from the excited pipeline, and the probe types. If liquid should leak from the excited pipeline, the escaping liquid will modify the induced potential of the soil surrounding the excited pipeline. This will change the response of the quiescent soil characteristics and cause the voltage level on the detecting probes in the area of the leak to increase. This voltage increase will indicate a soil anomaly. However, the liquid does not have to reach the detection probe to reveal an anomalous soil condition. Several different detection probes were used and evaluated for sensitivity and response time. Although not evaluated during this test, results indicate that a wire laid parallel to the pipe axis may be the best probe configuration. A general sensitivity figure for any of the probes cannot be made from these tests; however, the technique used will reliably detect a pipeline leak of ten gallons. An additional test was performed using the Boeing pipeline leak detection technique to locate the position and depth of an underground pipeline. This test showed that the location and depth of an excited pipeline could be determined from above the ground where other methods for pipeline location had previously failed

  4. Modeling leaks from liquid hydrogen storage systems.

    Energy Technology Data Exchange (ETDEWEB)

    Winters, William Stanley, Jr.

    2009-01-01

    This report documents a series of models for describing intended and unintended discharges from liquid hydrogen storage systems. Typically these systems store hydrogen in the saturated state at approximately five to ten atmospheres. Some of models discussed here are equilibrium-based models that make use of the NIST thermodynamic models to specify the states of multiphase hydrogen and air-hydrogen mixtures. Two types of discharges are considered: slow leaks where hydrogen enters the ambient at atmospheric pressure and fast leaks where the hydrogen flow is usually choked and expands into the ambient through an underexpanded jet. In order to avoid the complexities of supersonic flow, a single Mach disk model is proposed for fast leaks that are choked. The velocity and state of hydrogen downstream of the Mach disk leads to a more tractable subsonic boundary condition. However, the hydrogen temperature exiting all leaks (fast or slow, from saturated liquid or saturated vapor) is approximately 20.4 K. At these temperatures, any entrained air would likely condense or even freeze leading to an air-hydrogen mixture that cannot be characterized by the REFPROP subroutines. For this reason a plug flow entrainment model is proposed to treat a short zone of initial entrainment and heating. The model predicts the quantity of entrained air required to bring the air-hydrogen mixture to a temperature of approximately 65 K at one atmosphere. At this temperature the mixture can be treated as a mixture of ideal gases and is much more amenable to modeling with Gaussian entrainment models and CFD codes. A Gaussian entrainment model is formulated to predict the trajectory and properties of a cold hydrogen jet leaking into ambient air. The model shows that similarity between two jets depends on the densimetric Froude number, density ratio and initial hydrogen concentration.

  5. Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle: implications for exercise hyperemia

    Science.gov (United States)

    Al-Khazraji, Baraa K.; Mortensen, Stefan P.; Jackson, Dwayne N.; Ellis, Christopher G.; Hellsten, Ylva

    2013-01-01

    During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However, interstitial ATP has solely been described to induce vasoconstriction in skeletal muscle. To examine whether interstitial ATP induces vasodilation in skeletal muscle and to what extent this vasoactive effect is mediated by formation of nitric oxide (NO) and prostanoids, three different experimental models were studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1–10 μM) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition of NO and prostanoid formation. Inhibition of these systems abolished the vasodilator effect of ATP. Cell-culture experiments verified ATP-induced formation of NO and prostacyclin in rat skeletal muscle microvascular endothelial cells, and ATP-induced formation of NO in rat skeletal muscle cells. To confirm these findings in humans, ATP was infused into skeletal muscle interstitium of healthy subjects via microdialysis probes and found to increase muscle interstitial concentrations of NO and prostacyclin by ∼60% and ∼40%, respectively. Collectively, these data suggest that a physiologically relevant elevation in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation. PMID:23761642

  6. Non-linear leak currents affect mammalian neuron physiology

    Directory of Open Access Journals (Sweden)

    Shiwei eHuang

    2015-11-01

    Full Text Available In their seminal works on squid giant axons, Hodgkin and Huxley approximated the membrane leak current as Ohmic, i.e. linear, since in their preparation, sub-threshold current rectification due to the influence of ionic concentration is negligible. Most studies on mammalian neurons have made the same, largely untested, assumption. Here we show that the membrane time constant and input resistance of mammalian neurons (when other major voltage-sensitive and ligand-gated ionic currents are discounted varies non-linearly with membrane voltage, following the prediction of a Goldman-Hodgkin-Katz-based passive membrane model. The model predicts that under such conditions, the time constant/input resistance-voltage relationship will linearize if the concentration differences across the cell membrane are reduced. These properties were observed in patch-clamp recordings of cerebellar Purkinje neurons (in the presence of pharmacological blockers of other background ionic currents and were more prominent in the sub-threshold region of the membrane potential. Model simulations showed that the non-linear leak affects voltage-clamp recordings and reduces temporal summation of excitatory synaptic input. Together, our results demonstrate the importance of trans-membrane ionic concentration in defining the functional properties of the passive membrane in mammalian neurons as well as other excitable cells.

  7. Acoustic leak detection at complicated topologies using neural netwoks

    International Nuclear Information System (INIS)

    Hessel, G.; Schmitt, W.; Weiss, F.P.

    1994-01-01

    Considering the shortcomings of all the existing leak detecting principles, a new method again based on the measurement of the leak induced sound but also applying pattern recognition is being developed. The capability of neural networks to localize leaks at the reactor pressure vessel (RPV) head of VVER-440 reactors is discussed. (orig./DG)

  8. 49 CFR 195.444 - CPM leak detection.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 false CPM leak detection. 195.444 Section 195.444... PIPELINE Operation and Maintenance § 195.444 CPM leak detection. Each computational pipeline monitoring (CPM) leak detection system installed on a hazardous liquid pipeline transporting liquid in single...

  9. 49 CFR 195.134 - CPM leak detection.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 false CPM leak detection. 195.134 Section 195.134... PIPELINE Design Requirements § 195.134 CPM leak detection. This section applies to each hazardous liquid... computational pipeline monitoring (CPM) leak detection system and each replaced component of an existing CPM...

  10. Leak Detection and Location Technology Assessment for Aerospace Applications

    Science.gov (United States)

    Wilson, William C.; Coffey, Neil C.; Madaras, Eric I.

    2008-01-01

    Micro Meteoroid and Orbital Debris (MMOD) and other impacts can cause leaks in the International Space Station and other aerospace vehicles. The early detection and location of leaks is paramount to astronaut safety. Therefore this document surveys the state of the art in leak detection and location technology for aerospace vehicles.

  11. 1999 Leak Detection, Monitoring, and Mitigation Strategy Update

    International Nuclear Information System (INIS)

    OHL, P.C.

    1999-01-01

    This document is a complete revision of WHC-SD-WM-ES-378, Rev 1. This update includes recent developments in Leak Detection, Leak Monitoring, and Leak Mitigation technologies, as well as, recent developments in single-shell tank retrieval technologies. In addition, a single-shell tank retrieval release protection strategy is presented

  12. Double Shell Tank AY-102 Radioactive Waste Leak Investigation

    International Nuclear Information System (INIS)

    Washenfelder, Dennis J.

