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Sample records for cell atp leaking

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

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    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. 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

  3. 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.

  4. 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, ou...... may be important in pancreas physiology and potentially in pancreas pathophysiology....... aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan...

  5. Renal epithelial cells can release ATP by vesicular fusion

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    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.

  6. The ATP/DNA Ratio Is a Better Indicator of Islet Cell Viability Than the ADP/ATP Ratio

    Science.gov (United States)

    Suszynski, T.M.; Wildey, G.M.; Falde, E.J.; Cline, G.W.; Maynard, K. Stewart; Ko, N.; Sotiris, J.; Naji, A.; Hering, B.J.; Papas, K.K.

    2009-01-01

    Real-time, accurate assessment of islet viability is critical for avoiding transplantation of nontherapeutic preparations. Measurements of the intracellular ADP/ATP ratio have been recently proposed as useful prospective estimates of islet cell viability and potency. However, dead cells may be rapidly depleted of both ATP and ADP, which would render the ratio incapable of accounting for dead cells. Since the DNA of dead cells is expected to remain stable over prolonged periods of time (days), we hypothesized that use of the ATP/DNA ratio would take into account dead cells and may be a better indicator of islet cell viability than the ADP/ATP ratio. We tested this hypothesis using mixtures of healthy and lethally heat-treated (HT) rat insulinoma cells and human islets. Measurements of ATP/DNA and ADP/ATP from the known mixtures of healthy and HT cells and islets were used to evaluate how well these parameters correlated with viability. The results indicated that ATP and ADP were rapidly (within 1 hour) depleted in HT cells. The fraction of HT cells in a mixture correlated linearly with the ATP/DNA ratio, whereas the ADP/ADP ratio was highly scattered, remaining effectively unchanged. Despite similar limitations in both ADP/ADP and ATP/DNA ratios, in that ATP levels may fluctuate significantly and reversibly with metabolic stress, the results indicated that ATP/DNA was a better measure of islet viability than the ADP/ATP ratio. PMID:18374063

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

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    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.

  8. Red blood cell dynamics: from cell deformation to ATP release.

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    Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

    2011-10-01

    The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release. This journal is © The Royal Society of Chemistry 2011

  9. Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions.

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    Harper, M E; Brand, M D

    1994-08-01

    Thyroid hormones have well-known effects on oxidative phosphorylation, but there is little quantitative information on their important sites of action. We have used top-down elasticity analysis, an extension of metabolic control analysis, to identify the sites of action of thyroid hormones on oxidative phosphorylation in rat hepatocytes. We divided the oxidative phosphorylation system into three blocks of reactions: the substrate oxidation subsystem, the phosphorylating subsystem, and the mitochondrial proton leak subsystem and have identified those blocks of reactions whose kinetics are significantly changed by hyperthyroidism. Our results show significant effects on the kinetics of the proton leak and the phosphorylating subsystems. Quantitative analyses revealed that 43% of the increase in resting respiration rate in hyperthyroid hepatocytes compared with euthyroid hepatocytes was due to differences in the proton leak and 59% was due to differences in the activity of the phosphorylating subsystem. There were no significant effects on the substrate oxidation subsystem. Changes in nonmitochondrial oxygen consumption accounted for -2% of the change in respiration rate. Top-down control analysis revealed that the distribution of control over the rates of mitochondrial oxygen consumption, ATP synthesis and consumption, and proton leak and over mitochondrial membrane potential (delta psi m) was similar in hepatocytes from hyperthyroid and littermate-paired euthyroid controls. The results of this study include the first complete top-down elasticity and control analyses of oxidative phosphorylation in hepatocytes from hyperthyroid rats.

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

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    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.

  11. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

    International Nuclear Information System (INIS)

    Shin, Youn Ho; Lee, Seo Jin; Jung, Junyang

    2013-01-01

    Highlights: ► ATP-treated sciatic explants shows the decreased expression of p75NGFR. ► Extracellular ATP inhibits the expression of phospho-ERK1/2. ► Lysosomal exocytosis is involved in Schwann cell dedifferentiation. ► Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

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

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    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.

  13. Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women.

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    Reinl, Erin L; Cabeza, Rafael; Gregory, Ismail A; Cahill, Alison G; England, Sarah K

    2015-10-01

    Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 μM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. 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-

  15. 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.)

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

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    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.

  17. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes.

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    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-02-11

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1-3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca(2+)-dependent lysosomal exocytosis.

  18. 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

  19. An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

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    Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

    2012-12-01

    Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

  20. ATP secretion from nerve trunks and Schwann cells mediated by glutamate.

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    Liu, Guo Jun; Bennett, Max R

    2003-11-14

    ATP release from rat sciatic nerves and from cultured Schwann cells isolated from the nerves was investigated using an online bioluminescence technique. ATP was released in relatively large amounts from rat sciatic nerve trunks during electrical stimulation. This release was blocked by the sodium channel inhibitor tetrodotoxin and the non-NMDA glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Schwann cells isolated from the nerve trunks did not release ATP when electrically stimulated but did in response to glutamate in a concentration-dependent manner. Glutamate-stimulated ATP release was inhibited by specific non-competitive AMPA receptor antagonist GYKI 52466 and competitive non-NMDA receptor antagonist CNQX. Glutamate-stimulated ATP release was decreased by inhibition of anion transporter inhibitors by furosemide, cystic fibrosis transmembrane conductance regulator by glibenclamide and exocytosis by botulinum toxin A, indicating that anion transporters and exocytosis provide the main secretion mechanisms for ATP release from the Schwann cells.

  1. ATP synthase--a marvellous rotary engine of the cell.

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    Yoshida, M; Muneyuki, E; Hisabori, T

    2001-09-01

    ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

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

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    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.

  3. The effects of membrane cholesterol and simvastatin on red blood cell deformability and ATP release.

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    Forsyth, Alison M; Braunmüller, Susanne; Wan, Jiandi; Franke, Thomas; Stone, Howard A

    2012-05-01

    It is known that deformation of red blood cells (RBCs) is linked to ATP release from the cells. Further, membrane cholesterol has been shown to alter properties of the cell membrane such as fluidity and bending stiffness. Membrane cholesterol content is increased in some cardiovascular diseases, for example, in individuals with acute coronary syndromes and chronic stable angina, and therefore, because of the potential clinical relevance, we investigated the influence of altered RBC membrane cholesterol levels on ATP release. Because of the correlation between statins and reduced membrane cholesterol in vivo, we also investigated the effects of simvastatin on RBC deformation and ATP release. We found that reducing membrane cholesterol increases cell deformability and ATP release. We also found that simvastatin increases deformability by acting directly on the membrane in the absence of the liver, and that ATP release was increased for cells with enriched cholesterol after treatment with simvastatin. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release.

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    Forsyth, Alison M; Wan, Jiandi; Owrutsky, Philip D; Abkarian, Manouk; Stone, Howard A

    2011-07-05

    RBCs are known to release ATP, which acts as a signaling molecule to cause dilation of blood vessels. A reduction in the release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. Furthermore, reduced deformation of RBCs has been correlated with myocardial infarction and coronary heart disease. Because ATP release has been linked to cell deformation, we undertook a multiscale approach to understand the links between single RBC dynamics, ATP release, and macroscopic viscosity all at physiological shear rates. Our experimental approach included microfluidics, ATP measurements using a bioluminescent reaction, and rheology. Using microfluidics technology with high-speed imaging, we visualize the deformation and dynamics of single cells, which are known to undergo motions such as tumbling, swinging, tanktreading, and deformation. We report that shear thinning is not due to cellular deformation as previously believed, but rather it is due to the tumbling-to-tanktreading transition. In addition, our results indicate that ATP release is constant at shear stresses below a threshold (3 Pa), whereas above the threshold ATP release is increased and accompanied by large cellular deformations. Finally, performing experiments with well-known inhibitors, we show that the Pannexin 1 hemichannel is the main avenue for ATP release both above and below the threshold, whereas, the cystic fibrosis transmembrane conductance regulator only contributes to deformation-dependent ATP release above the stress threshold.

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

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    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.

  6. 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.

  7. Dynamics of shear-induced ATP release from red blood cells.

    Science.gov (United States)

    Wan, Jiandi; Ristenpart, William D; Stone, Howard A

    2008-10-28

    Adenosine triphosphate (ATP) is a regulatory molecule for many cell functions, both for intracellular and, perhaps less well known, extracellular functions. An important example of the latter involves red blood cells (RBCs), which help regulate blood pressure by releasing ATP as a vasodilatory signaling molecule in response to the increased shear stress inside arterial constrictions. Although shear-induced ATP release has been observed widely and is believed to be triggered by deformation of the cell membrane, the underlying mechanosensing mechanism inside RBCs is still controversial. Here, we use an in vitro microfluidic approach to investigate the dynamics of shear-induced ATP release from human RBCs with millisecond resolution. We demonstrate that there is a sizable delay time between the onset of increased shear stress and the release of ATP. This response time decreases with shear stress, but surprisingly does not depend significantly on membrane rigidity. Furthermore, we show that even though the RBCs deform significantly in short constrictions (duration of increased stress <3 ms), no measurable ATP is released. This critical timescale is commensurate with a characteristic membrane relaxation time determined from observations of the cell deformation by using high-speed video. Taken together our results suggest a model wherein the retraction of the spectrin-actin cytoskeleton network triggers the mechanosensitive ATP release and a shear-dependent membrane viscosity controls the rate of release.

  8. The Contribution of Red Blood Cell Dynamics to Intrinsic Viscosity and Functional ATP Release

    Science.gov (United States)

    Forsyth, Alison; Abkarian, Manouk; Wan, Jiandi; Stone, Howard

    2010-11-01

    In shear flow, red blood cells (RBCs) exhibit a variety of behaviors such as rouleaux formation, tumbling, swinging, and tank-treading. The physiological consequences of these dynamic behaviors are not understood. In vivo, ATP is known to signal vasodilation; however, to our knowledge, no one has deciphered the relevance of RBC microrheology to the functional release of ATP. Previously, we correlated RBC deformation and ATP release in microfluidic constrictions (Wan et al., 2008). In this work, a cone-plate rheometer is used to shear a low hematocrit solution of RBCs at varying viscosity ratios (λ) between the inner cytoplasmic hemoglobin and the outer medium, to determine the intrinsic viscosity of the suspension. Further, using a luciferin-luciferase enzymatic reaction, we report the relative ATP release at varying shear rates. Results indicate that for λ = 1.6, 3.8 and 11.1, ATP release is constant up to 500 s-1, which suggests that the tumbling-tanktreading transition does not alter ATP release in pure shear. For lower viscosity ratios, λ = 1.6 and 3.8, at 500 s-1 a change in slope occurs in the intrinsic viscosity data and is marked by an increase in ATP release. Based on microfluidic observations, this simultaneous change in viscosity and ATP release occurs within the tank-treading regime.

  9. Extracellular ATP reduces HIV-1 transfer from immature dendritic cells to CD4+ T lymphocytes

    Directory of Open Access Journals (Sweden)

    Barat Corinne

    2008-03-01

    Full Text Available Abstract Background Dendritic cells (DCs are considered as key mediators of the early events in human immunodeficiency virus type 1 (HIV-1 infection at mucosal sites. Previous studies have shown that surface-bound virions and/or internalized viruses found in endocytic vacuoles of DCs are efficiently transferred to CD4+ T cells. Extracellular adenosine triphosphate (ATP either secreted or released from necrotic cells induces a distorted maturation of DCs, transiently increases their endocytic capacity and affects their migratory capacity. Knowing that high extracellular ATP concentrations are present in situations of tissue injury and inflammation, we investigated the effect of ATP on HIV-1 transmission from DCs to CD4+ T lymphocytes. Results In this study, we show that extracellular ATP reduces HIV-1 transfer from immature monocyte-derived DCs (iDCs to autologous CD4+ T cells. This observed decrease in viral replication was related to a lower proportion of infected CD4+ T cells following transfer, and was seen with both X4- and R5-tropic isolates of HIV-1. Extracellular ATP had no effect on direct CD4+ T cell infection as well as on productive HIV-1 infection of iDCs. These observations indicate that extracellular ATP affects HIV-1 infection of CD4+ T cells in trans with no effect on de novo virus production by iDCs. Additional experiments suggest that extracellular ATP might modulate the trafficking pathway of internalized virions within iDCs leading to an increased lysosomal degradation, which could be partly responsible for the decreased HIV-1 transmission. Conclusion These results suggest that extracellular ATP can act as a factor controlling HIV-1 propagation.

  10. 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, ident......, identified as ATP-ase positive dendritic cells, have almost disappeared from the transplanted epithelium whereas at day 21 after transplantation such cells were abundant. It is suggested that the ATP-ase positive cells which reappear in the transplanted epithelium are of mouse origin....

  11. Maitotoxin-induced liver cell death involving loss of cell ATP following influx of calcium

    International Nuclear Information System (INIS)

    Kutty, R.K.; Singh, Y.; Santostasi, G.; Krishna, G.