    2014-01-01

    PowerPoint. The objectives of this presentation are to: Describe Effort to Determine Whether Tank AY-102 Leaked; Review Probable Causes of the Tank AY-102 Leak; and, Discuss Influence of Leak on Hanford's Double-Shell Tank Integrity Program

  13. Automated leak localization performance without detailed demand distribution data

    NARCIS (Netherlands)

    Moors, Janneke; Scholten, L.; van der Hoek, J.P.; den Besten, J.

    2018-01-01

    Automatic leak localization has been suggested to reduce the time and personnel efforts needed to localize
    (small) leaks. Yet, the available methods require a detailed demand distribution model for successful
    calibration and good leak localization performance. The main aim of this work was

  14. Vehicle-based Methane Mapping Helps Find Natural Gas Leaks and Prioritize Leak Repairs

    Science.gov (United States)

    von Fischer, J. C.; Weller, Z.; Roscioli, J. R.; Lamb, B. K.; Ferrara, T.

    2017-12-01

    Recently, mobile methane sensing platforms have been developed to detect and locate natural gas (NG) leaks in urban distribution systems and to estimate their size. Although this technology has already been used in targeted deployment for prioritization of NG pipeline infrastructure repair and replacement, one open question regarding this technology is how effective the resulting data are for prioritizing infrastructure repair and replacement. To answer this question we explore the accuracy and precision of the natural gas leak location and emission estimates provided by methane sensors placed on Google Street View (GSV) vehicles. We find that the vast majority (75%) of methane emitting sources detected by these mobile platforms are NG leaks and that the location estimates are effective at identifying the general location of leaks. We also show that the emission rate estimates from mobile detection platforms are able to effectively rank NG leaks for prioritizing leak repair. Our findings establish that mobile sensing platforms are an efficient and effective tool for improving the safety and reducing the environmental impacts of low-pressure NG distribution systems by reducing atmospheric methane emissions.

  15. Modulation of Central Synapses by Astrocyte-Released ATP and Postsynaptic P2X Receptors

    Science.gov (United States)

    Pankratov, Yuriy

    2017-01-01

    Communication between neuronal and glial cells is important for neural plasticity. P2X receptors are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons and/or glia. Recent data show that postsynaptic P2X receptors underlie slow neuromodulatory actions rather than fast synaptic transmission at brain synapses. Here, we review these findings with a particular focus on the release of ATP by astrocytes and the diversity of postsynaptic P2X-mediated modulation of synaptic strength and plasticity in the CNS. PMID:28845311

  16. Modulation of Central Synapses by Astrocyte-Released ATP and Postsynaptic P2X Receptors

    Directory of Open Access Journals (Sweden)

    Eric Boué-Grabot

    2017-01-01

    Full Text Available Communication between neuronal and glial cells is important for neural plasticity. P2X receptors are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons and/or glia. Recent data show that postsynaptic P2X receptors underlie slow neuromodulatory actions rather than fast synaptic transmission at brain synapses. Here, we review these findings with a particular focus on the release of ATP by astrocytes and the diversity of postsynaptic P2X-mediated modulation of synaptic strength and plasticity in the CNS.

  17. Variation in Metabolic Rate among Individuals Is Related to Tissue-Specific Differences in Mitochondrial Leak Respiration.

    Science.gov (United States)

    Salin, Karine; Auer, Sonya K; Rudolf, Agata M; Anderson, Graeme J; Selman, Colin; Metcalfe, Neil B

    Standard metabolic rate (SMR) and maximum metabolic rate (MMR) typically vary two- or threefold among conspecifics, with both traits assumed to significantly impact fitness. However, the underlying mechanisms that determine such intraspecific variation are not well understood. We examined the influence of mitochondrial properties on intraspecific variation in SMR and MMR and hypothesized that if SMR supports the cost of maintaining the metabolic machinery required for MMR, then the mitochondrial properties underlying these traits should be shared. Mitochondrial respiratory capacity (leak and phosphorylating respiration) and mitochondrial content (cytochrome c oxidase activity) were determined in the liver and white muscle of brown trout Salmo trutta of similar age and maintenance conditions. SMR and MMR were uncorrelated across individuals and were not associated with the same mitochondrial properties, suggesting that they are under the control of separate physiological processes. Moreover, tissue-specific relationships between mitochondrial properties and whole-organism metabolic traits were observed. Specifically, SMR was positively associated with leak respiration in liver mitochondria, while MMR was positively associated with muscle mitochondrial leak respiration and mitochondrial content. These results suggest that a high SMR or MMR, rather than signaling a higher ability for respiration-driven ATP synthesis, may actually reflect greater dissipation of energy, driven by proton leak across the mitochondrial inner membrane. Knowledge of these links should aid interpretation of the potential fitness consequences of such variation in metabolism, given the importance of mitochondria in the utilization of resources and their allocation to performance.

  18. ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

    OpenAIRE

    Bernardi, H; De Weille, J R; Epelbaum, J; Mourre, C; Amoroso, S; Slama, A; Fosset, M; Lazdunski, M

    1993-01-01

    The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by an...

  19. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system.

    Science.gov (United States)

    Tinker, Andrew; Aziz, Qadeer; Thomas, Alison

    2014-01-01

    ATP-sensitive potassium channels (K(ATP)) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated K(ATP) channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system. © 2013 The British Pharmacological Society.

  20. Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients

    Science.gov (United States)

    Lahner, Edith; Brigatti, Cristina; Marzinotto, Ilaria; Carabotti, Marilia; Scalese, Giulia; Davidson, Howard W; Wenzlau, Janet M; Bosi, Emanuele; Piemonti, Lorenzo; Annibale, Bruno; Lampasona, Vito

    2017-01-01

    Objectives: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls. Methods: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies. Results: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (Pgastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa. PMID:28102858

  1. The effect of growth phase and medium on the use of the firefly adenosine triphosphate (ATP) assay for the quantitation of bacteria

    Science.gov (United States)

    Bush, V. N.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was used as a rapid method to determine the number of bacteria in a urine sample after nonbacterial components were removed. Accurate cellular ATP values, determined when bacteria were grown in an environment similar to that in which they were found, were necessary for the calculation of bacterial titer in urine. Cellular ATP values vary depending on the extraction method, the cell growth phase, and cell growth conditions. ATP per cell values of stationary E. coli grown in urine were two times greater than ATP per cell values of cells grown in trypticase soy broth. Glucose and urea were examined as possible components responsible for the cellular ATP variation.

  2. Laser-based sensor for a coolant leak detection in a nuclear reactor

    Science.gov (United States)

    Kim, T.-S.; Park, H.; Ko, K.; Lim, G.; Cha, Y.-H.; Han, J.; Jeong, D.-Y.

    2010-08-01

    Currently, the nuclear industry needs strongly a reliable detection system to continuously monitor a coolant leak during a normal operation of reactors for the ensurance of nuclear safety. In this work, we propose a new device for the coolant leak detection based on tunable diode laser spectroscopy (TDLS) by using a compact diode laser. For the feasibility experiment, we established an experimental setup consisted of a near-IR diode laser with a wavelength of about 1392 nm, a home-made multi-pass cell and a sample injection system. The feasibility test was performed for the detection of the heavy water (D2O) leaks which can happen in a pressurized heavy water reactor (PWHR). As a result, the device based on the TDLS is shown to be operated successfully in detecting a HDO molecule, which is generated from the leaked heavy water by an isotope exchange reaction between D2O and H2O. Additionally, it is suggested that the performance of the new device, such as sensitivity and stability, can be improved by adapting a cavity enhanced absorption spectroscopy and a compact DFB diode laser. We presume that this laser-based leak detector has several advantages over the conventional techniques currently employed in the nuclear power plant, such as radiation monitoring, humidity monitoring and FT-IR spectroscopy.

  3. Application of acoustic leak detection technology for the detection and location of leaks in light water reactors

    International Nuclear Information System (INIS)

    Kupperman, D.S.; Prine, D.; Mathieson, T.

    1988-10-01

    This report presents the results of a study to evaluate the adequacy of leak detection systems in light water reactors. The sources of numerous reported leaks and methods of detection have been documented. Research to advance the state of the art of acoustic leak detection is presented, and procedures for implementation are discussed. 14 refs., 70 figs., 10 tabs

  4. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

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

  5. Predictive value of BRCA1, ERCC1, ATP7B, PKM2, TOPOI, TOPΟ-IIA, TOPOIIB and C-MYC genes in patients with small cell lung cancer (SCLC) who received first line therapy with cisplatin and etoposide.

    Science.gov (United States)

    Karachaliou, Niki; Papadaki, Chara; Lagoudaki, Eleni; Trypaki, Maria; Sfakianaki, Maria; Koutsopoulos, Anastasios; Mavroudis, Dimitris; Stathopoulos, Efstathios; Georgoulias, Vassilis; Souglakos, John

    2013-01-01

    The aim of the study was to evaluate the predictive value of genes involved in the action of cisplatin-etoposide in Small Cell Lung Cancer (SCLC). 184 SCLC patients' primary tumour samples were analyzed for ERCCI, BRCA1, ATP7B, PKM2 TOPOI, TOPOIIA, TOPOIIB and C-MYC mRNA expression. All patients were treated with cisplatin-etoposide. The patients' median age was 63 years and 120 (65%) had extended stage, 75 (41%) had increased LDH serum levels and 131 (71%) an ECOG performance status was 0-1. Patients with limited stage, whose tumours expressed high ERCC1 (p=0.028), PKM2 (p=0.046), TOPOI (p=0.008), TOPOIIA (p=0.002) and TOPOIIB (p<0.001) mRNA had a shorter Progression Free Survival (PFS). In limited stage patients, high expression of ERCC1 (p=0.014), PKM2 (p=0.026), TOPOIIA (p=0.021) and TOPOIIB (p=0.019) was correlated with decreased median overall survival (mOS) while in patients with extended stage, only high TOPOIIB expression had a negative impact on Os (p=0.035). The favorable expression signature expression signature (low expression of ERCC1, PKM2, TOPOIIA and TOPOIIB) was correlated with significantly better PFS and Os in both LS-SCLC (p<0.001 and p=0.007, respectively) and ES-SCLC (p=0.007 and (p=0.011, respectively) group. The unfavorable expression signature was an independent predictor for poor PFS (HR: 3.18; p=0.002 and HR: 3.14; p=0.021) and Os (HR: 4.35; p=0.001and HR: 3.32; p=0.019) in both limited and extended stage, respectively. Single gene's expression analysis as well as the integrated analysis of ERCC1, PKM2, TOPOIIA and TOPOIIB may predict treatment outcome in patients with SCLC. These findings should be further validated in a prospective study.

  6. Steam generator leak detection at Bruce A Unit 1

    International Nuclear Information System (INIS)

    Maynard, K.J.; McInnes, D.E.; Singh, V.P.

    1997-01-01

    A new steam generator leak detection system was recently developed and utilized at Bruce A. The equipment is based on standard helium leak detection, with the addition of moisture detection and several other capability improvements. All but 1% of the Unit 1 Boiler 03 tubesheet was inspected, using a sniffer probe which inspected tubes seven at a time and followed by individual tube inspections. The leak search period was completed in approximately 24 hours, following a prerequisite period of several days. No helium leak indications were found anywhere on the boiler. A single water leak indication was found, which was subsequently confirmed as a through-wall defect by eddy current inspection. (author)

  7. Fluid pipeline leak detection and location with miniature RF tags

    Science.gov (United States)

    McIntyre, Timothy J.

    2017-05-16

    Sensors locate troublesome leaks in pipes or conduits that carry a flowing medium. These sensors, through tailored physical and geometric properties, preferentially seek conduit leaks or breaches due to flow streaming. The sensors can be queried via transceivers outside the conduit or located and interrogated inside by submersible unmanned vehicle to identify and characterize the nature of a leak. The sensors can be functionalized with other capabilities for additional leak and pipeline characterization if needed. Sensors can be recovered from a conduit flow stream and reused for future leak detection activities.

  8. Development of humidity leak detection system for pipelines of WWER

    International Nuclear Information System (INIS)

    Gonor, V.K.; Morozov, S.A.; Polionov, V.P.; Portyanoj, A.G.; Shvetsov, D.M.; Konoplev, N.I.; Molyavkin, A.N.

    2007-01-01

    With allowance made for the basic requirements for the NPP pipeline leak detection system and for the accumulated experience one analyzed the basic engineering approaches to design a humidity leak detection system for the WWER primary pipelines. Paper presents the results of the feasibility study of the diagnostic algorithm service ability and the leak detection humidity system characteristics. Making use of the Kupol-M code and of the thermal bench simulating the Kalinin NPP 3-rd power unit dull basalt heat insulation pipeline leak the system is shown to meet the basic requirements stipulated in the present-day leak-before-break concept [ru

  9. Redox regulation of ATP sulfurylase in microalgae

    Czech Academy of Sciences Publication Activity Database

    Prioretti, L.; Lebrun, R.; Gontero, B.; Giordano, Mario

    2016-01-01

    Roč. 478, č. 4 (2016), s. 1555-1562 ISSN 0006-291X Institutional support: RVO:61388971 Keywords : ATP sulfurylase * cysteine * Sulfur metabolism Subject RIV: EE - Microbiology, Virology Impact factor: 2.466, year: 2016

  10. Evaluation of methods for leak detection in reactor primary systems

    International Nuclear Information System (INIS)

    Kupperman, D.S.; Claytor, T.N.

    1985-01-01

    No currently available, single leak-detection method combines optimal leakage detection sensitivity, leak-locating ability, and leakage measurement accuracy. Technology is available to improve leak detection capability at specific sites by use of acoustic monitoring. However, current acoustic monitoring techniques provide no source discrimination (e.g., to distinguish between leaks from pipe cracks and valves) and no leak-rate information (a small leak may saturate the system). Seven cracks, including three field-induced IGSCC specimens and two thermal-fatigue cracks, have been installed in a laboratory acoustic leak detection facility. The IGSCC specimens produce stronger acoustic signals than the thermal-fatigue cracks at equivalent leak rates. Despite significant differences in crack geometry, the acoustic signals from the three IGSCC specimens, tested at the same leak rate, are virtually identical in the frequency range from 300 to 400 kHz. Thus, the quantitative correlations between the acoustic signals and leak rate in the 300-400 kHz band are very similar for the IGSCC specimens. Also, acoustic background data have been acquired during a hot functional test at the Watts Bar PWR. With these data, it is now possible to estimate the sensitivity of acoustic leak detection techniques. (orig.)