    1989-01-01

    Maitotoxin, one of the most potent marine toxins known, produced cell death in cultures of rat hepatocytes with a TD50 of 80 pM at 24 hr. The cell death, as indicated by a dose- and time-dependent leakage of lactate dehydrogenase (LDH), was also associated with the leakage of [14C]adenine nucleotides from hepatocytes prelabeled with [14C]-adenine. The toxic effect of maitotoxin was completely abolished by the omission of calcium from the culture medium. The cell death induced by maitotoxin increased with increasing concentrations of calcium in the medium. Treatment of hepatocytes with low concentrations of the toxin (less than 0.5 ng/ml) resulted in increases in 45Ca influx into the cells. At higher concentrations of maitotoxin (greater than 1ng/ml), the initial increase in 45Ca influx was followed by the release of the 45Ca from the cells into the medium. Since the 45Ca release paralleled the LDH leakage, the release of calcium was due to cell death. The 45Ca influx, [14C]adenine nucleotide leakage, and LDH leakage were effectively inhibited by verapamil, a calcium channel blocker. Maitotoxin also induced a time- and dose-dependent loss of ATP from hepatocytes, which preceded the [14C]adenine nucleotide and LDH leakage. Thus, it appears that the cell death resulting from maitotoxin treatment is caused by the elevated intracellular calcium, which in turn inhibits mitochondrial oxidative phosphorylation causing depletion of cell ATP. Loss of cell ATP may be the causative event in the maitotoxin-induced cell death

  12. Use of luciferase probes to measure ATP in living cells and animals.

    Science.gov (United States)

    Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

    2017-08-01

    ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

  13. 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

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

    Directory of Open Access Journals (Sweden)

    Reedijk Jan

    2008-06-01

    Full Text Available Abstract Background 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. Methods 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 Results 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. Conclusion 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

  15. ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

    Science.gov (United States)

    Bohlken, A; Cheung, B B; Bell, J L; Koach, J; Smith, S; Sekyere, E; Thomas, W; Norris, M; Haber, M; Lovejoy, D B; Richardson, D R; Marshall, G M

    2009-01-01

    Increased retinoic acid receptor β (RARβ2) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARβ2 expression is a common feature of many human cancers, suggesting that RARβ2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARβ2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARβ2 protein alone was sufficient for the growth inhibitory effects of RARβ2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARβ2. The ectopic overexpression of the RARβ2 ABC domain was sufficient to induce ATP7A expression, whereas, RARβ2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor β and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism. PMID:19127267

  16. ATP-gamma-S shifts the operating point of outer hair cell transduction towards scala tympani.

    Science.gov (United States)

    Bobbin, Richard P; Salt, Alec N

    2005-07-01

    ATP receptor agonists and antagonists alter cochlear mechanics as measured by changes in distortion product otoacoustic emissions (DPOAE). Some of the effects on DPOAEs are consistent with the hypothesis that ATP affects mechano-electrical transduction and the operating point of the outer hair cells (OHCs). This hypothesis was tested by monitoring the effect of ATP-gamma-S on the operating point of the OHCs. Guinea pigs anesthetized with urethane and with sectioned middle ear muscles were used. The cochlear microphonic (CM) was recorded differentially (scala vestibuli referenced to scala tympani) across the basal turn before and after perfusion (20 min) of the perilymph compartment with artificial perilymph (AP) and ATP-gamma-S dissolved in AP. The operating point was derived from the cochlear microphonics (CM) recorded in response low frequency (200 Hz) tones at high level (106, 112 and 118 dB SPL). The analysis procedure used a Boltzmann function to simulate the CM waveform and the Boltzmann parameters were adjusted to best-fit the calculated waveform to the CM. Compared to the initial perfusion with AP, ATP-gamma-S (333 microM) enhanced peak clipping of the positive peak of the CM (that occurs during organ of Corti displacements towards scala tympani), which was in keeping with ATP-induced displacement of the transducer towards scala tympani. CM waveform analysis quantified the degree of displacement and showed that the changes were consistent with the stimulus being centered on a different region of the transducer curve. The change of operating point meant that the stimulus was applied to a region of the transducer curve where there was greater saturation of the output on excursions towards scala tympani and less saturation towards scala vestibuli. A significant degree of recovery of the operating point was observed after washing with AP. Dose response curves generated by perfusing ATP-gamma-S (333 microM) in a cumulative manner yielded an EC(50) of 19.8 micro

  17. 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.

  18. 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.

  19. Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

    Science.gov (United States)

    Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E; Spurgeon, Harold A; Sollott, Steven J; Lakatta, Edward G

    2011-11-01

    In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. Published by Elsevier Ltd.

  20. ATP Binding cassette transporter gene expression in rat liver progenitor cells

    NARCIS (Netherlands)

    Ros, J.E.; Roskams, T.A.D.; Geuken, M.; Havinga, R.; Splinter, P.L.; Petersen, B.E.; LaRusso, N.F.; Kolk, van der D.M.; Kuipers, F.; Faber, K.N.; Müller, M.R.; Jansen, P.L.M.

    2003-01-01

    Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

  1. ATP binding cassette transporter gene expression in rat liver progenitor cells

    NARCIS (Netherlands)

    Ros, J. E.; Roskams, T. A. D.; Geuken, M.; Havinga, R.; Splinter, P. L.; Petersen, B. E.; LaRusso, N. F.; van der Kolk, D. M.; Kuipers, F.; Faber, K. N.; Müller, M.; Jansen, P. L. M.

    2003-01-01

    BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

  2. ATP binding cassette transporter gene expression in rat liver progenitor cells

    NARCIS (Netherlands)

    Ros, J.E.; Roskams, TAD; Geuken, M; Havinga, R; Splinter, PL; Petersen, BE; LaRusso, NF; van der Kolk, D.M.; Kuipers, F; Faber, KN; Muller, M; Jansen, PLM

    Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

  3. 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

    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...

  4. Interlaboratory comparison of red-cell ATP, 2,3-diphosphoglycerate and haemolysis measurements.

    Science.gov (United States)

    Hess, J R; Kagen, L R; van der Meer, P F; Simon, T; Cardigan, R; Greenwalt, T J; AuBuchon, J P; Brand, A; Lockwood, W; Zanella, A; Adamson, J; Snyder, E; Taylor, H L; Moroff, G; Hogman, C

    2005-07-01

    Red blood cell (RBC) storage systems are licensed based on their ability to prevent haemolysis and maintain RBC 24-h in vivo recovery. Preclinical testing includes measurement of RBC ATP as a surrogate for recovery, 2,3-diphosphoglycerate (DPG) as a surrogate for oxygen affinity, and free haemoglobin, which is indicative of red cell lysis. The reproducibility of RBC ATP, DPG and haemolysis measurements between centres was investigated. Five, 4-day-old leucoreduced AS-1 RBC units were pooled, aliquotted and shipped on ice to 14 laboratories in the USA and European Union (EU). Each laboratory was to sample the bag twice on day 7 and measure RBC ATP, DPG, haemoglobin and haemolysis levels in triplicate on each sample. The variability of results was assessed by using coefficients of variation (CV) and analysis of variance. Measurements were highly reproducible at the individual sites. Between sites, the CV was 16% for ATP, 35% for DPG, 2% for total haemoglobin and 54% for haemolysis. For ATP and total haemoglobin, 94 and 80% of the variance in measurements was contributed by differences between sites, and more than 80% of the variance for DPG and haemolysis measurements came from markedly discordant results from three sites and one site, respectively. In descending order, mathematical errors, unvalidated analytical methods, a lack of shared standards and fluid handling errors contributed to the variability in measurements from different sites. While the methods used by laboratories engaged in RBC storage system clinical trials demonstrated good precision, differences in results between laboratories may hinder comparative analysis. Efforts to improve performance should focus on developing robust methods, especially for measuring RBC ATP.

  5. Pluripotent Stem Cell Metabolism and Mitochondria: Beyond ATP

    Directory of Open Access Journals (Sweden)

    Jarmon G. Lees

    2017-01-01

    Full Text Available Metabolism is central to embryonic stem cell (ESC pluripotency and differentiation, with distinct profiles apparent under different nutrient milieu, and conditions that maintain alternate cell states. The significance of altered nutrient availability, particularly oxygen, and metabolic pathway activity has been highlighted by extensive studies of their impact on preimplantation embryo development, physiology, and viability. ESC similarly modulate their metabolism in response to altered metabolite levels, with changes in nutrient availability shown to have a lasting impact on derived cell identity through the regulation of the epigenetic landscape. Further, the preferential use of glucose and anaplerotic glutamine metabolism serves to not only support cell growth and proliferation but also minimise reactive oxygen species production. However, the perinuclear localisation of spherical, electron-poor mitochondria in ESC is proposed to sustain ESC nuclear-mitochondrial crosstalk and a mitochondrial-H2O2 presence, to facilitate signalling to support self-renewal through the stabilisation of HIFα, a process that may be favoured under physiological oxygen. The environment in which a cell is grown is therefore a critical regulator and determinant of cell fate, with metabolism, and particularly mitochondria, acting as an interface between the environment and the epigenome.

  6. 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

  7. Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells.

    Science.gov (United States)

    Buravkova, L B; Rylova, Y V; Andreeva, E R; Kulikov, A V; Pogodina, M V; Zhivotovsky, B; Gogvadze, V

    2013-10-01

    Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs. Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide. We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs. Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state. These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. 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.

  9. Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

    Science.gov (United States)

    De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

    2014-01-01

    Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

  10. 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.

  11. 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

  12. Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.

    Science.gov (United States)

    Liu, Ying; Geng, Yue-Hang; Yang, Hui; Yang, Han; Zhou, Yan-Ting; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

    2018-05-04

    Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis. Copyright © 2018. Published by Elsevier B.V.

  13. 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......Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium...... stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied...

  14. 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.

  15. Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

    Science.gov (United States)

    Kozgunova, Elena; Higashiyama, Tetsuya; Kurihara, Daisuke

    2016-10-02

    Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. Analysis of various tracts of mastoid air cells related to CSF leak after the anterior transpetrosal approach.

    Science.gov (United States)

    Tamura, Ryota; Tomio, Ryosuke; Mohammad, Farrag; Toda, Masahiro; Yoshida, Kazunari

    2018-03-16

    OBJECTIVE The anterior transpetrosal approach (ATPA) was established in 1984 and has been particularly effective for petroclival tumors. Although some complications associated with this approach, such as venous hemorrhage in the temporal lobe and nervous disturbances, have been resolved over the years, the incidence rate of CSF leaks has not greatly improved. In this study, some varieties of air cell tracts that are strongly related to CSF leaks are demonstrated. In addition, other pre- and postoperative risk factors for CSF leakage after ATPA are discussed. METHODS Preoperative and postoperative target imaging of the temporal bone was performed in a total of 117 patients who underwent ATPA, and various surgery-related parameters were analyzed. RESULTS The existence of air cells at the petrous apex, as well as fluid collection in the mastoid antrum detected by a postoperative CT scan, were possible risk factors for CSF leakage. Tracts that directly connected to the antrum from the squamous part of the temporal bone and petrous apex, rather than through numerous air cells, were significantly related to CSF leak and were defined as "direct tract." All patients with a refractory CSF leak possessed "unusual tracts" that connected to the attic, tympanic cavity, or eustachian tube, rather than through the mastoid antrum. CONCLUSIONS Preoperative assessment of petrous pneumatization types is necessary to prevent CSF leaks. Direct and unusual tracts are particularly strong risk factors for CSF leaks.

  2. Determination of glucose deficiency-induced cell death by mitochondrial ATP generation-driven proton homeostasis

    Institute of Scientific and Technical Information of China (English)

    Yanfen Cui; Yuanyuan Wang; Miao Liu; Li Qiu; Pan Xing; Xin Wang; Guoguang Ying; Binghui Li

    2017-01-01

    Glucose is one of major nutrients and its catabolism provides energy and/or building bricks for cell proliferation.Glucose deficiency results in cell death.However,the underlying mechanism still remains elusive.By using our recently developed method to monitor real-time cellular apoptosis and necrosis,we show that glucose deprivation can directly elicit necrosis,which is promoted by mitochondrial impairment,depending on mitochondrial adenosine triphosphate (ATP) generation instead of ATP depletion.We demonstrate that glucose metabolism is the major source to produce protons.Glucose deficiency leads to lack of proton provision while mitochondrial electron transfer chain continues consuming protons to generate energy,which provokes a compensatory iysosomal proton effiux and resultant increased lysosomal pH.This lysosomal alkalinization can trigger apoptosis or necrosis depending on the extent of alkalinization.Taken together,our results build up a metabolic connection between glycolysis,mitochondrion,and lysosome,and reveal an essential role of glucose metabolism in maintaining proton homeostasis to support cell survival.

  3. Local ATP generation by brain-type creatine kinase (CK-B facilitates cell motility.

    Directory of Open Access Journals (Sweden)

    Jan W P Kuiper

    Full Text Available BACKGROUND: Creatine Kinases (CK catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics. CONCLUSION/SIGNIFICANCE: Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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...

  10. ATP signals

    DEFF Research Database (Denmark)

    Novak, Ivana

    2016-01-01

    The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas......The Department of Biology at the University of Copenhagen explains the function of ATP signalling in the pancreas...

  11. Release of ATP from Marginal Cells in the Cochlea of Neonatal Rats Can Be Induced by Changes in Extracellular and Intracellular Ion Concentrations

    Science.gov (United States)

    Peng, Yating; Chen, Jie; He, Shan; Yang, Jun; Wu, Hao

    2012-01-01

    Background Adenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. Methods Sprague-Dawley rats aged 1–3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. Results Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K+ and intra- and extracellular Ca2+. Furthermore, changes in the concentration of intracellular Ca2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. Conclusion We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A2. PMID:23071731

  12. ATP Release Channels

    Directory of Open Access Journals (Sweden)

    Akiyuki Taruno

    2018-03-01

    Full Text Available Adenosine triphosphate (ATP has been well established as an important extracellular ligand of autocrine signaling, intercellular communication, and neurotransmission with numerous physiological and pathophysiological roles. In addition to the classical exocytosis, non-vesicular mechanisms of cellular ATP release have been demonstrated in many cell types. Although large and negatively charged ATP molecules cannot diffuse across the lipid bilayer of the plasma membrane, conductive ATP release from the cytosol into the extracellular space is possible through ATP-permeable channels. Such channels must possess two minimum qualifications for ATP permeation: anion permeability and a large ion-conducting pore. Currently, five groups of channels are acknowledged as ATP-release channels: connexin hemichannels, pannexin 1, calcium homeostasis modulator 1 (CALHM1, volume-regulated anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR anion channels, and maxi-anion channels (MACs. Recently, major breakthroughs have been made in the field by molecular identification of CALHM1 as the action potential-dependent ATP-release channel in taste bud cells, LRRC8s as components of VRACs, and SLCO2A1 as a core subunit of MACs. Here, the function and physiological roles of these five groups of ATP-release channels are summarized, along with a discussion on the future implications of understanding these channels.