  11. Probabilistic pipe fracture evaluations for leak-rate-detection applications

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, S.; Ghadiali, N.; Paul, D.; Wilkowski, G. [Battelle, Columbus, OH (United States)

    1995-04-01

    Regulatory Guide 1.45, {open_quotes}Reactor Coolant Pressure Boundary Leakage Detection Systems,{close_quotes} was published by the U.S. Nuclear Regulatory Commission (NRC) in May 1973, and provides guidance on leak detection methods and system requirements for Light Water Reactors. Additionally, leak detection limits are specified in plant Technical Specifications and are different for Boiling Water Reactors (BWRs) and Pressurized Water Reactors (PWRs). These leak detection limits are also used in leak-before-break evaluations performed in accordance with Draft Standard Review Plan, Section 3.6.3, {open_quotes}Leak Before Break Evaluation Procedures{close_quotes} where a margin of 10 on the leak detection limit is used in determining the crack size considered in subsequent fracture analyses. This study was requested by the NRC to: (1) evaluate the conditional failure probability for BWR and PWR piping for pipes that were leaking at the allowable leak detection limit, and (2) evaluate the margin of 10 to determine if it was unnecessarily large. A probabilistic approach was undertaken to conduct fracture evaluations of circumferentially cracked pipes for leak-rate-detection applications. Sixteen nuclear piping systems in BWR and PWR plants were analyzed to evaluate conditional failure probability and effects of crack-morphology variability on the current margins used in leak rate detection for leak-before-break.

  12. Development of leak detector by radiation. 2

    International Nuclear Information System (INIS)

    Suzuki, Takashi; Okano, Yasuhiro; Chisaka, Haruo

    1997-01-01

    Leak detector by radiation has been developed by cooperative research between Water Authority and us. In his fiscal year, the most suitable arrangement of detector system was simulated by Monte Carlo method. The first, the experimental values were compared with the results of simulation. The second, calculation was carried out by changing the quality of reflective materials and distance between radiation source and detector. The simulation results were agreed with the experimental results. On the basis of the rate of presence of leak, the most suitable arrangement of detector system was obtained under the conditions that both radiation source and detector covered with graphite or iron of 5 cm thickness and separated each other 3 cm apart. However, by comparing FOM (figure of merit), the suitable arrangement was that radiation source and detector adjoined each other and covered by graphite or iron of 20 cm thickness. (S.Y.)

  13. Heavy water leak detection using diffusion sampler

    International Nuclear Information System (INIS)

    Joshi, M.L.; Hussain, S.A.

    1990-01-01

    In the Pressurrised Heavy Water Reactors (PHWRs) detection of the sources of heavy water leaks is importent both for the purpose of radiation hazard control as well as for the reduction of escape/loss of heavy water which, is an expensive nuclear material. This paper describes an application of tritium diffusion sampler for heavy water leak detection. The diffusion sampler comprises an usual tritium counting glass vial with a special orifice. The counting vial has water vapour, deficient in HTO concentration. The HTO present outside diffuses in the vial through the orifice, gets exchanged with water of the wet filter paper kept at the bottom and the moisture in the vial atmosphere which has HTO concentration lower than that outside. This results in continuation of net movement of HTO in the vial. The exchanged tritium is counted in liquid scintillation spectrometer. The method has a sensitivity of 10000 dpm/DAC-h. (author). 2 figs., 2 ta bs

  14. Hydrogen leak detection in the Space Shuttle

    Science.gov (United States)

    Barile, Ronald G

    1992-01-01

    This study focuses on a helium gas jet flowing into room air. Measurements of helium concentration and velocity in the jet-air mixture are reported. The objective is to learn about jet characteristics so that dynamically similar hydrogen leaks may be located in the Space Shuttle. The hazardous gas detection system (HGDS) in the mobile launch pad uses mass spectrometers to monitor the shuttle environment for leaks. The mass spectrometers are fed by long sample tubes which draw gas from the payload bay, mid body, aft engine compartment and external tank. The overall purpose of this study is to improve the HGDS especially in its potential for locating hydrogen leaks. A rapid-response leak detection experiment was designed, built, and tested, following on the work done in this program last summer. The apparatus included a Perkin Elmer MGA-1200 mass spectrometer and air velocity transducer, both monitored by a Macintosh IIFX computer using LabVIEW software. A jet of helium flowing into the lab air simulated a gas leak. Steady helium or hydrogen-nitrogen jets were logged for concentration and velocity, and the power spectral density of each was computed. Last year, large eddies and vortices were visually seen with Schlieren imaging, and they were detected in the time plots of the various instruments. The response time of the MGA-1200 was found in the range of 0.05 to 0.1 sec. Pulsed concentration waves were clearly detected at 25 cycles per sec by spectral analysis of MGA data. No peaks were detected in the power spectrum, so in the present study, 10 Hz bandwidth-averaged power levels were examined at regular frequency intervals. The practical consequences of last year's study are as follows: sampling frequency should be increased above the present rate of 1 sample per second so that transients could be observed and analyzed with frequency response methods. Many more experiments and conditions were observed in this second summer, including the effects of orifice diameter

  15. NCEP ATP III dan Framingham score

    OpenAIRE

    Hasan, Refli; Fahila, Reny

    2016-01-01

    Laporan ini merupakan Program Pendidikan Kolesterol National yang diperbaharui yaitu pedoman klinis untuk melakukan pengujian kolesterol dan manajemen. ATP III dibuat berdasarkan bukti dan laporan ekstensif yang akan menjadi referensi dan rekomendasi ilmiah. Laporan ATP III dapat dijadikan pedoman untuk pemberian terapi penurun kolesterol yang intensif dalam praktek. Pedoman ini hanya sebagai informasi , tidak dapat mempengaruhi secara mutlak dalam penilaian klinis dokter yang akhirnya menent...

  16. ATP-ase activity in the human oral mucous membrane, the guinea pig and the rabbit epidermis. A light- and electronmicroscopical investigation

    DEFF Research Database (Denmark)

    Zelander, T; Kirkeby, S

    1984-01-01

    The activity for ATP-ase was investigated in cells of rabbit and guinea pig epidermis and human oral mucosa. Observations both in the light- and electron microscope indicate that the ATP-ase positive cells of guinea pig and human epithelia are Langerhans cells while in the rabbit epidermis...

  17. Feedback regarding leaks in pipe work

    International Nuclear Information System (INIS)

    Guerville, C.; Boudouin, E.

    2011-01-01

    Contaminated effluent from nuclear medicine departments is stored in decay tanks before being discharged via the sewage system. Generally speaking, these tanks are located outside hospital wards, within the hospital's maintenance rooms. Pipes leading from the evacuation points (e.g. toilets in isolation wards) to the tanks may nonetheless pass through various other parts of the hospital premises (wards, corridors, offices, etc.). Should a leak occur in any of these pipes, this may impact on the public, workers or the environment. (author)

  18. Assessment of leak detection systems for LWRs

    International Nuclear Information System (INIS)

    Kupperman, D.S.

    1987-01-01

    This topical report summarizes work performed by the Argonne National Laboratory as subcontractor on on-line leak monitoring of LWRs during the 12 months from October 1985 to September 1986. The use of at least three different methods is recommended for leakage detection in reactors. Monitoring of sump-flow and airborne particulate radioactivity are suggested. A third method could involve monitoring condensate flow rate from air coolers or monitoring of airborne gaseous radioactivity

  19. Anastomotic leak detection by electrolyte electrical resistance.

    Science.gov (United States)

    DeArmond, Daniel T; Cline, Adam M; Johnson, Scott B

    2010-08-01

    To characterize a new method of postoperative gastrointestinal leak detection based on electrical resistance changes due to extravasated electrolyte contrast. Postoperative gastrointestinal leak results in increased patient morbidity, mortality, and hospital costs that can be mitigated by early diagnosis. A sensitive and specific diagnostic test that could be performed at the bedside has the potential to shorten the time to diagnosis and thereby improve the quality of treatment. Anaesthetized rats underwent celiotomy and creation of a 5-mm gastrotomy. In experimental animals, electrical resistance changes were measured with a direct current ohmmeter after the introduction of 5 cc of 23.4% NaCl electrolyte solution via gavage and measured with a more sensitive alternating current ohmmeter after the gavage of 1-5 cc of 0.9% NaCl. Comparison was made to negative controls and statistical analysis was performed. Leakage from the gastrotomy induced by as little as 1 cc of gavage-delivered 0.9% NaCl contrast solution was detectable as a statistically significant drop in electrical resistance when compared to results from negative controls. Electrical resistance change associated with electrolyte-gated leak detection is highly sensitive and specific and has the potential to be rapidly translated into clinical settings.

  20. Epidural block and neostigmine cause anastomosis leak

    Directory of Open Access Journals (Sweden)

    Ataro G

    2016-05-01

    Full Text Available Getu Ataro Department of Anesthesia, Jimma University, Jimma, EthiopiaI read the article by Phillips entitled, “Reducing gastrointestinal anastomotic leak rates: review of challenges and solutions”, published in the journal of Open Access Surgery with enthusiasm and found it crucial for perioperative management of patients with gastrointestinal (GI surgery, particularly anastomosis. I appreciate the author’s exhaustive search of literature and discussion with some limitation on review basics like methodology, which may affect the reliability of the review findings. The effects of risk factors for anastomosis leak, such as malnutrition, smoking, steroid use, bowel preparation, chemotherapy, duration of surgery, use of pressors, intravenous fluid administration, blood transfusion, and surgical anastomotic technique, were well discussed.1 However, from anesthesia perspective, there are some other well-studied risk factors that can affect healing of anastomosis wound and cause anastomosis leak. Among others, the effect of neostigmine and epidural block has been reported in many studies since half a century ago. View the original paper by Phillips

  1. Waste Transfer Leaks Control Decision Record

    International Nuclear Information System (INIS)

    RYAN, G.W.

    2000-01-01

    Control decision meetings for Waste Transfer Leaks were held on April 24,25,26, and 27, 2000. The agenda for the control decision meetings is included in Appendix A, and attendee lists are included in Appendix B. The purpose of the control decision meetings was to review and revise previously selected controls for the prevention or mitigation of waste transfer leak accidents. Re-evaluation of the controls is warranted due to revisions in the hazard and accident analysis for these Tank Farm events. In particular, calculated radiological consequences are significantly reduced from those currently reported in the Final Safety Analysis Report (FSAR). Revised hazard and accident analysis and a revised control recommendation will be reflected in an Authorization Basis Amendment to be submitted at the Department of Energy, Office of River Protection's (ORP's) request by June 30, 2000 to satisfy ORP Performance Incentive (PI) 2.1.1, Revision 1, ''Authorization Basis Management Process Efficiency Improvement''. The scope of the control decision meetings was to address all waste transfer leak-related hazardous conditions identified in the Tank Farm hazard analysis database, excluding those associated with the use of the Replacement Cross-Site Transfer System (RCSTS) slurry line and sluicing of Tank 241-C-106, which is addressed in FSAR Addendum 1. The scope of this control decision process does include future waste feed delivery waste transfer operations

  2. Color Changing Material for Hydrogen Leak Detection

    Science.gov (United States)

    Victor, Megan E.

    2014-01-01

    Kennedy Space Center scientists developed a hydrogen leak sensor utilizing a combination of chemochromic pigment and polymer that can be molded or fiber spun into rigid or flexible shapes such as tape. The sensor turns a dark color when exposed to hydrogen gas. This sensor has proven to be very effective for pinpointing the exact location of leaks in hydrogen gas lines and fittings at launch pads. Kennedy Space Center exclusively licensed this technology to the University of Central Florida (UCF), who also holds patents that are complimentary to KSC's. UCF has bundled the patents and exclusively licensed the portfolio to HySense Technology LLC, a startup company founded by a UCF professor who supports the UCF Florida Solar Energy Center (FSEC). HySense has fully developed its product (known as Intellipigment"TM"), and currently has five commercial customers. The company recently won the $100,000 first-place award at the CAT5 innovation competition at the Innovation Concourse of the Southeast: Safety & Manufacturing event in Orlando, FL. Commercial production and sales of this technology by HySense Technology will make this leak sensor widely available for use by NASA, DoD, and industries that utilize hydrogen gas.

  3. Mathematical models for explaining the Warburg effect: a review focussed on ATP and biomass production.

    Science.gov (United States)

    Schuster, Stefan; Boley, Daniel; Möller, Philip; Stark, Heiko; Kaleta, Christoph

    2015-12-01

    For producing ATP, tumour cells rely on glycolysis leading to lactate to about the same extent as on respiration. Thus, the ATP synthesis flux from glycolysis is considerably higher than in the corresponding healthy cells. This is known as the Warburg effect (named after German biochemist Otto H. Warburg) and also applies to striated muscle cells, activated lymphocytes, microglia, endothelial cells and several other cell types. For similar phenomena in several yeasts and many bacteria, the terms Crabtree effect and overflow metabolism respectively, are used. The Warburg effect is paradoxical at first sight because the molar ATP yield of glycolysis is much lower than that of respiration. Although a straightforward explanation is that glycolysis allows a higher ATP production rate, the question arises why cells do not re-allocate protein to the high-yield pathway of respiration. Mathematical modelling can help explain this phenomenon. Here, we review several models at various scales proposed in the literature for explaining the Warburg effect. These models support the hypothesis that glycolysis allows for a higher proliferation rate due to increased ATP production and precursor supply rates. © 2015 Authors; published by Portland Press Limited.

  4. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria.

    Science.gov (United States)

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P; Yi, Ling; Kaler, Stephen G; Lutsenko, Svetlana; Ralle, Martina

    2016-08-05

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Polyacrylamide gel electrophoresis and subspeciation of total cell proteins from multi-antibiotic-resistant skin diphtheroids labelled with [35S]methionine or [35S]thioATP and of coagulase negative staphylococci labelled with [35S]methionine

    International Nuclear Information System (INIS)

    Asante, M.; Holton, J.; Jackson, F.R.; Smith, I.

    1987-01-01

    The authors demonstrate that [ 35 S]methionine labelling of proteins followed by PAGE can be used to distinguish biochemically similar, multi-antibiotic-resistant skin diphtheroids; thirty-one isolates fell into four subgroups. The method also distinguished ten separate electrophoretypes of coagulase-negative staphylococci which correlated approximately with eight biotypes; the fifty-one isolates of biotypes SII, the commonest clinical isolate, were electrophoretically identical, thus suggesting that they are, indeed, members of a single subgroup. The authors also report a novel method of radiolabelling the phosphoproteins using [ 35 S]thioATP and have demonstrated the method using the above diphtheroids. These were again distinguished into the same four subgroups although the patterns of phosphoproteins were qualitatively and quantitatively different from those of the proteins. This thioATP labelling method should have wide application also. 14 refs.; 7 figs.; 4 tabs

  6. Single rod leak detection and repair of leaking or damaged fuel assemblies

    International Nuclear Information System (INIS)

    Beuneche, D.