  13. 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.

  14. 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.

  15. 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.

  16. Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

    International Nuclear Information System (INIS)

    Mistafa, Oras; Hoegberg, Johan; Stenius, Ulla

    2008-01-01

    Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells

  17. Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mistafa, Oras; Hoegberg, Johan [Institute of Environmental Medicine, Karolinska Institutet, Box 210, 17177 Stockholm (Sweden); Stenius, Ulla [Institute of Environmental Medicine, Karolinska Institutet, Box 210, 17177 Stockholm (Sweden)

    2008-01-04

    Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells.

  18. Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity

    Directory of Open Access Journals (Sweden)

    Francesca Schena

    2013-06-01

    Full Text Available Immunoglobulin (Ig isotype diversification by class switch recombination (CSR is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID. Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

  19. A new approach for helium backfilling and leak testing seal-welded capsules in a hot cell

    International Nuclear Information System (INIS)

    Strasslsund, E.K.; Berger, D.N.

    1992-05-01

    Gamma irradiation sources containing radioactive 137 Cesium Chloride are being produced at the US Department of Energy's Hanford Site as part of a Westinghouse Hanford company/Pacific Northwest Laboratory cooperative program. New equipment was developed to leak test the double-encapsulated sources in a hot cell. The equipment, which includes a helium backfill chamber and end cap press , a vacuum chamber, and a helium mass spectrometer, has provided technicians with the capability to detect leaks in sealed sources as small as 1. 0x10 -7 atm cm 3 /S helium

  20. Surface-Enhanced Raman Spectroscopy Study of 4-ATP on Gold Nanoparticles for Basal Cell Carcinoma Fingerprint Detection

    Science.gov (United States)

    Quynh, Luu Manh; Nam, Nguyen Hoang; Kong, K.; Nhung, Nguyen Thi; Notingher, I.; Henini, M.; Luong, Nguyen Hoang

    2016-05-01

    The surface-enhanced Raman signals of 4-aminothiophenol (4-ATP) attached to the surface of colloidal gold nanoparticles with size distribution of 2 to 5 nm were used as a labeling agent to detect basal cell carcinoma (BCC) of the skin. The enhanced Raman band at 1075 cm-1 corresponding to the C-S stretching vibration in 4-ATP was observed during attachment to the surface of the gold nanoparticles. The frequency and intensity of this band did not change when the colloids were conjugated with BerEP4 antibody, which specifically binds to BCC. We show the feasibility of imaging BCC by surface-enhanced Raman spectroscopy, scanning the 1075 cm-1 band to detect the distribution of 4-ATP-coated gold nanoparticles attached to skin tissue ex vivo.

  1. Low-level light therapy potentiates NPe6-mediated photodynamic therapy in a human osteosarcoma cell line via increased ATP.

    Science.gov (United States)

    Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R

    2015-03-01

    Low-level light therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-l-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5J/cm(2) of 810nm near infrared radiation (NIR) followed by addition of 10μM NPe6 and after 2h incubation by 1.5J/cm(2) of 652nm red light for PDT. PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Proposal of leak path passivation for InGaN solar cells to reduce the leakage current

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ke, E-mail: ke.wang@chiba-u.jp; Imai, Daichi; Kusakabe, Kazuhide [Center for SMART Green Innovation Research, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Yoshikawa, Akihiko, E-mail: yoshi@faculty.chiba-u.jp [Center for SMART Green Innovation Research, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Department of Information and Communication Engineering, Graduate School of Engineering, Kogakuin University, Nakano-cho, Hachioji, Tokyo 2665-1 (Japan)

    2016-01-25

    We propose some general ways to passivate the leak paths in InGaN solar cells and report some experimental evidences of its effectiveness. By adopting an AlOx passivation process, the photovoltaic performances of GaN pn-junctions and InGaN solar cells, grown by molecular beam epitaxy, have been significantly improved. The open circuit voltage under 1 sun illumination increases from 1.46 to 2.26 V for a GaN pn junction, and from 0.95 to 1.27 V for an InGaN solar cell, demonstrating evidence of leak path passivation (LPP) by AlOx. The proposed LPP is expected to be a realistic way to exploit the potential of thick and relaxed but defective InGaN for solar cell applications.

  3. Osteoblast-like MC3T3-E1 Cells Prefer Glycolysis for ATP Production but Adipocyte-like 3T3-L1 Cells Prefer Oxidative Phosphorylation.

    Science.gov (United States)

    Guntur, Anyonya R; Gerencser, Akos A; Le, Phuong T; DeMambro, Victoria E; Bornstein, Sheila A; Mookerjee, Shona A; Maridas, David E; Clemmons, David E; Brand, Martin D; Rosen, Clifford J

    2018-06-01

    Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes, and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of preosteoblast MC3T3-E1 cells and calvarial osteoblasts with preadipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met adenosine triphosphate (ATP) demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas nondifferentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared with nondifferentiated osteoblasts. In contrast, both nondifferentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that nondifferentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts, they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes, they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. © 2018 American Society for Bone and Mineral Research. © 2018 American Society for Bone and Mineral Research.

  4. 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

  5. Cation depletion by the sodium pump in red cells with pathologic cation leaks. Sickle cells and xerocytes.

    Science.gov (United States)

    Joiner, C H; Platt, O S; Lux, S E

    1986-12-01

    The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blocked by ouabain, removal of external potassium, or pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonate, which blocks the increase in cation permeability induced by deoxygenation. The loss of cation exhibited by oxygenated xerocytes similarly incubated was also blocked by ouabain. These data support the hypothesis that the elevated "passive" cation fluxes of xerocytes and deoxygenated sickle cells are not directly responsible for cation depletion of these cells; rather, these pathologic leaks interact with the sodium pump to produce a net loss of cellular cation.

  6. 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.

  7. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    Science.gov (United States)

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  8. [Mg2+, ATP-dependent plasma membrane calcium pump of smooth muscle cells. I. Structural organization and properties].

    Science.gov (United States)

    Veklich, T O; Mazur, Iu Iu; Kosterin, S O

    2015-01-01

    Tight control of cytoplasm Ca2+ concentration is essential in cell functioning. Changing of Ca2+ concentration is thorough in smooth muscle cells, because it determines relaxation/constraint process. One of key proteins which control Ca2+ concentration in cytoplasm is Mg2+, ATP-dependent plasma membrane calcium pump. Thus, it is important to find compoumds which allowed one to change Mg2+, ATP-dependent plasma membrane calcium pump activity, as long as this topic is of current interest in biochemical research which regards energy and pharmacomechanical coupling mechanism of muscle excitation and contraction. In this article we generalized literatute and own data about properties of smooth muscle cell plasma membrane Ca(2+)-pump. Stuctural oganization, kinetical properties and molecular biology are considered.

  9. 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.

  10. 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.

  11. Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

    Science.gov (United States)

    Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H; Bhakta, Sanjib

    2013-01-01

    ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

  12. Change in ATP-ase activity and transport of rna of liver cell nuclei of pregnant rats and embryos following irradiation

    International Nuclear Information System (INIS)

    Shamsutdinova, G.T.; Mirkhamidova, P.; Ibragimkhodzhaeva, M.P.; Mirakhmedov, A.K.

    1990-01-01

    The effect of radiation on ATP-ase activity in rat liver nuclei and RNA transport of isolated liver nuclei in vitro is studied. It is shown that irradiation changes RNA transport from isolated liver cell nuclei of maternal organism and embryos. Irradiation during prefetus and fetus periods changes ATP-ase activity of embryon and maternal organism nuclei

  13. 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

  14. 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

  15. 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.

  16. ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

    Science.gov (United States)

    Gomes, P; Chevalier, J; Boesmans, W; Roosen, L; van den Abbeel, V; Neunlist, M; Tack, J; Vanden Berghe, P

    2009-08-01

    The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

  17. 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.

  18. 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

  19. 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

  20. Transcription factor FOXO1 promotes cell migration toward exogenous ATP via controlling P2Y1 receptor expression in lymphatic endothelial cells.

    Science.gov (United States)

    Niimi, Kenta; Ueda, Mizuha; Fukumoto, Moe; Kohara, Misaki; Sawano, Toshinori; Tsuchihashi, Ryo; Shibata, Satoshi; Inagaki, Shinobu; Furuyama, Tatsuo

    2017-08-05

    Sprouting migration of lymphatic endothelial cell (LEC) is a pivotal step in lymphangiogenic process. However, its molecular mechanism remains unclear including effective migratory attractants. Meanwhile, forkhead transcription factor FOXO1 highly expresses in LEC nuclei, but its significance in LEC migratory activity has not been researched. In this study, we investigated function of FOXO1 transcription factor associated with LEC migration toward exogenous ATP which has recently gathered attentions as a cell migratory attractant. The transwell membrane assay indicated that LECs migrated toward exogenous ATP, which was impaired by FOXO1 knockdown. RT-PCR analysis showed that P2Y1, a purinergic receptor, expression was markedly reduced by FOXO1 knockdown in LECs. Moreover, P2Y1 blockage impaired LEC migration toward exogenous ATP. Western blot analysis revealed that Akt phosphorylation contributed to FOXO1-dependent LEC migration toward exogenous ATP and its blockage affected LEC migratory activity. Furthermore, luciferase reporter assay and ChIP assay suggested that FOXO1 directly bound to a conserved binding site in P2RY1 promoter and regulated its activity. These results indicated that FOXO1 serves a pivotal role in LEC migration toward exogenous ATP via direct transcriptional regulation of P2Y1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Tulika Munshi

    Full Text Available ATP-dependent Mur ligases (Mur synthetases play essential roles in the biosynthesis of cell wall peptidoglycan (PG as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

  2. Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity.

    Science.gov (United States)

    Nowrousian, M; Masloff, S; Pöggeler, S; Kück, U

    1999-01-01

    During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL

  3. 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

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

    International Nuclear Information System (INIS)

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa

    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

  5. Antitumor activity of zoledronic acid in primary breast cancer cells determined by the ATP tumor chemosensitivity assay

    International Nuclear Information System (INIS)

    Fehm, Tanja; Zwirner, Manfred; Wallwiener, Diethelm; Seeger, Harald; Neubauer, Hans

    2012-01-01

    The NeoAzure study has demonstrated that the use of the bisphosphonate zoledronic acid (Zol) in the neoadjuvant setting increases the rate of complete response in primary breast cancer and therefore indicates direct antitumor activity. The purpose of this study was to compare the antitumor effect of Zol with standard chemotherapy in primary breast cancer cells using ATP-tumor chemosensitivity assay (ATP-TCA). Breast cancer specimens were obtained from patients with breast cancer who underwent primary breast cancer surgery at the Department of Obstetrics and Gynecology, Tübingen, Germany, between 2006 through 2009. Antitumor effects of Zol, TAC (Docetaxel, Adriamycin, Cyclophosphamide) and FEC (5-Fluorouracil, Epirubicin, Cyclophosphamide) were tested in 116 fresh human primary breast cancer specimens using ATP-TCA. ATP-TCA results were analyzed with different cut-off levels for the half maximal inhibitory concentration (IC50), for IC90 and for the sensitivity index (IndexSUM). Each single agent or combination was tested at six doubling dilutions from 6.25, 12.5, 25, 50, 100, and 200% of test drug concentrations (TDC) derived from the plasma peak concentrations determined by pharmacokinetic data. The assay was carried out in duplicate wells with positive and negative controls. The median IndexSUM value was lower for Zol than for the combined regimen FEC (36.8%) and TAC (12.9%), respectively, indicating increased antitumor activity of Zol in primary breast cancer cells. The difference regarding Zol and FEC was significant (p < 0.05). The median IC50 value for Zol (8.03% TDC) was significantly lower than the IC50 values for FEC (33.5% TDC) and TAC (19.3% TDC) treatment (p < 0.05). However, the median IC90 value for Zol (152.5% TDC) was significantly higher than the IC90 value obtained with TAC (49.5% TDC; p < 0.05), but similar to the IC90 value for FEC (180.9% TDC). In addition a significant positive correlation was observed for the IndexSum of Zol and the ER status

  6. 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....

  7. The examination of urine samples for pathogenic microbes by the luciferase assay for ATP. 1: The effect of the presence of fungi, fungal like bacteria and kidney cells in urine samples

    Science.gov (United States)

    Bush, V. N.

    1973-01-01

    A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.

  8. Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

    Science.gov (United States)

    Gazzerro, Elisabetta; Baldassari, Simona; Assereto, Stefania; Fruscione, Floriana; Pistorio, Angela; Panicucci, Chiara; Volpi, Stefano; Perruzza, Lisa; Fiorillo, Chiara; Minetti, Carlo; Traggiai, Elisabetta; Grassi, Fabio; Bruno, Claudio

    2015-12-01

    Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-β and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

    Science.gov (United States)

    Matsuda, Shuichi; Takano, Sho; Sato, Moeko; Furukawa, Kaoru; Nagasawa, Hidetaka; Yoshikawa, Shoko; Kasuga, Jun; Tokuji, Yoshihiko; Yazaki, Kazufumi; Nakazono, Mikio; Takamure, Itsuro; Kato, Kiyoaki

    2016-03-07

    Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  10. 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.

  11. ATP Release and Effects in Pancreas

    DEFF Research Database (Denmark)

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

    2003-01-01

    ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock...