    1986-01-01

    In some circumstances, it is necessary to perform rework operations on some fuel assemblies in order to make them reusable in reactors, movable, transportable or consistent with fuel reprocessor specifications, depending on the plant utility policy. These rework operations are of two types: - Those which consist in restoring the leak tightness of the fuel assemblies. They are made after a series of tests allowing the localization of the failed fuel rods: at first, overall leak detection is provided by monitoring primary coolant activity during reactor operation; then, during refuelling, leaking assemblies are identified by subjecting each of the assemblies scheduled for reloading to a sipping test; finally individual leaking fuel rods are singled out before the defective assemblies can be repaired, i.e. failed rods can be replaced. - Those which involve replacement of part of or the whole assembly structure (combined or not with replacement of failed fuel rods). In order to meet these two needs for rework operations, FRAGEMA has developed a full range of test and tooling systems for detecting single leaking rods in irradiated fuel assemblies and for restoring fuel assemblies to be used in PWR nuclear power plants. As an illustration of the means available, two of these systems are described

  7. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5⿲ triphosphate (ATP)

    Science.gov (United States)

    Hammami, Khaled; El-Feki, Hafed; Marsan, Olivier; Drouet, Christophe

    2016-01-01

    ATP is a well-known energy supplier in cells. The idea to associate ATP to pharmaceutical formulations/biotechnological devices to promote cells activity by potentially modulating their microenvironment thus appears as an appealing novel approach. Since biomimetic nanocrystalline apatites have shown great promise for biomedical applications (bone regeneration, cells diagnostics/therapeutics, ⿦), thanks to a high surface reactivity and an intrinsically high biocompatibility, the present contribution was aimed at exploring ATP/apatite interactions. ATP adsorption on a synthetic carbonated nanocrystalline apatite preliminarily characterized (by XRD, FTIR, Raman, TG-DTA and SEM-EDX) was investigated in detail, pointing out a good agreement with Sips isothermal features. Adsorption characteristics were compared to those previously obtained on monophosphate nucleotides (AMP, CMP), unveiling some specificities. ATP was found to adsorb effectively onto biomimetic apatite: despite smaller values of the affinity constant KS and the exponential factor m, larger adsorbed amounts were reached for ATP as compared to AMP for any given concentration in solution. m guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard οGads ° was estimated to only ⿿4 kJ/mol, the large value of Nmax led to significantly negative effective οGads values down to ⿿33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, ⿦).

  8. Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth

    Energy Technology Data Exchange (ETDEWEB)

    Ashino, T.; Varadarajan, S.; Urao, N.; Oshikawa, J.; Chen, G. -F.; Wang, H.; Huo, Y.; Finney, L.; Vogt, S.; McKinney, R. D.; Maryon, E. B.; Kaplan, J. H.; Ushio-Fukai, M.; Fukai, T. (Biosciences Division); ( XSD); ( PSC-USR); (Univ. of Illinois at Chicago); (Univ. of Minnesota)

    2010-09-09

    Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

  9. The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Westerhoff, H.V.; Snoep, J.L.

    2002-01-01

    Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch...... cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis...... (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells....

  10. Evaluation of advanced and current leak detection systems

    International Nuclear Information System (INIS)

    Kupperman, D.S.

    1988-01-01

    U.S. Nuclear Regulatory Commission Guide 1.45 recommends the use of at least three different detection methods in reactors to detect leakage. Monitoring of both sump-flow and airborne particulate radioactivity is mandatory. A third method can involve either monitoring of condensate flow rate from air coolers or monitoring of airborne gaseous radioactivity. Although the methods currently used for leak detection reflect the state of the art, other techniques may be developed and used. Since the recommendations of Regulatory Guide 1.45 are not mandatory, Licensee Event Report Compilations have been reviewed to help establish actual capabilities for leak detection. The review of event reports, which had previously covered the period of June 1985 to August 1986 has been extended, and now covers events to June 1987. The total number of significant events is now 83. These reports have provided documented, sometimes detailed, summaries of reactor leaks. They have helped establish the capabilities of existing systems to detect and locate leaks. Differences between PWRs and BWRs with regard to leak detection have now been analyzed. With regard to detection methods, the greatest differences between reactor types are as follows: (a) The sump pump is reported as the detection method more frequently in BWRs than in PWRs (64% vs. 35%). (b) The radiation monitor is reported as the detection method (excluding false alarms) more frequently in PWRs. Current efforts at Argonne National Laboratory (ANL) to evaluate advanced acoustic leak detection methods are directed toward the generation and analysis of acoustic data from large (0.5 to 10 gal/min) leaks and modification of the software of the GARD/ANL advanced acoustic leak detection system. In order to reach the goal of 10 gal/min leaks, the Steam Generator Test Facility at ANL has been modified to carry out the leak testing. Tests were carried out with water at 525 deg. F and 1100 psi leaking through a fatigue crack in a 4-in

  11. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

    Science.gov (United States)

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-01-01

    HIV type 1 (HIV-1) infects CD4+ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

  12. Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

    Science.gov (United States)

    Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

    2015-06-23

    HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

  13. Environmental risk comparisons with internal methods of UST leak detection

    International Nuclear Information System (INIS)

    Durgin, P.B.

    1993-01-01

    The past five years have seen a variety of advances in how leaks can be detected from within underground storage tanks. Any leak-detection approach employed within a storage tanks must be conducted at specific time intervals and meet certain leak-rate criteria according to federal and state regulations. Nevertheless, the potential environmental consequences of leak detection approaches differ widely. Internal, volumetric UST monitoring techniques have developed over time including: (1) inventory control with stick measurements, (2) precision tank testing, (3) automatic tank gauging (ATG), (4) statistical inventory reconciliation (SIR), and (5) statistical techniques with automatic tank gauging. An ATG focuses on the advantage of precise data but measured for only a brief period. On the other hand, stick data has less precision but when combined with SIR over extended periods it too can detect low leak rates. Graphs demonstrate the comparable amounts of fuel than can leak out of a tank before being detected by these techniques. The results indicate that annual tank testing has the greatest potential for large volumes of fuel leaking without detection while new statistical approaches with an ATG have the least potential. The environmental implications of the volumes of fuel leaked prior to detection are site specific. For example, if storage tank is surrounded by a high water table and in a sole-source aquifer even small leaks may cause problems. The user must also consider regulatory risks. The level of environmental and regulatory risk should influence selection of the UST leak detection method

  14. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation.

    Science.gov (United States)

    Jimenez, Laure; Laporte, Damien; Duvezin-Caubet, Stephane; Courtout, Fabien; Sagot, Isabelle

    2014-02-15

    Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities.

  15. ATP as a Multi-target Danger Signal in the Brain

    Directory of Open Access Journals (Sweden)

    Ricardo J Rodrigues

    2015-04-01

    Full Text Available ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR, which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection.

  16. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Michelle Barbi de Moura

    Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  17. The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

    Science.gov (United States)

    Nath, Sunil

    2016-12-01

    As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. LEAK: A source term generator for evaluating release rates from leaking vessels

    International Nuclear Information System (INIS)

    Clinton, J.H.

    1994-01-01

    An interactive computer code for estimating the rate of release of any one of several materials from a leaking tank or broken pipe leading from a tank is presented. It is generally assumed that the material in the tank is liquid. Materials included in the data base are acetonitrile, ammonia, carbon tetrachloride, chlorine, chlorine trifluoride, fluorine, hydrogen fluoride, nitric acid, nitrogen tetroxide, sodium hydroxide, sulfur hexafluoride, sulfuric acid, and uranium hexafluoride. Materials that exist only as liquid and/or vapor over expected ranges of temperature and pressure can easily be added to the data base file. The Fortran source code for LEAK and the data file are included with this report

  19. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid...

  20. Acoustic detection for water/steam leak from a tube of LMFBR steam generator

    International Nuclear Information System (INIS)

    Sonoda, Masataka; Shindo, Yoshihisa

    1989-01-01

    Acoustic leak detector is useful for detecting more quickly intermediate leak than the existing hydrogen detector and is available for identification of leak location on the accident of water/steam leak from a tube of LMFBR steam generator. This paper presents the overview of HALD (High frequency Acoustics Leak Detection) system, which is more sensitive for leak detection and lower cost of equipment for identification of leak location than a low frequency type detector. (author)

  1. Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

    Directory of Open Access Journals (Sweden)

    Leigh A. Swayne

    2017-08-01

    Full Text Available Pannexin 1 (Panx1 channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs. Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

  2. Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

    Science.gov (United States)

    Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

    2014-01-01

    The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

  3. Leak detection and location in MONJU steam generators

    International Nuclear Information System (INIS)

    Saito, T.; Kosugi, T.

    1978-01-01

    Leak detection system of MONJU steam generator depends mostly on in-sodium hydrogen detectors. The requirements on leak detector performance are determined from the point of view of protecting tube leak propagation due to wastage, and the process of determining the performance is shown briefly. Research and development activities on in-sodium hydrogen detectors are described and the specifications of leak detectors for MONJU are also presented. In-cover-gas hydrogen detector and acoustic detector are under development. Research and development activities on the leak location after steam generator shutdown by such methods as an electromagnetic method and ultrasonic method are described. The results of the research and development work on inserting the test probes into tubes are described also. An idea for finding the condition of tubes in the neighbourhood of the leak is also presented. (author)

  4. A Hydrogen Leak Detection System for Aerospace and Commercial Applications

    Science.gov (United States)

    Hunter, Gary W.; Makel, D. B.; Jansa, E. D.; Patterson, G.; Cova, P. J.; Liu, C. C.; Wu, Q. H.; Powers, W. T.

    1995-01-01

    Leaks on the space shuttle while on the launch pad have generated interest in hydrogen leak monitoring technology. Microfabricated hydrogen sensors are being fabricated at Case Western Reserve University (CWRU) and tested at NASA Lewis Research Center (LeRC). These sensors have been integrated into hardware and software designed by Aerojet. This complete system allows for multipoint leak monitoring designed to provide leak source and magnitude information in real time. The monitoring system processes data from the hydrogen sensors and presents the operator with a visual indication of the leak location and magnitude. Although the leak monitoring system was designed for hydrogen propulsion systems, the possible applications of this monitoring system are wide ranged. This system is in operation in an automotive application which requires high sensitivity to hydrogen.

  5. Leak Detection of High Pressure Feedwater Heater Using Empirical Models

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Song Kyu; Kim, Eun Kee [Korea Power Engineering Company, Daejeon (Korea, Republic of); Heo, Gyun Young [Kyung Hee University, Yongin (Korea, Republic of); An, Sang Ha [Korea Advanced Institute of Science and Technology, Daejeon (Korea, Republic of)

    2009-10-15

    Even small leak from tube side or pass partition within the high pressure feedwater heater (HPFWH) causes a significant deficiency in its performance. Plant operation under the HPFWH leak condition for long time will result in cost increase. Tube side leak within HPFWH can produce the high velocity jet of water and it can cause neighboring tube failures. However, most of plants are being operated without any information for internal leaks of HPFWH, even though it is prone to be damaged under high temperature and high pressure operating conditions. Leaks from tubes and/or pass partition of HPFWH occurred in many nuclear power plants, for example, Mihama PS-2, Takahama PS-2 and Point Beach Nuclear Plant Unit 1. If the internal leaks of HPFWH are monitored, the cost can be reduced by inexpensive repairs relative to loss in performance and moreover plant shutdown as well as further tube damages can be prevented.

  6. IR analyzer spots heavy water leaks

    International Nuclear Information System (INIS)

    Anon.

    1978-01-01

    A correlation spectrometer developed by Barringer Research Ltd. (in collaboration with Atomic Energy of Canada and Ontario Hydro) is used to measure HDO concentrations in DTO in the final (distillation) stage of heavy-water production. A unit has been installed at Bruce Heavy Water Plant. Previously, such spectrometers had been installed to detect heavy-water leaks in CANDU reactors. The principle on which the instrument works is explained, with illustrations. It works by comparing the absorption at 2.9 μm, due to HDO, with that at 2.6 μm, due to both HDO and D 2 O. (N.D.H.)

  7. Cerebrospinal fluid leak mimicking allergic rhinitis.

    Science.gov (United States)

    Ricketti, Anthony J; Cleri, Dennis J; Porwancher, Richard B; Panesar, Mandip; Villota, Francisco J; Seelagy, Marc M

    2005-01-01

    Rhinitis and rhinorrhea are common clinical complaints that may be allergic or nonallergic in etiology. Distinguishing between allergic and nonallergic etiologies can be difficult but necessary for treatment. Here, we present a case of a 50-year-old woman with > 20 years of rhinorrhea before a diagnosis of cerebrospinal fluid leak and a life-threatening complication occurred. It is essential that no symptom, especially that which persists and resists treatment, is trivialized. Here, we establish how a careful history and evaluation will direct the clinician to the correct diagnosis.

  8. One-Piece Leak-Proof Battery

    Science.gov (United States)

    Verhoog, Roelof

    1999-03-23

    The casing of a leak-proof one-piece battery is made of a material comprising a mixture of at least a matrix based on polypropylene and an alloy of a polyamide and a polypropylene. The ratio of the matrix to the alloy is in the range 0.5 to 6 by weight. The alloy forms elongate arborescent inclusions in the matrix such that, on average, the largest dimension of a segment of the arborescence is at least twenty times the smallest dimension of the segment.

  9. Pathway of processive ATP hydrolysis by kinesin

    Science.gov (United States)

    Gilbert, Susan P.; Webb, Martin R.; Brune, Martin; Johnson, Kenneth A.

    1995-02-01

    Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin - ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.

  10. Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors.

    Science.gov (United States)

    Stoop, R; Surprenant, A; North, R A

    1997-10-01

    The effect of changing extracellular pH was studied on the currents induced by ATP or alphabeta-methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X1, P2X3, or P2X4 receptors, the effect of ATP was decreased by acidification. In cells expressing P2X2 receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X2 and P2X3 receptors. At P2X2 receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pKa for this effect was 7.3. At P2X4 receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pKa for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons.

  11. Telescope sipping the optimum fuel leak detection system

    International Nuclear Information System (INIS)

    Deleryd, R.