  12. Docetaxel modulates the delayed rectifier potassium current (IK) and ATP-sensitive potassium current (IKATP) in human breast cancer cells.

    Science.gov (United States)

    Sun, Tao; Song, Zhi-Guo; Jiang, Da-Qing; Nie, Hong-Guang; Han, Dong-Yun

    2015-04-01

    Ion channel expression and activity may be affected during tumor development and cancer growth. Activation of potassium (K(+)) channels in human breast cancer cells is reported to be involved in cell cycle progression. In this study, we investigated the effects of docetaxel on the delayed rectifier potassium current (I K) and the ATP-sensitive potassium current (I KATP) in two human breast cancer cell lines, MCF-7 and MDA-MB-435S, using the whole-cell patch-clamp technique. Our results show that docetaxel inhibited the I K and I KATP in both cell lines in a dose-dependent manner. Compared with the control at a potential of +60 mV, treatment with docetaxel at doses of 0.1, 1, 5, and 10 µM significantly decreased the I K in MCF-7 cells by 16.1 ± 3.5, 30.2 ± 5.2, 42.5 ± 4.3, and 46.4 ± 9% (n = 5, P < 0.05), respectively and also decreased the I KATP at +50 mV. Similar results were observed in MDA-MB-435S cells. The G-V curves showed no significant changes after treatment of either MCF-7 or MDA-MB-435S cells with 10 μM docetaxel. The datas indicate that the possible mechanisms of I K and I KATP inhibition by docetaxel may be responsible for its effect on the proliferation of human breast cancer cells.

  13. 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.

  14. Adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided platinum-based 2-drug chemotherapy for unresectable nonsmall-cell lung cancer.

    Science.gov (United States)

    Moon, Yong Wha; Choi, Sung Ho; Kim, Yong Tai; Sohn, Joo Hyuk; Chang, Joon; Kim, Se Kyu; Park, Moo Suk; Chung, Kyung Young; Lee, Hyoun Ju; Kim, Joo-Hang

    2007-05-01

    The study investigated correlations between adenosine triphosphate / chemotherapy response assay (ATP-CRA) and clinical outcomes after ATP-CRA-guided platinum-based chemotherapy for unresectable nonsmall-cell lung cancer (NSCLC). The authors performed an in vitro chemosensitivity test, ATP-CRA, to evaluate the chemosensitivities of anticancer drugs such as cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, and vinorelbine for chemonaive, unresectable NSCLC. The cell death rate was determined by measuring the intracellular ATP levels of drug-exposed cells compared with untreated controls. A sensitive drug was defined as a drug producing 30% or more reduction in ATP compared with untreated controls. Assay-guided platinum-based 2-drug chemotherapy was given to patients with pathologically confirmed NSCLC. Thirty-four patients were enrolled. Thirty tumor specimens were obtained by bronchoscopic biopsies and 4 obtained surgically. The median age was 61 years and 27 patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. The response rate was 43.8%. At a median follow-up period of 16.9 months, the median progression-free and overall survivals were 3.6 and 11.2 months, respectively. Patients were dichotomized into the platinum-sensitive (S; 20 patients) and resistant (R; 14 patients) groups. The positive/negative predictive values were 61.1% and 78.6% with a predictive accuracy of 68.8%. Although without significant differences in pretreatment parameters, the S-group showed better clinical response (P=.036), longer progression-free survival (P=.060), and longer overall survival (P=.025). Despite using bronchoscopic biopsied specimens, ATP-CRA and clinical outcomes correlated well after assay-guided platinum-based 2-drug chemotherapy for unresectable NSCLC. There was a favorable response and survival in the platinum-sensitive vs resistant groups. Copyright (c) 2007 American Cancer Society

  15. Adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided versus empirical chemotherapy in unresectable non-small cell lung cancer.

    Science.gov (United States)

    Moon, Yong Wha; Sohn, Joo Hyuk; Kim, Yong Tai; Chang, Hyun; Jeong, Jae Heon; Lee, Young Joo; Chang, Joon; Kim, Se Kyu; Jung, Minkyu; Hong, Soojung; Choi, Sung Ho; Kim, Joo-Hang

    2009-10-01

    We retrospectively compared adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided and empirical chemotherapies for unresectable non-small cell lung cancer (NSCLC) in this case-control study. Unresectable NSCLC patients receiving ATP-CRA-guided platinum-based doublets as first-line therapy were enrolled as cases (n=27; 14 platinum-sensitive and 13 platinum-resistant patients). Performance status, stage, and chemotherapeutic regimen-matched patients receiving empirical chemotherapy were selected from the retrospective database as controls (n=93) in a case to control ratio of approximately 1:3. Response rate and survival (progression-free; overall) in both groups were not significantly different. However, the platinum-sensitive subgroup by ATP-CRA showed a higher response rate than the empirical group (71 versus 38%; p=0.023) with a trend toward longer progression-free survival (8.7 versus 4.8 months for platinum-sensitive versus empirical; p=0.223) and overall survival (not reached versus 12.6 months for platinum-sensitive versus empirical for p=0.134). ATP-CRA may be helpful in selecting platinum-responsive patients in unresectable NSCLC. We consider that nonplatinum doublets in platinum-resistant patients by ATP-CRA may be a more adapted approach than platinum-based doublets in future clinical trials.

  16. Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion

    NARCIS (Netherlands)

    Roelofsen, H; Wolters, H; Van Luyn, MJA; Miura, N; Kuipers, F; Vonk, RJ

    Background & Aims: Mutations in the ATP7B gene, encoding a copper-transporting P-type adenosine triphosphatase, lead to excessive hepatic copper accumulation because of impaired biliary copper excretion in Wilson's disease. In human liver, ATP7B is predominantly localized to the trans-Golgi network,

  17. 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.

  18. 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}.

  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. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. Cation depletion by the sodium pump in red cells with pathologic cation leaks. Sickle cells and xerocytes.

    OpenAIRE

    Joiner, C H; Platt, O S; Lux, S E

    1986-01-01

    The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blo...

  8. Optimisation of ATP determination in drinking water

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution,...... be separated from the water phase by filtration.......Adenosine Triphosphate (ATP) can be used as a relative measure of cell activity, and is measured by the light output from the reaction between luciferin and ATP catalyzed by firefly luciferase. The measurement has potential as a monitoring and surveillance tool within drinking water distribution...... 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...

  9. The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells

    Science.gov (United States)

    Castella, Barbara; Kopecka, Joanna; Sciancalepore, Patrizia; Mandili, Giorgia; Foglietta, Myriam; Mitro, Nico; Caruso, Donatella; Novelli, Francesco; Riganti, Chiara; Massaia, Massimo

    2017-01-01

    Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation. PMID:28580927

  10. 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.

  11. Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

    Science.gov (United States)

    Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

    2013-02-01

    We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival.

    NARCIS (Netherlands)

    Hucthagowder, V.; Morava, E.; Kornak, U.; Lefeber, D.J.; Fischer, B.; Dimopoulou, A.; Aldinger, A.; Choi, J.; Davis, E.C.; Abuelo, D.N.; Adamowicz, M.; Al-Aama, J.Y.; Basel-Vanagaite, L.; Fernandez, B.; Greally, M.T.; Gillessen-Kaesbach, G.; Kayserili, H.; Lemyre, E.; Tekin, M.; Turkmen, S.; Tuysuz, B.; Yuksel-Konuk, B.; Mundlos, S.; Maldergem, L. van; Wevers, R.A.; Urban, Z.

    2009-01-01

    Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue

  13. 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.)

  14. 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.

  15. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb’s cycle flux independent of ATP synthesis

    International Nuclear Information System (INIS)

    Cline, Gary W.; Pongratz, Rebecca L.; Zhao, Xiaojian; Papas, Klearchos K.

    2011-01-01

    Highlights: ► We studied media effects on mechanisms of insulin secretion of INS-1 cells. ► Insulin secretion was higher in DMEM than KRB despite identical ATP synthesis rates. ► Insulin secretion rates correlated with rates of anaplerosis and TCA cycle. ► Mitochondria metabolism and substrate cycles augment secretion signal of ATP. -- Abstract: Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with 31 P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by 13 C NMR isotopomer analysis of the fate of [U- 13 C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM

  16. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb's cycle flux independent of ATP synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Cline, Gary W., E-mail: gary.cline@yale.edu [The Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520 (United States); Department of Surgery, University of Minnesota-Twin Cities, Minneapolis, MN 55455 (United States); Pongratz, Rebecca L.; Zhao, Xiaojian [The Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520 (United States); Papas, Klearchos K. [Department of Surgery, University of Minnesota-Twin Cities, Minneapolis, MN 55455 (United States)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer We studied media effects on mechanisms of insulin secretion of INS-1 cells. Black-Right-Pointing-Pointer Insulin secretion was higher in DMEM than KRB despite identical ATP synthesis rates. Black-Right-Pointing-Pointer Insulin secretion rates correlated with rates of anaplerosis and TCA cycle. Black-Right-Pointing-Pointer Mitochondria metabolism and substrate cycles augment secretion signal of ATP. -- Abstract: Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with {sup 31}P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by {sup 13}C NMR isotopomer analysis of the fate of [U-{sup 13}C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15 mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found

  17. Water leaks in sodium-heated fast reactor boilers

    International Nuclear Information System (INIS)

    Hayes, D.J.

    1978-01-01

    Constraints on plant design which may result from considerations of leak behaviour and leak detection limits are briefly considered. The sodium-water interface and reactions, the behaviour of small leaks, hydrogen bubbles and detection methods, including galvanic cell methods, are included. (UK)

  18. 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

  19. Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

    Directory of Open Access Journals (Sweden)

    Lenartowicz Malgorzata

    2016-08-01

    Full Text Available Deficiency of one of the copper transporters ATP7A and ATP7B leads to the rare X-linked disorder Menkes Disease (MD or the rare autosomal disorder Wilson disease (WD, respectively. In order to investigate whether the ATP7A and the ATP7B genes may be transcriptionally regulated, we measured 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 and10 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 allele had a selective growth advantage at high copper concentrations, whereas more surprisingly, cells that express the mutant ATP7A allele had a selective growth advantage at low copper concentrations. Thus, although the transcription of ATP7A is regulated by copper, clonal growth selection in mosaic cell cultures is affected by the level of copper. Female carriers of MD are rarely affected probably due to a skewed inactivation of the X-chromosome bearing the ATP7A mutation.

  20. 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.

  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. Permanent underwater leak detector

    International Nuclear Information System (INIS)

    Costello, L; McStay, D; Moodie, D; Kane, D

    2009-01-01

    A new optoelectronic sensor for the real time monitoring of key components such as valves and connectors within the subsea production equipment for leaks of hydraulic fluid is reported. The sensor is capable of detecting low concentrations of such fluids, allowing the early detection of small leaks, and the ability to monitor the evolution of the leak-rate with time, hence providing an important new tool in complying with environmental requirements, enabling early intervention and optimising subsea production

  3. 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

  4. Extracellular ATP in the Exocrine Pancreas – ATP Release, Signalling and Metabolism

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena

    release. So far, the contribution of duct cells in purinergic signalling has never been studied. This work presents that both acinar and duct cells are sources of extracellular ATP in the exocrine pancreas. Here we show that duct cells release ATP in response to several physiological......ATP plays an important role as an autocrine/paracrine signalling molecule, being released from a number of tissues, in response to physiological and pathophysiological stimuli. Released ATP induces Ca2+ - and/or cAMP - dependent cellular responses via activation of ubiquitously expressed P2X and P2......, particularly during Ca2+ stress conditions. In conclusion, these studies demonstrate a complex regulation of purinergic signalling in exocrine pancreas. A crucial role for duct cells in mediating extracellular nucleotides homeostasis, involving ATP release, subsequent hydrolysis and conversion via...

  5. Chemochromic Hydrogen Leak Detectors

    Science.gov (United States)

    Roberson, Luke; Captain, Janine; Williams, Martha; Smith, Trent; Tate, LaNetra; Raissi, Ali; Mohajeri, Nahid; Muradov, Nazim; Bokerman, Gary

    2009-01-01

    At NASA, hydrogen safety is a key concern for space shuttle processing. Leaks of any level must be quickly recognized and addressed due to hydrogen s lower explosion limit. Chemo - chromic devices have been developed to detect hydrogen gas in several embodiments. Because hydrogen is odorless and colorless and poses an explosion hazard, there is an emerging need for sensors to quickly and accurately detect low levels of leaking hydrogen in fuel cells and other advanced energy- generating systems in which hydrogen is used as fuel. The device incorporates a chemo - chromic pigment into a base polymer. The article can reversibly or irreversibly change color upon exposure to hydrogen. The irreversible pigment changes color from a light beige to a dark gray. The sensitivity of the pigment can be tailored to its application by altering its exposure to gas through the incorporation of one or more additives or polymer matrix. Furthermore, through the incorporation of insulating additives, the chemochromic sensor can operate at cryogenic temperatures as low as 78 K. A chemochromic detector of this type can be manufactured into any feasible polymer part including injection molded plastic parts, fiber-spun textiles, or extruded tapes. The detectors are simple, inexpensive, portable, and do not require an external power source. The chemochromic detectors were installed and removed easily at the KSC launch pad without need for special expertise. These detectors may require an external monitor such as the human eye, camera, or electronic detector; however, they could be left in place, unmonitored, and examined later for color change to determine whether there had been exposure to hydrogen. In one type of envisioned application, chemochromic detectors would be fabricated as outer layers (e.g., casings or coatings) on high-pressure hydrogen storage tanks and other components of hydrogen-handling systems to provide visible indications of hydrogen leaks caused by fatigue failures or

  6. Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

    Science.gov (United States)

    Jastreboff, M; Kedzierska, B; Rode, W

    1982-01-15

    Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.