    1998-01-01

    The TELESCOPE Sipping technology is an evolutionary development from previous ABB fuel leak systems used in LWR reactors. The system utilizes the existing dynamics that cause numerous fission products to leak from a failed fuel rod when the fuel assembly is raised from a reactor core during core fuel alterations. The system can also be used by repair work in pool side inspection in order to detect leaking rods or to verify reconstituted assemblies as non leakers. (author)

  12. Detection of steam generator tube leaks in pressurized water reactors

    International Nuclear Information System (INIS)

    Roach, W.H.

    1984-11-01

    This report addresses the early detection of small steam generator tube leaks in pressurized water reactors. It identifies physical parameters, establishes instrumentation performance goals, and specifies sensor types and locations. It presents a simple algorithm that yields the leak rate as a function of known or measurable quantities. Leak rates of less than one-tenth gram per second should be detectable with existing instrumentation

  13. Design of a Novel In-Pipe Reliable Leak Detector

    OpenAIRE

    Chatzigeorgiou, Dimitrios; Youcef-Toumi, Kamal; Ben-Mansour, Rached

    2013-01-01

    Leakage is the major factor for unaccounted losses in every pipe network around the world (oil, gas, or water). In most cases, the deleterious effects associated with the occurrence of leaks may present serious economical and health problems. Therefore, leaks must be quickly detected, located, and repaired. Unfortunately, most state-of-the-art leak detection systems have limited applicability, are neither reliable nor robust, while others depend on the user experience. In this paper, we prese...

  14. Leak noise detection and characterisation using statistical features

    International Nuclear Information System (INIS)

    Srinivasan, G.S.; Singh, Om Pal; Prabhakar, R.

    2000-01-01

    Covariance Matrix based statistical features have been used to detect leak, identify noise source and characterise noise transmission off-line on data from PFR Steam Generator Units. The methods show promise in their ability to detect leak in actual Steam Generator Units from the noise data with very low S/N ratios. Further analysis of data have yielded useful information on the potential of acoustic leak detection system

  15. Use of a steam leak simulator in EBR-II

    International Nuclear Information System (INIS)

    McKee, J.M.; Osterhout, M.M.; Batten, R.L.

    1984-01-01

    A steam leak simulator has been installed on EBR-II to periodically test and calibrate the steam-generator leak detection system. Measured amounts of molten anhydrous sodium hydroxide are injected at controlled rates simulating leaks in the range of 0.024 to 0.16 g H 2 O/s. Experience with 11 injections over an 18 month period is described

  16. Rosiglitazone Improves Stallion Sperm Motility, ATP Content, and Mitochondrial Function.

    Science.gov (United States)

    Swegen, Aleona; Lambourne, Sarah Renay; Aitken, R John; Gibb, Zamira

    2016-11-01

    Media used for equine sperm storage often contain relatively high concentrations of glucose, even though stallion spermatozoa preferentially utilize oxidative phosphorylation (OXPHOS) over glycolysis to generate ATP and support motility. Rosiglitazone is an antidiabetic compound that enhances metabolic flexibility and glucose utilization in various cell types, but its effects on sperm metabolism are unknown. This study investigated the effects of rosiglitazone on stallion sperm function in vitro, along with the possible role of AMP-activated protein kinase (AMPK) in mediating these effects. Spermatozoa were incubated with or without rosiglitazone, GW9662 (an antagonist of peroxisome proliferator-activating receptor-gamma), and compound C (CC; an AMPK inhibitor). Sperm motility, viability, reactive oxygen species production, mitochondrial membrane potential (mMP), ATP content, and glucose uptake capacity were measured. Samples incubated with rosiglitazone displayed significantly higher motility, percentage of cells with normal mMP, ATP content, and glucose uptake capacity, while sperm viability was unaffected. The percentage of spermatozoa positive for mitochondrial ROS was also significantly lower in rosiglitazone-treated samples. AMPK localized to the sperm midpiece, and its phosphorylation, was increased in rosiglitazone-treated spermatozoa. CC decreased sperm AMPK phosphorylation and reduced sperm motility, and successfully inhibited the effects of rosiglitazone. Inclusion of rosiglitazone in a room temperature sperm storage medium maintained sperm motility above 60% for 6 days, attaining significantly higher motility than sperm stored in control media. The ability of rosiglitazone to substantially alleviate the time-dependent deterioration of stallion spermatozoa by diverting metabolism away from OXPHOS and toward glycolysis has novel implications for the long-term, functional preservation of these cells. © 2016 by the Society for the Study of Reproduction

  17. Gd3+ and calcium sensitive, sodium leak currents are features of weak membrane-glass seals in patch clamp recordings.

    Directory of Open Access Journals (Sweden)

    Adrienne N Boone

    Full Text Available The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE and calcium (EEEE selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM was lowered to 0.1 mM and were inhibited (>40% to >90% with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (<30% to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 µM mibefradil or 100 µM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.

  18. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    Czech Academy of Sciences Publication Activity Database

    Čížková, Alena; Stránecký, V.; Ivánek, Robert; Hartmannová, H.; Nosková, L.; Piherová, L.; Tesařová, M.; Hansíková, H.; Honzík, T.; Zeman, J.; Divina, Petr; Potocká, A.; Paul, J.; Sperl, W.; Mayr, J. A.; Seneca, S.; Houštěk, J.; Kmoch, S.

    2008-01-01

    Roč. 9, - (2008), s. 38-38 ISSN 1471-2164 R&D Projects: GA ČR(CZ) GD303/03/H065; GA ČR(CZ) GA303/07/0781 Grant - others:GA MZd(CZ) NR8069 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50520514 Keywords : microarray * mitochondria * F1Fo ATP synthase deficiency Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.926, year: 2008

  19. Equivalent leak definitions for Smart LDAR (leak detection and repair) when using optical imaging technology.

    Science.gov (United States)

    Epperson, David; Lev-On, Miriam; Taback, Hal; Siegell, Jeffrey; Ritter, Karin

    2007-09-01

    Controlling fugitive emissions from leaks in petrochemical industry process equipment now requires periodic monitoring of valves, flanges, pumps etc., typically on a quarterly basis. Previous studies have shown that over 90% of the reducible emissions come from approximately 0.1% of the components, i.e. the large leakers. A new, and more cost-effective approach for controlling these large leakers would entail more frequent monitoring of process equipment, allowing for the detection and repair of the highly leaking components that contribute the most to emissions. This approach has been called "Smart LDAR." New optical imaging instruments, which significantly reduce monitoring costs, are now available to implement such an alternative work practice. This work describes the determination of the leak detection sensitivity (equivalency threshold) that an optical imaging instrument must achieve to ensure that it will provide at least the equivalent emission control of the current leak detection and repair practice. Equivalency thresholds were developed for various monitoring intervals. The U.S. Environment Protection Agency's Monte Carlo simulation approach was used to perform the analysis and to demonstrate that optical imaging, which is capable of identifying all of the largest leakers, can provide better control of fugitive emissions.

  20. Margins in high temperature leak-before-break assessments

    International Nuclear Information System (INIS)

    Budden, P.J.; Hooton, D.G.

    1997-01-01

    Developments in the defect assessment procedure R6 to include high-temperature mechanisms in Leak-before-Break arguments are described. In particular, the effect of creep on the ti