  7. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Masaki Nakano

    2017-08-01

    Full Text Available Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances, which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

  8. 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.

  9. Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts

    International Nuclear Information System (INIS)

    Hu, Fen; Hui, Zhenhai; Wei, Wei; Yang, Jianyu; Chen, Ziyuan; Guo, Bu; Xing, Fulin; Zhang, Xinzheng; Pan, Leiting; Xu, Jingjun

    2017-01-01

    Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca 2+ ] c ) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects were almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca 2+ channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca 2+ entry through activation of TRPV4 channel in synoviocytes. - Highlights: • Hypotonic stress evokes Ca 2+ entry in rheumatoid arthritis synovial fibroblasts. • Hypotonic stress induces rapid ATP release and ROS production in synoviocytes. • Hypotonic stimulation promotes the proliferation of synovial fibroblasts. • TRPV4 controls hypotonic-induced responses in synoviocytes.

  10. Rates of insulin secretion in INS-1 cells are enhanced by coupling to anaplerosis and Kreb's cycle flux independent of ATP synthesis.

    Science.gov (United States)

    Cline, Gary W; Pongratz, Rebecca L; Zhao, Xiaojian; Papas, Klearchos K

    2011-11-11

    Mechanistic models of glucose stimulated insulin secretion (GSIS) established in minimal media in vitro, may not accurately describe the complexity of coupling metabolism with insulin secretion that occurs in vivo. As a first approximation, we have evaluated metabolic pathways in a typical growth media, DMEM as a surrogate in vivo medium, for comparison to metabolic fluxes observed under the typical experimental conditions using the simple salt-buffer of KRB. Changes in metabolism in response to glucose and amino acids and coupling to insulin secretion were measured in INS-1 832/13 cells. Media effects on mitochondrial function and the coupling efficiency of oxidative phosphorylation were determined by fluorometrically measured oxygen consumption rates (OCRs) combined with (31)P NMR measured rates of ATP synthesis. Substrate preferences and pathways into the TCA cycle, and the synthesis of mitochondrial 2nd messengers by anaplerosis were determined by (13)C NMR isotopomer analysis of the fate of [U-(13)C] glucose metabolism. Despite similar incremental increases in insulin secretion, the changes of OCR in response to increasing glucose from 2.5 to 15mM were blunted in DMEM relative to KRB. Basal and stimulated rates of insulin secretion rates were consistently higher in DMEM, while ATP synthesis rates were identical in both DMEM and KRB, suggesting greater mitochondrial uncoupling in DMEM. The relative rates of anaplerosis, and hence synthesis and export of 2nd messengers from the mitochondria were found to be similar in DMEM to those in KRB. And, the correlation of total PC flux with insulin secretion rates in DMEM was found to be congruous with the correlation in KRB. Together, these results suggest that signaling mechanisms associated with both TCA cycle flux and with anaplerotic flux, but not ATP production, may be responsible for the enhanced rates of insulin secretion in more complex, and physiologically-relevant media. Copyright © 2011 Elsevier Inc. All

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

    Energy Technology Data Exchange (ETDEWEB)

    Rocafull, Miguel A., E-mail: mrocaful@ivic.ve [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    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{sup 2+}, Na{sup +}, K{sup +} and H{sup +}), have been reported. They include reticulum and plasma-membrane Ca{sup 2+}-ATPases, Na{sup +}/K{sup +}-ATPase and H{sup +}/K{sup +}-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{sup 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{sup +}/K{sup +}-ATPase {alpha}1-isoform, H{sup +}/K{sup +}-ATPase {alpha}2-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{sup +}/K{sup +}-ATPase {alpha}2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  12. Leak detection method

    International Nuclear Information System (INIS)

    1978-01-01

    This invention provides a method for removing nuclear fuel elements from a fabrication building while at the same time testing the fuel elements for leaks without releasing contaminants from the fabrication building or from the fuel elements. The vacuum source used, leak detecting mechanism and fuel element fabrication building are specified to withstand environmental hazards. (UK)

  13. 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...... analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol....

  14. SALL4, a stem cell factor, affects the side population by regulation of the ATP-binding cassette drug transport genes.

    Directory of Open Access Journals (Sweden)

    Ha-Won Jeong

    2011-04-01

    Full Text Available Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2-4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP, quantitative reverse transcription polymerase chain reaction (qRT-PCR and electrophoretic mobility shift assay(EMSA, we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.

  15. 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.

  16. ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation.

    Directory of Open Access Journals (Sweden)

    Shin-Ei Cheng

    Full Text Available BACKGROUND: Up-regulation of cyclooxygenase (COX-2 and its metabolite prostaglandin E(2 (PGE(2 are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2 release remain unclear. PRINCIPAL FINDINGS: Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin, PKC (Gö6983, Gö6976, Ro318220, and Rottlerin, ROS (Edaravone, NADPH oxidase [diphenyleneiodonium chloride (DPI and apocynin], Jak2 (AG490, and STAT3 [cucurbitacin E (CBE] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47(phox, Jak2, STAT3, and cPLA(2 markedly reduced ATPγS-induced COX-2 expression and PGE(2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47(phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. SIGNIFICANCE: Taken together, these results showed that ATPγS induced COX-2 expression and PGE(2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

  17. Corticotropin-Releasing Hormone (CRH Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1.

    Directory of Open Access Journals (Sweden)

    Wonkyoung Cho

    Full Text Available Atherosclerosis, the major pathology of cardiovascular disease, is caused by multiple factors involving psychological stress. Corticotropin-releasing hormone (CRH, which is released by neurosecretory cells in the hypothalamus, peripheral nerve terminals and epithelial cells, regulates various stress-related responses. Our current study aimed to verify the role of CRH in macrophage foam cell formation, the initial critical stage of atherosclerosis. Our quantitative real-time reverse transcriptase PCR (qRT-PCR, semi-quantitative reverse transcriptase PCR, and Western blot results indicate that CRH down-regulates ATP-binding cassette transporter-1 (ABCA1 and liver X receptor (LXR-α, a transcription factor for ABCA1, in murine peritoneal macrophages and human monocyte-derived macrophages. Oil-red O (ORO staining and intracellular cholesterol measurement of macrophages treated with or without oxidized LDL (oxLDL and with or without CRH (10 nM in the presence of apolipoprotein A1 (apoA1 revealed that CRH treatment promotes macrophage foam cell formation. The boron-dipyrromethene (BODIPY-conjugated cholesterol efflux assay showed that CRH treatment reduces macrophage cholesterol efflux. Western blot analysis showed that CRH-induced down-regulation of ABCA1 is dependent on phosphorylation of Akt (Ser473 induced by interaction between CRH and CRH receptor 1(CRHR1. We conclude that activation of this pathway by CRH accelerates macrophage foam cell formation and may promote stress-related atherosclerosis.

  18. 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

  19. Fuel leak testing performance at NPP Jaslovske Bohunice

    International Nuclear Information System (INIS)

    Slugen, V.; Krnac, S.; Smiesko, I.

    1995-01-01

    The NPP Bohunice VVER-440 fuel leak testing experience are relatively extensive in comparison with other VVER-440 users. As the first Europe NPP was adapted Siemens (KWU) in core-sipping equipment to VVER-440 units and since this time were have done these tests also for NPP Paks (Hungary) and NPP Dukovany (Czech Republic). The occurrence of leaking fuel assemblies in NPP is in the last 5 years relatively stabilised and low. A significant difference can be observed between type V-230 (31 leaks) and type V-213 (1 leak). None of of the indicated leaking fuel assemblies has been investigated in the hot cell. Therefore cannot be confirm the effective causes of leak occurrence. Nevertheless, the fuel failure rate and the performance of leak testing in NPP Bohunice are comparable to the world standard at PWR's. 1 tab., 2 figs., 3 refs

  20. Fuel leak testing performance at NPP Jaslovske Bohunice

    Energy Technology Data Exchange (ETDEWEB)

    Slugen, V; Krnac, S [Slovak Technical Univ., Bratislava (Slovakia); Smiesko, I [Nuclear Powr Plant EBO, Jaslovske Bohuce (Slovakia)

    1996-12-31

    The NPP Bohunice VVER-440 fuel leak testing experience are relatively extensive in comparison with other VVER-440 users. As the first Europe NPP was adapted Siemens (KWU) in core-sipping equipment to VVER-440 units and since this time were have done these tests also for NPP Paks (Hungary) and NPP Dukovany (Czech Republic). The occurrence of leaking fuel assemblies in NPP is in the last 5 years relatively stabilised and low. A significant difference can be observed between type V-230 (31 leaks) and type V-213 (1 leak). None of of the indicated leaking fuel assemblies has been investigated in the hot cell. Therefore cannot be confirm the effective causes of leak occurrence. Nevertheless, the fuel failure rate and the performance of leak testing in NPP Bohunice are comparable to the world standard at PWR`s. 1 tab., 2 figs., 3 refs.

  1. Analysis of small leaks

    International Nuclear Information System (INIS)

    Frisch, W.; Hofmann, K.

    1979-01-01

    Problems associated with 'small leaks' are described and requirements are derived for experimental facilities and computer codes. Based on these requirements, a valuation of the existing experimental facilities and codes is presented. Facilities for integral tests in relatively large scale (ex. LOFT) are suitable for small leak test in principle, however minor changes (instrumentation, secondary side) are necessary for the evaluation of certain phenomena. The 'advanced blowdown codes' are capable of describing most of the phenomena occurring during small leak events, however a substantial amount of code development and verification is still needed. In addition, the use of transient codes in small leak analysis is demonstrated. There are some areas (neutronics feedback, influence of control system) in which the use of transient codes is possible and advantageous. (orig.) 891 HP/orig. 892 BRE [de

  2. Traumatic orbital CSF leak

    Science.gov (United States)

    Borumandi, Farzad

    2013-01-01

    Compared to the cerebrospinalfluid (CSF) leak through the nose and ear, the orbital CSF leak is a rare and underreported condition following head trauma. We present the case of a 49-year-old woman with oedematous eyelid swelling and ecchymosis after a seemingly trivial fall onto the right orbit. Apart from the above, she was clinically unremarkable. The CT scan revealed a minimally displaced fracture of the orbital roof with no emphysema or intracranial bleeding. The fractured orbital roof in combination with the oedematous eyelid swelling raised the suspicion for orbital CSF leak. The MRI of the neurocranium demonstrated a small-sized CSF fistula extending from the anterior cranial fossa to the right orbit. The patient was treated conservatively and the lid swelling resolved completely after 5 days. Although rare, orbital CSF leak needs to be included in the differential diagnosis of periorbital swelling following orbital trauma. PMID:24323381

  3. 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

  4. Small liquid sodium leaks

    International Nuclear Information System (INIS)

    Dufresne, J.; Rochedereux, Y.; Antonakas, D.; Casselman, C.; Malet, J.C.

    1986-05-01

    Usually, pessimistic considerations inassessing the safety of secondary sodium loops in LMFBR reactor lead to assume guillotine rupture releasing a large amount of sodium estimate the consequences of large sodium fires. In order to reduce these consequences, one has to detect the smallest leak as soon as possible and to evaluate the future of an initial small leak. Analysis of the relationship between crack size and sodium outflow rate; Analysis of a sodium pipe with a small open crack

  5. Pipeline Leak Detection Techniques

    OpenAIRE

    Chis, Timur

    2009-01-01

    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.

  6. 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.

  7. Low Level Leaks

    Science.gov (United States)

    1998-01-01

    NASA has transferred the improved portable leak detector technology to UE Systems, Inc.. This instrument was developed to detect leaks in fluid systems of critical launch and ground support equipment. This system incorporates innovative electronic circuitry, improved transducers, collecting horns, and contact sensors that provide a much higher degree of reliability, sensitivity and versatility over previously used systems. Potential commercial uses are pipelines, underground utilities, air-conditioning systems, petrochemical systems, aerospace, power transmission lines and medical devices.

  8. 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+...

  9. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

    Science.gov (United States)

    MacDonald, Patrick E; De Marinis, Yang Zhang; Ramracheya, Reshma; Salehi, Albert; Ma, Xiaosong; Johnson, Paul R V; Cox, Roger; Eliasson, Lena; Rorsman, Patrik

    2007-06-01

    Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

  10. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans.

    Directory of Open Access Journals (Sweden)

    Patrick E MacDonald

    2007-06-01

    Full Text Available Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+ responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+ signalling was blocked, but was reversed by low concentrations (1-20 muM of the ATP-sensitive K(+ (KATP channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM. Higher diazoxide concentrations (>/=30 muM decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (10 muM were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM, glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+ (TTX and N-type Ca(2+ channels (omega-conotoxin, but not L-type Ca(2+ channels (nifedipine, prevented glucagon secretion. Both the N-type Ca(2+ channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

  11. A taste for ATP: neurotransmission in taste buds

    Science.gov (United States)

    Kinnamon, Sue C.; Finger, Thomas E.

    2013-01-01

    Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat, and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells. PMID:24385952

  12. A taste for ATP: neurotransmission in taste buds

    Directory of Open Access Journals (Sweden)

    Thomas E. Finger

    2013-12-01

    Full Text Available Not only is ATP a ubiquitous source of energy but it is also used widely as an intercellular signal. For example, keratinocytes release ATP in response to numerous external stimuli including pressure, heat and chemical insult. The released ATP activates purinergic receptors on nerve fibers to generate nociceptive signals. The importance of an ATP signal in epithelial-to-neuronal signaling is nowhere more evident than in the taste system. The receptor cells of taste buds release ATP in response to appropriate stimulation by tastants and the released ATP then activates P2X2 and P2X3 receptors on the taste nerves. Genetic ablation of the relevant P2X receptors leaves an animal without the ability to taste any primary taste quality. Of interest is that release of ATP by taste receptor cells occurs in a non-vesicular fashion, apparently via gated membrane channels. Further, in keeping with the crucial role of ATP as a neurotransmitter in this system, a subset of taste cells expresses a specific ectoATPase, NTPDase2, necessary to clear extracellular ATP which otherwise will desensitize the P2X receptors on the taste nerves. The unique utilization of ATP as a key neurotransmitter in the taste system may reflect the epithelial rather than neuronal origins of the receptor cells.

  13. 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.

  14. 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-...

  15. Ammonia Leak Locator Study

    Science.gov (United States)

    Dodge, Franklin T.; Wuest, Martin P.; Deffenbaugh, Danny M.

    1995-01-01

    The thermal control system of International Space Station Alpha will use liquid ammonia as the heat exchange fluid. It is expected that small leaks (of the order perhaps of one pound of ammonia per day) may develop in the lines transporting the ammonia to the various facilities as well as in the heat exchange equipment. Such leaks must be detected and located before the supply of ammonia becomes critically low. For that reason, NASA-JSC has a program underway to evaluate instruments that can detect and locate ultra-small concentrations of ammonia in a high vacuum environment. To be useful, the instrument must be portable and small enough that an astronaut can easily handle it during extravehicular activity. An additional complication in the design of the instrument is that the environment immediately surrounding ISSA will contain small concentrations of many other gases from venting of onboard experiments as well as from other kinds of leaks. These other vapors include water, cabin air, CO2, CO, argon, N2, and ethylene glycol. Altogether, this local environment might have a pressure of the order of 10(exp -7) to 10(exp -6) torr. Southwest Research Institute (SwRI) was contracted by NASA-JSC to provide support to NASA-JSC and its prime contractors in evaluating ammonia-location instruments and to make a preliminary trade study of the advantages and limitations of potential instruments. The present effort builds upon an earlier SwRI study to evaluate ammonia leak detection instruments [Jolly and Deffenbaugh]. The objectives of the present effort include: (1) Estimate the characteristics of representative ammonia leaks; (2) Evaluate the baseline instrument in the light of the estimated ammonia leak characteristics; (3) Propose alternative instrument concepts; and (4) Conduct a trade study of the proposed alternative concepts and recommend promising instruments. The baseline leak-location instrument selected by NASA-JSC was an ion gauge.

  16. 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

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

    Science.gov (United States)

    Rhodes, David A.; Chen, Hung-Chang; Williamson, James C.; Hill, Alfred; Yuan, Jack; Smith, Sam; Rhodes, Harriet; Trowsdale, John; Lehner, Paul J.; Herrmann, Thomas; Eberl, Matthias

    2018-01-01

    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 with

  18. 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

  19. 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

    in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug......-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack...... of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant...

  20. 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

  1. 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.

  2. 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.

  3. Contribution of proton leak to oxygen consumption in skeletal muscle during intense exercise is very low despite large contribution at rest.

    Directory of Open Access Journals (Sweden)

    Bernard Korzeniewski

    Full Text Available A computer model was used to simulate the dependence of protonmotive force (Δp, proton leak and phenomenological (involving proton leak ATP/O2 ratio on work intensity in skeletal muscle. Δp, NADH and proton leak decreased with work intensity. The contribution of proton leak to oxygen consumption ([Formula: see text] decreased from about 60% at rest to about 3 and 1% at moderate and heavy/severe exercise, respectively, while the ATP/O2 ratio increased from 2.1 to 5.5 and 5.7. A two-fold increase in proton leak activity or its decrease to zero decreased/increased the ATP/O2 ratio by only about 3 and 1% during moderate and heavy/severe exercise, respectively. The low contribution of proton leak to [Formula: see text] in intensively working skeletal muscle was mostly caused by a huge increase in ATP usage intensity during rest-to-work transition, while OXPHOS, and thus oxidative ATP supply and [Formula: see text] related to it, was mostly stimulated by high each-step activation (ESA of OXPHOS complexes. The contribution of proton leak to [Formula: see text] and ATP/O2 ratio in isolated mitochondria should not be directly extrapolated to working muscle, as mitochondria lack ESA, at least in the absence of Ca2+, and therefore [Formula: see text] cannot be elevated as much as in intact muscle.

  4. 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.

  5. 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

  6. 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

  7. PFR evaporator leak

    Energy Technology Data Exchange (ETDEWEB)

    Smedley, J A

    1975-07-01

    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{sup -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{sup -7} to 10{sup -6} g/s equivalent water leak could be

  8. 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 ...

  9. 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.

  10. 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)

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

    OpenAIRE

    T. О. Veklich; Iu. Iu. Mazur; S. О. Kosterin

    2015-01-01

    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 (U...

  12. Insulin-like Growth Factor 1 Regulates the Expression of ATP-Binding Cassette Transporter A1 in Pancreatic Beta Cells.

    Science.gov (United States)

    Lyu, J; Imachi, H; Iwama, H; Zhang, H; Murao, K

    2016-05-01

    ATP-binding cassette transporter A1 (ABCA1) in pancreatic beta cells influences insulin secretion and cholesterol homeostasis. The present study investigates whether insulin-like growth factor 1 (IGF-1), which mediates stimulation of ABCA1 gene expression, could also interfere with the phosphatidylinositol 3-kinase (PI3-K) cascade.ABCA1 expression was examined by real-time polymerase chain reaction (PCR), Western blot analysis, and a reporter gene assay in rat insulin-secreting INS-1 cells incubated with IGF-1. The binding of forkhead box O1 (FoxO1) protein to the ABCA1 promoter was assessed by a chromatin immunoprecipitation (ChIP) assay. ABCA1 protein levels increased in response to rising concentrations of IGF-1. Real-time PCR analysis showed a significant increase in ABCA1 mRNA expression. However, both effects were suppressed after silencing the IGF-1 receptor. In parallel with its effect on endogenous ABCA1 mRNA levels, IGF-1 induced the activity of a reporter construct containing the ABCA1 promoter, while it was abrogated by LY294002, a specific inhibitor of PI3-K. Constitutively active Akt stimulated activity of the ABCA1 promoter, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element in the ABCA1 promoter abolished the ability of IGF-1 to stimulate promoter activity. A ChIP assay showed that FoxO1 mediated its transcriptional activity by directly binding to the ABCA1 promoter region. The knockdown of FoxO1 disrupted the effect of IGF-1 on ABCA1 expression. Furthermore, IGF-1 promoted cholesterol efflux and reduced the pancreatic lipotoxicity. These results demonstrate that the PI3-K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF-1 stimulation. © Georg Thieme Verlag KG Stuttgart · New York.

  13. 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

  14. 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

  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. 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.

  17. Natural gas leak mapper

    Science.gov (United States)

    Reichardt, Thomas A [Livermore, CA; Luong, Amy Khai [Dublin, CA; Kulp, Thomas J [Livermore, CA; Devdas, Sanjay [Albany, CA

    2008-05-20

    A system is described that is suitable for use in determining the location of leaks of gases having a background concentration. The system is a point-wise backscatter absorption gas measurement system that measures absorption and distance to each point of an image. The absorption measurement provides an indication of the total amount of a gas of interest, and the distance provides an estimate of the background concentration of gas. The distance is measured from the time-of-flight of laser pulse that is generated along with the absorption measurement light. The measurements are formated into an image of the presence of gas in excess of the background. Alternatively, an image of the scene is superimosed on the image of the gas to aid in locating leaks. By further modeling excess gas as a plume having a known concentration profile, the present system provides an estimate of the maximum concentration of the gas of interest.

  18. Does a dyon leak

    International Nuclear Information System (INIS)

    Grossman, B.

    1983-01-01

    In the presence of a CP-nonconservation measured by an angle theta, the ground state of a point magnetic monopole is shown to have an electric charge of value -etheta/2π which changes discontinuously to zero for massless fermions. A new version of Levinson's theorem is also given. The latter effect as well as the S-wave helicity flip of a dyon can be interpreted as a leak at the origin

  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. Method of forming a leak proof plasma sprayed interconnection layer on an electrode of an electrochemical cell

    Science.gov (United States)

    Kuo, Lewis J. H.; Vora, Shailesh D.

    1995-01-01

    A dense, substantially gas-tight, electrically conductive interconnection layer is formed on an electrode structure of an electrochemical cell by: (A) providing an electrode structure; (B) forming on a selected portion of the electrode surface, an interconnection layer having the general formula La.sub.1-x M.sub.x Cr.sub.1-y N.sub.y O.sub.3, where M is a dopant selected from the group of Ca, Sr, Ba, and mixtures thereof, and where N is a dopant selected from the group of Mg, Co, Ni, Al, and mixtures thereof, and where x and y are each independently about 0.075-0.25, by thermally spraying, preferably plasma arc spraying, a flux added interconnection spray powder, preferably agglomerated, the flux added powder comprising flux particles, preferably including dopant, preferably (CaO).sub.12. (Al.sub.2 O.sub.3).sub.7 flux particles including Ca and Al dopant, and LaCrO.sub.3 interconnection particles, preferably undoped LaCrO.sub.3, to form a dense and substantially gas-tight interconnection material bonded to the electrode structure by a single plasma spraying step; and, (C) heat treating the interconnection layer at from about 1200.degree. to 1350.degree. C. to further densify and heal the micro-cracks and macro-cracks of the thermally sprayed interconnection layer. The result is a substantially gas-tight, highly doped, electrically conductive interconnection material bonded to the electrode structure. The electrode structure can be an air electrode, and a solid electrolyte layer can be applied to the unselected portion of the air electrode, and further a fuel electrode can be applied to the solid electrolyte, to form an electrochemical cell for generation of electrical power.

  2. Novel Methods of Hydrogen Leak Detection

    International Nuclear Information System (INIS)

    Pushpinder S Puri

    2006-01-01

    With the advent of the fuel cell technology and a drive for clean fuel, hydrogen gas is emerging as a leading candidate for the fuel of choice. For hydrogen to become a consumer fuel for automotive and domestic power generation, safety is paramount. It is, therefore, desired to have a method and system for hydrogen leak detection using odorant which can incorporate a uniform concentration of odorant in the hydrogen gas, when odorants are mixed in the hydrogen storage or delivery means. It is also desired to develop methods where the odorant is not added to the bulk hydrogen, keeping it free of the odorization additives. When odorants are not added to the hydrogen gas in the storage or delivery means, methods must be developed to incorporate odorant in the leaking gas so that leaks can be detected by small. Further, when odorants are not added to the stored hydrogen, it may also be desirable to observe leaks by sight by discoloration of the surface of the storage or transportation vessels. A series of novel solutions are proposed which address the issues raised above. These solutions are divided into three categories as follows: 1. Methods incorporating an odorant in the path of hydrogen leak as opposed to adding it to the hydrogen gas. 2. Methods where odorants are generated in-situ by chemical reaction with the leaking hydrogen 3. Methods of dispensing and storing odorants in high pressure hydrogen gas which release odorants to the gas at a uniform and predetermined rates. Use of one or more of the methods described here in conjunction with appropriate engineering solutions will assure the ultimate safety of hydrogen use as a commercial fuel. (authors)

  3. 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

  4. 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

  5. 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.

  6. Mobile leak testing system

    International Nuclear Information System (INIS)

    Ungr, F.

    The design and implementation are described of a mobile testing unit ULTRATEST M for helium leak tests. The equipment has been developed by Leybold-Heraeus GmbH in Cologne and is in-built in a Mercedes-Benz 208 van. The equipment is designed for the operative use in assembly and construction of nuclear power plants and its throughput is sufficient for checking the whole upper reactor block. It may also be used for removing defects of vacuum equipment requiring a high level of tightness or equally demanding equipment used in the chemical industry. Experience with the equipment is described. (B.S.)

  7. 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

  8. Deconstructing Gender Stereotypes in Leak

    Directory of Open Access Journals (Sweden)

    Nengah Bawa Atmadja

    2015-05-01

    Full Text Available The belief of Balinese people towards leak still survive. Leak is a magic based on durgaism that can transform a person from human to another form, such as apes, pigs, etc. People tend to regard leak as evil. In general, the evilness is constructed in gender stereotypes, so it is identified that leak are always women. This idea is a power game based on the ideology of patriarchy that provides legitimacy for men to dominate women with a plea for social harmony. As a result, women are marginalized in the Balinese society. Women should be aware of so it would provide encouragement for them to make emancipatory changes dialogically. Kepercayaan orang Bali terhadap leak tetap bertahan sampai saat ini. Leak adalah sihir yang berbasiskan durgaisme yang dapat mengakibatkan seseorang bisa merubah bentuk dari manusia ke wujud yang lain, misalnya kera, babi, dll. Leak termasuk magi hitam sehingga dinilai bersifat jelek. Pada umumnya perempuan diidentikkan dengan leak sehingga melahirkan asumsi yang bermuatan steriotip gender bahwa leak = perempuan. Gagasan ini merupakan permainan kekuasaan berbasis ideologi patriarkhi dan sekaligus memberikan legitimasi bagi laki-laki untuk menguasai perempuan dengan dalih demi keharmonisan sosial. Akibatnya, perempuan menjadi termarginalisasi pada masyarakat Bali.  Perempuan harus menyadarinya sehingga memberikan dorongan bagi mereka untuk melakukan perubahan secara dialogis emansipatoris.

  9. Radiation leaking protection device

    International Nuclear Information System (INIS)

    Sunami, Yoshio; Mitsumori, Kojiro

    1980-01-01

    Purpose: To prevent radioactivity from leaking outside of a reactor container by way of pipeways passing therethrough, by supplying pressurized fluid between each of a plurality of valves for separating the pipeways. Constitution: Pressurized fluid is supplied between each of a plurality of valves for separating pipeways. For instance, water in a purified water tank is pressurized by a pressure pump and the pressure of the pressurized water is controlled by a differential pressure detector, a pressure controller and a pressure control valve. In the case if a main steam pipe is ruptured outside of the reactor container or to be repaired, the separation valves are wholly closed and then the pressurizing device is actuated to supply pressurized water containing no radioactivity from the purified water tank to the position between the valves. The pressure in the pressurized water is controlled such that it is always higher by a predetermined level than the pressure in the reactor. This prevents the radioacitivity in the reactor core from leaking outside of the container passing through the valves, whereby radiation exposure in the working can be reduced and the circumferential contamination upon accident of pipeway rupture can be decreased. (Kawakami, Y.)

  10. ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

    Science.gov (United States)

    Nakano, Masaki; Imamura, Hiromi; Sasaoka, Norio; Yamamoto, Masamichi; Uemura, Norihito; Shudo, Toshiyuki; Fuchigami, Tomohiro; Takahashi, Ryosuke; Kakizuka, Akira

    2017-08-01

    Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

    Directory of Open Access Journals (Sweden)

    Ip Virginia

    2010-09-01

    Full Text Available Abstract Background ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg or drug vehicle twice weekly for 8 weeks. Results In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H. High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals. Conclusions In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non

  12. 40 CFR 63.1005 - Leak repair.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 10 2010-07-01 2010-07-01 false Leak repair. 63.1005 Section 63.1005... Standards for Equipment Leaks-Control Level 1 § 63.1005 Leak repair. (a) Leak repair schedule. The owner or operator shall repair each leak detected no later than 15 calendar days after it is detected, except as...

  13. 40 CFR 63.1024 - Leak repair.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 10 2010-07-01 2010-07-01 false Leak repair. 63.1024 Section 63.1024... Standards for Equipment Leaks-Control Level 2 Standards § 63.1024 Leak repair. (a) Leak repair schedule. The owner or operator shall repair each leak detected as soon as practical, but not later than 15 calendar...

  14. 40 CFR 65.105 - Leak repair.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 15 2010-07-01 2010-07-01 false Leak repair. 65.105 Section 65.105... FEDERAL AIR RULE Equipment Leaks § 65.105 Leak repair. (a) Leak repair schedule. The owner or operator shall repair each leak detected as soon as practical but not later than 15 calendar days after it is...

  15. Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

    Science.gov (United States)

    Xiong, Yingfei; Teng, Sasa; Zheng, Lianghong; Sun, Suhua; Li, Jie; Guo, Ning; Li, Mingli; Wang, Li; Zhu, Feipeng; Wang, Changhe; Rao, Zhiren; Zhou, Zhuan

    2018-02-28

    Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca 2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca 2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca 2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca 2+ independent single large non-quantal ATP release occurred, in contrast to the Ca 2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

  16. 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.

  17. Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

    Science.gov (United States)

    Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2013-03-01

    Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. © 2013 The Authors Journal compilation © 2013 FEBS.

  18. 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

  19. The Leaking Pipeline

    DEFF Research Database (Denmark)

    Henningsen, Inge; Højgaard, Lis

    2002-01-01

    negotiations of cultural prescriptions of gendered subjectivity and identities, organizational understandings and procedures embedded in specific university cultures, traditional of different science disciplines, and the systemic logic and political rationale of the education and research system....... these positions, and one that maintains that a closer look at the statistics does not support this optimism because women’s percentage in recruitment positions is not increasing as the pool of potential female researchers increases, or to put it metaphorically, “the pipeline is leaking women all along” (Alper...... it identifies and describes a Danish verion of ‘the leaky pipeline’ from analyses of the ratios of women in science from high school through tenured positions. Finally it illustrates the cultural mechanisms at play in this process, based on the results of three studies. The first two analyze the educational...

  20. 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.

  1. Small amounts of functional ATP7A protein permit mild phenotype

    DEFF Research Database (Denmark)

    Møller, Lisbeth Birk

    2015-01-01

    concentrations, ATP7A shifts to the post-Golgi compartments or to the plasma membrane to export copper out of the cell. Impaired copper-regulation trafficking has been observed for ATP7A mutants, but its impact on the clinical outcome is not clear. The major problem in patients with MD seems to be insufficient...... of missense mutations on structural models of the ATP7A protein suggests that affected conserved residues generally lead to a severe phenotype. The ATP7A protein traffics within the cells. At low copper levels, ATP7A locates to the Trans-Golgi Network (TGN) to load cuproenzymes with copper, whereas at higher...

  2. Novel Methods of Hydrogen Leak Detection

    International Nuclear Information System (INIS)

    Pushpinder S Puri

    2006-01-01

    For hydrogen to become a consumer fuel for automotive and domestic power generation, safety is paramount. Today's hydrogen systems are built with inherent safety measures and multiple levels of protection. However, human senses, in particular, the sense of smell, is considered the ultimate safeguards against leaks. Since hydrogen is an odorless gas, use of odorants to detect leaks, as is done in case of natural gas, is obvious solution. The odorants required for hydrogen used in fuel cells have a unique requirement which must be met. This is because almost all of the commercial odorants used in gas leak detection contain sulfur which acts as poison for the catalysts used in hydrogen based fuel cells, most specifically for the PEM (polymer electrolyte membrane or proton exchange membrane) fuel cells. A possible solution to this problem is to use non-sulfur containing odorants. Chemical compounds based on mixtures of acrylic acid and nitrogen compounds have been adopted to achieve a sulfur-free odorization of a gas. It is, therefore, desired to have a method and system for hydrogen leak detection using odorant which can incorporate a uniform concentration of odorant in the hydrogen gas, when odorants are mixed in the hydrogen storage or delivery means. It is also desired to develop methods where the odorant is not added to the bulk hydrogen, keeping it free of the odorization additives. A series of novel solutions are proposed which address the issues raised above. These solutions are divided into three categories as follows: 1. Methods incorporating an odorant in the path of hydrogen leak as opposed to adding it to the hydrogen gas. 2. Methods where odorants are generated in-situ by chemical reaction with the leaking hydrogen 3. Methods of dispensing and storing odorants in high pressure hydrogen gas which release odorants to the gas at a uniform and predetermined rates. Use of one or more of the methods described here in conjunction with appropriate engineering

  3. Imbalance of plasma membrane ion leak and pump relationship as a new aetiological basis of certain disease states.

    Science.gov (United States)

    Ronquist, G; Waldenström, A

    2003-12-01

    The basis for life is the ability of the cell to maintain ion gradients across biological membranes. Such gradients are created by specific membrane-bound ion pumps [adenosine triphosphatases (ATPases)]. According to physicochemical rules passive forces equilibrate (dissipate) ion gradients. The cholesterol/phospholipid ratio of the membrane and the degree of saturation of phospholipid fatty acids are important factors for membrane molecular order and herewith a determinant of the degree of non-specific membrane leakiness. Other operative principles, i.e. specific ion channels can be opened and closed according to mechanisms that are specific to the cell. Certain compounds called ionophores can be integrated in the plasma membrane and permit specific inorganic ions to pass. Irrespective of which mechanism ions leak across the plasma membrane the homeostasis may be kept by increasing ion pumping (ATPase activity) in an attempt to restore the physiological ion gradient. The energy source for this work seems to be glycolytically derived ATP formation. Thus an increase in ion pumping is reflected by increased ATP hydrolysis and rate of glycolysis. This can be measured as an accumulation of breakdown products of ATP and end-products of anaerobic glycolysis (lactate). In certain disease entities, the balance between ATP formation and ion pumping may be disordered resulting in a decrease in inter alia (i.a.) cellular energy charge, and an increase in lactate formation and catabolites of adenylates. Cardiac syndrome X is proposed to be due to an excessive leakage of potassium ions, leading to electrocardiographic (ECG) changes, abnormal Tl-scintigraphy of the heart and anginal pain (induced by adenosine). Cocksackie B3 infections, a common agent in myocarditis might also induce an ionophore-like effect. Moreover, Alzheimer's disease is characterized by the formation of extracellular amyloid deposits in the brain of patients. Perturbation of cellular membranes by the

  4. Experiments on leak-selfwastage and leak-propagation

    International Nuclear Information System (INIS)

    Voss, J.; Vagt, P.; Westenbrugge, J.K. van; Joziasse, J.

    1984-01-01

    During the last years a considerable number of selfwastage experiments with small leaks of different shape and size and for different ferritic materials (2 1/4% Cr - and 12% Cr-steel) were performed by TNO and by INTERATOM, using several sodium test facilities. Many fabrication-methods of artificial micro-leaks were applied and examined. Selfplugging-, selfwastage- and reopening-effects were observed and evaluated during different time periods and under various test conditions. The main results will be discussed. Concerning the leak propagation program of INTERATOM, the first series of experiments was carried out this year. A short status report and some first results will be given. (author)

  5. 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.

  6. Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

    Science.gov (United States)

    Pissarek, M; Garcia de Arriba, S; Schäfer, M; Sieler, D; Nieber, K; Illes, P

    1998-10-01

    In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri

  7. 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.

  8. Aerospace Payloads Leak Test Methodology

    Science.gov (United States)

    Lvovsky, Oleg; Grayson, Cynthia M.

    2010-01-01

    Pressurized and sealed aerospace payloads can leak on orbit. When dealing with toxic or hazardous materials, requirements for fluid and gas leakage rates have to be properly established, and most importantly, reliably verified using the best Nondestructive Test (NDT) method available. Such verification can be implemented through application of various leak test methods that will be the subject of this paper, with a purpose to show what approach to payload leakage rate requirement verification is taken by the National Aeronautics and Space Administration (NASA). The scope of this paper will be mostly a detailed description of 14 leak test methods recommended.

  9. Tube leak detector

    International Nuclear Information System (INIS)

    Morita, Bunji; Takamura, Koichi; Matsuda, Shigehiro; Kiyosawa, Shun-ichi; Asami, Toru; Yamada, Hiroshi; Naruse, Shin-ichi.

    1995-01-01

    The device of the present invention detects occurrence of leakage in a steam generator, a steam heating tube, or a heat exchanger of a nuclear power plant. Namely, an vibration sensor is disposed at the rear end of a rod-like supersonic resonance member. A node portion for the vibrations of the resonance member is held by a holding member and attached to a wall surface of a can such as a boiler. With such a constitution, the resonance member is resonated by supersonic waves generated upon leakage of the tube. The vibrations are measured by the vibration sensor at the rear end. Presence of leakage is detected by utilizing one or more of resonance frequencies. Since the device adopts a resonance phenomenon, a conduction efficiency of the vibrations is high, thereby enabling to detect leakage at high sensitivity. In addition, the supersonic wave resonance member has its top end directly protruded into a pressure vessel such as a boiler by using a metal or a ceramic which is excellent in heat and pressure resistance. Accordingly, the sound of leak can be detected efficiently. (I.S.)

  10. Analysis of SX farm leak histories - Historical leak model (HLM)

    International Nuclear Information System (INIS)

    Fredenburg, E.A.

    1998-01-01

    This report uses readily available historical information to better define the volume, chemical composition, and Cs-137/Sr-90 amounts for leaks that have occurred in the past for tanks SX-108, SX-109, SX-111, and SX-112. In particular a Historical Leak Model (HLM) is developed that is a month by month reconciliation of tank levels, fill records, and calculated boil-off rates for these tanks. The HLM analysis is an independent leak estimate that reconstructs the tank thermal histories thereby deriving each tank's evaporative volume loss and by difference, its unaccounted losses as well. The HLM analysis was meant to demonstrate the viability of its approach, not necessarily to establish the HLM leak estimates as being definitive. Past leak estimates for these tanks have invariably resorted to soil wetting arguments but the extent of soil contaminated by each leak has always been highly uncertain. There is also a great deal of uncertainty with the HLM that was not quantified in this report, but will be addressed later. These four tanks (among others) were used from 1956 to 1975 for storage of high-level waste from the Redox process at Hanford. During their operation, tank waste temperatures were often as high as 150 C (300 F), but were more typically around 130 C. The primary tank cooling was by evaporation of tank waste and therefore periodic replacement of lost volume with water was necessary to maintain each tank's inventory. This active reflux of waste resulted in very substantial turnovers in tank inventory as well as significant structural degradation of these tanks. As a result of the loss of structural integrity, each of these tanks leaked during their active periods of operation. Unfortunately, the large turnover in tank volume associated with their reflux cooling has made a determination of leak volumes very difficult. During much of these tanks operational histories, inventory losses because of evaporative cooling could have effectively masked any volume

  11. Excessive extracellular ATP desensitizes P2Y2 and P2X4 ATP receptors provoking surfactant impairment ending in ventilation-induced lung injury

    NARCIS (Netherlands)

    D. Hasan (Djo); Satalin, J. (Joshua); van der Zee, P. (Philip); Kollisch-Singule, M. (Michaela); P. Blankman (Paul); Shono, A. (Atsuko); P. Somhorst (Peter); C.A. den Uil (Corstiaan); H.J. Meeder (Han); Kotani, T. (Toru); G.F. Nieman (Gary F.)

    2018-01-01

    textabstractStretching 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,

  12. Concentration device for leak liquids

    International Nuclear Information System (INIS)

    Matsumoto, Kaname; Matsuda, Ken; Takabori, Ken-ichi.

    1987-01-01

    Purpose: To improve radioactivity recovery and volume-reducing rates, as well as enable safety and easy handling for leak liquids resulted from reptures in coolant circuits. Constitution: The device of the invention comprises an evaporation vessel filled with leak fluids to a predetermined level, an airtight vessel disposed in the evaporation vessel containing hydrophilic porous material partially immersed in the leak fluids and means for heating the hydrophilic material. In this device, leak liquids are absorbed in the hydrophilic porous material, a great amount of water is evaporated from the outer surface of the hydrophilic porous material exposed above the liquid surface, and salts and radioactive material are remained on the inside and the outer surface of the porous material. The evaporated water content is condensated and recovered in a cooler and the remaining salts, etc. are discarded together with the porous material. The volume-reducing property can be improved by constituting the porous material with burnable material. (Takahashi, M.)

  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. Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.

    Science.gov (United States)

    da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

    2014-07-01

    We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Piezo1 regulates mechanotransductive release of ATP from human RBCs.

    Science.gov (United States)

    Cinar, Eyup; Zhou, Sitong; DeCourcey, James; Wang, Yixuan; Waugh, Richard E; Wan, Jiandi

    2015-09-22

    Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

  16. Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase.

    Science.gov (United States)

    Shyng, S L; Barbieri, A; Gumusboga, A; Cukras, C; Pike, L; Davis, J N; Stahl, P D; Nichols, C G

    2000-01-18

    ATP-sensitive potassium channels (K(ATP) channels) regulate cell excitability in response to metabolic changes. K(ATP) channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K(+) channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), activate K(ATP) channels and antagonize ATP inhibition of K(ATP) channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP(2) levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K(ATP) channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K(1/2), the half maximal inhibitory concentration, approximately 60 microM) than the sensitivities from control cells (K(1/2) approximately 10 microM). An inactive form of the PIP5K had little effect on the K(1/2) of wild-type channels but increased the ATP-sensitivity of a mutant K(ATP) channel that has an intrinsically lower ATP sensitivity (from K(1/2) approximately 450 microM to K(1/2) approximately 100 microM), suggesting a decrease in membrane PIP(2) levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP(2) and PI-3,4,5-P(3) levels, is a significant determinant of the physiological nucleotide sensitivity of K(ATP) channels.

  17. Mapping urban pipeline leaks: Methane leaks across Boston

    International Nuclear Information System (INIS)

    Phillips, Nathan G.; Ackley, Robert; Crosson, Eric R.; Down, Adrian; Hutyra, Lucy R.; Brondfield, Max; Karr, Jonathan D.; Zhao Kaiguang; Jackson, Robert B.

    2013-01-01

    Natural gas is the largest source of anthropogenic emissions of methane (CH 4 ) in the United States. To assess pipeline emissions across a major city, we mapped CH 4 leaks across all 785 road miles in the city of Boston using a cavity-ring-down mobile CH 4 analyzer. We identified 3356 CH 4 leaks with concentrations exceeding up to 15 times the global background level. Separately, we measured δ 13 CH 4 isotopic signatures from a subset of these leaks. The δ 13 CH 4 signatures (mean = −42.8‰ ± 1.3‰ s.e.; n = 32) strongly indicate a fossil fuel source rather than a biogenic source for most of the leaks; natural gas sampled across the city had average δ 13 CH 4 values of −36.8‰ (±0.7‰ s.e., n = 10), whereas CH 4 collected from landfill sites, wetlands, and sewer systems had δ 13 CH 4 signatures ∼20‰ lighter (μ = −57.8‰, ±1.6‰ s.e., n = 8). Repairing leaky natural gas distribution systems will reduce greenhouse gas emissions, increase consumer health and safety, and save money. Highlights: ► We mapped 3356 methane leaks in Boston. ► Methane leaks in Boston carry an isotopic signature of pipeline natural gas. ► Replacing failing gas pipelines will provide safety, environmental, and economic benefits. - We identified 3356 methane leaks in Boston, with isotopic characteristics consistent with pipeline natural gas.

  18. 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.

  19. 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...

  20. 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

  1. 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.

  2. 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.

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

    International Nuclear Information System (INIS)

    HEY, B.E.

    1999-01-01

    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

  4. 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.[...

  5. Effect of hydrogen peroxide on the main kinetic parameters of ATP hydrolysis by ouabain sensitive Na+, K+-ATP-ase in spermatozoa of infertile men

    Directory of Open Access Journals (Sweden)

    Р. В. Фафула

    2017-12-01

    Full Text Available Background: It is known that Na+,K+-ATP-ase plays important role in physiology of spermatozoa including their motility. Na+,K+-ATP-ase is one of the targets for reactive oxygen species. Hyperproduction of reactive oxygen species can damage sperm cells and it is considered to be as one of the mechanisms of male infertility. Objectives: To evaluate the H2O2 effect on the main kinetic parameters of ATP hydrolysis by ouabain-sensitive Na+,K+-ATPase of spermatozoa of fertile (normozoospermia and infertility men (asthenozoospermia. Materials and methods: Na+, K+-ATP-ase activity was determined spectrophotometrically by production of Pi. Concentration dependencies ware linearized in Lineweaver-Burk plot. Results: Effective inhibitory effect of H2O2 on ouabain-sensitive Na+,K+-ATP-ase activity of sperm cells of fertile and infertile men was demonstrated. The effects of H2O2 on the main kinetic parameters of the ATP hydrolysis with the involvement of Na+, K+-ATP-ase was studied. In the whole range of studied concentrations of ATP the Na+, K+-ATP-ase activity of spermatozoa of fertile and infertile men was reduced in the presence of H2O2 in the incubation medium. However, the optimal activity of the Na+, K+-ATP-ase activity of sperm cells in both normozoospermic and asthenozoospermic men was observed in the presence of 5 mM ATP in the incubation medium. By linearization of concentration curves in Lineweaver-Burk plot the main kinetic parameters of Na+, K+-activated, Mg2+-dependent ATP hydrolysis in the sperm cells of fertile and infertile men were determined. Under the effect of H2O2, the affinity constant of enzyme to ATP in normozoospermic and asthenozoospermic men increases several times. The initial maximum rate of ATP hydrolysis was significantly reduced only in the spermatozoa of fertile men with normozoospermia. Conclusions: Under conditions of H2O2-induced oxidative stress the inhibition of ouabain-sensitive Na+,K+-ATP-ase activity in sperm cells

  6. CFTR mediates noradrenaline-induced ATP efflux from DRG neurons.

    Science.gov (United States)

    Kanno, Takeshi; Nishizaki, Tomoyuki

    2011-09-24

    In our earlier study, noradrenaline (NA) stimulated ATP release from dorsal root ganglion (DRG) neurons as mediated via β(3) adrenoceptors linked to G(s) protein involving protein kinase A (PKA) activation, to cause allodynia. The present study was conducted to understand how ATP is released from DRG neurons. In an outside-out patch-clamp configuration from acutely dissociated rat DRG neurons, single-channel currents, sensitive to the P2X receptor inhibitor PPADS, were evoked by approaching the patch-electrode tip close to a neuron, indicating that ATP is released from DRG neurons, to activate P2X receptor. NA increased the frequency of the single-channel events, but such NA effect was not found for DRG neurons transfected with the siRNA to silence the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the immunocytochemical study using acutely dissociated rat DRG cells, CFTR was expressed in neurons alone, but not satellite cells, fibroblasts, or Schwann cells. It is concluded from these results that CFTR mediates NA-induced ATP efflux from DRG neurons as an ATP channel.

  7. 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.

  8. LOCATING LEAKS WITH ACOUSTIC TECHNOLOGY

    Science.gov (United States)

    Many water distribution systems in this country are almost 100 years old. About 26 percent of piping in these systems is made of unlined cast iron or steel and is in poor condition. Many methods that locate leaks in these pipes are time-consuming, costly, disruptive to operations...

  9. 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...

  10. 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.

  11. Frequencies of leaks and probability of ignition sources in the H-area tank farm

    International Nuclear Information System (INIS)

    Cramer, D.S.

    1994-01-01

    Point estimates are developed for the probability of an ignition source for tetraphenylborate (TPB) solids in H-area which leak into the annulus of Tank 48 and/or in the Filter Cell. Additionally, leak frequencies and leak rates are estimated for: the inner cell wall of Tank 48; Hanford connectors and single-wall transfer lines in the Filter Cell of the In-Tank Precipitation (ITP) Facility; and the double-wall transfer lines between tank 48, the Filter Cell, Tank 49 and the 'Late Wash' Tank

  12. 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

  13. 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

  14. Stable shRNA Silencing of Lactate Dehydrogenase A (LDHA) in Human MDA-MB-231 Breast Cancer Cells Fails to Alter Lactic Acid Production, Glycolytic Activity, ATP or Survival.

    Science.gov (United States)

    Mack, Nzinga; Mazzio, Elizabeth A; Bauer, David; Flores-Rozas, Hernan; Soliman, Karam F A

    2017-03-01

    In the US, African Americans have a high death rate from triple-negative breast cancer (TNBC), characterized by lack of hormone receptors (ER, PR, HER2/ERRB2) which are otherwise valuable targets of chemotherapy. There is a need to identify novel targets that negatively impact TNBC tumorigenesis. TNBCs release an abundance of lactic acid, under normoxic, hypoxic and hyperoxic conditions; this referred to as the Warburg effect. Accumulated lactic acid sustains peri-cellular acidity which propels metastatic invasion and malignant aggressive transformation. The source of lactic acid is believed to be via conversion of pyruvate by lactate dehydrogenase (LDH) in the last step of glycolysis, with most studies focusing on the LDHA isoform. In this study, LDHA was silenced using long-term MISSION® shRNA lentivirus in human breast cancer MDA-MB-231 cells. Down-regulation of LDHA transcription and protein expression was confirmed by western blot, immunocytochemistry and qPCR. A number of parameters were measured in fully viable vector controls versus knock-down (KD) clones, including levels of lactic acid produced, glucose consumed, ATP and basic metabolic rates. The data show that lentivirus V-165 generated a knock-down clone most effective in reducing both gene and protein levels to less than 1% of vector controls. Stable KD showed absolutely no changes in cell viability, lactic acid production, ATP, glucose consumption or basic metabolic rate. Given the complete absence of impact on any observed parameter by LDH-A KD and this being somewhat contrary to findings in the literature, further analysis was required to determine why. Whole-transcriptome analytic profile on MDA-MB-231 for LDH subtypes using Agilent Human Genome 4×44k microarrays, where the data show the following component breakdown. Transcripts: 30.47 % LDHA, 69.36% LDHB, 0.12% LDHC and 0.05% LDHD. These findings underscore the importance of alternative isoforms of LDH in cancer cells to produce lactic acid

  15. 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

  16. Imaging review of cerebrospinal fluid leaks

    OpenAIRE

    Naga V Vemuri; Lakshmi S P Karanam; Venkatesh Manchikanti; Srinivas Dandamudi; Sampath K Puvvada; Vineet K Vemuri

    2017-01-01

    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-dimensiona...

  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. Calibration of a leak detection spectrometer

    International Nuclear Information System (INIS)

    Geller, R.

    1958-01-01

    This paper describes a study of the possible methods for calibrating a leak detection spectrometer, and the estimation of outputs from the leaks is considered. With this in mind the question of sensitivity of leak detection is tackled on a very general level; first the sensitivity of the isolated instrument is determined, and then the sensitivity of an instrument connected to an installation where leaks may be suspected. Finally, practical solutions are proposed. (author) [fr

  19. Intraoperative leak testing has no correlation with leak after laparoscopic sleeve gastrectomy.

    Science.gov (United States)

    Sethi, Monica; Zagzag, Jonathan; Patel, Karan; Magrath, Melissa; Somoza, Eduardo; Parikh, Manish S; Saunders, John K; Ude-Welcome, Aku; Schwack, Bradley F; Kurian, Marina S; Fielding, George A; Ren-Fielding, Christine J

    2016-03-01

    Staple line leak is a serious complication of sleeve gastrectomy. Intraoperative methylene blue and air leak tests are routinely used to evaluate for leak; however, the utility of these tests is controversial. We hypothesize that the practice of routine intraoperative leak testing is unnecessary during sleeve gastrectomy. A retrospective cohort study was designed using a prospectively collected database of seven bariatric surgeons from two institutions. All patients who underwent sleeve gastrectomy from March 2012 to November 2014 were included. The performance of intraoperative leak testing and the type of test (air or methylene blue) were based on surgeon preference. Data obtained included BMI, demographics, comorbidity, presence of intraoperative leak test, result of test, and type of test. The primary outcome was leak rate between the leak test (LT) and no leak test (NLT) groups. SAS version 9.4 was used for univariate and multivariate analyses. A total of 1550 sleeve gastrectomies were included; most were laparoscopic (99.8%), except for one converted and two open cases. Routine intraoperative leak tests were performed in 1329 (85.7%) cases, while 221 (14.3%) did not have LTs. Of the 1329 cases with LTs, there were no positive intraoperative results. Fifteen (1%) patients developed leaks, with no difference in leak rate between the LT and NLT groups (1 vs. 1%, p = 0.999). After adjusting for baseline differences between the groups with a propensity analysis, the observed lack of association between leak and intraoperative leak test remained. In this cohort, leaks presented at a mean of 17.3 days postoperatively (range 1-67 days). Two patients with staple line leaks underwent repeat intraoperative leak testing at leak presentation, and the tests remained negative. Intraoperative leak testing has no correlation with leak due to laparoscopic sleeve gastrectomy and is not predictive of the later development of staple line leak.

  20. 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

  1. 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.

  2. 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.

  3. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, D J [Berkeley Nuclear Laboratories, Berkeley, Gloucestershire (United Kingdom)

    1978-10-01

    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.

  5. Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK(1) cells result in phosphorylation of FKHR and FKHRL1.

    NARCIS (Netherlands)

    Andreucci, M.; Michael, A.; Kramers, C.; Park, K.M.; Chen, A.; Matthaeus, T.; Alessandrini, A.; Haq, S.; Force, T.; Bonventre, J.V.

    2003-01-01

    BACKGROUND: Cell death and survival pathways are critical determinants of epithelial cell fate after ischemia. Forkhead proteins have been implicated in the regulation of cellular survival. METHODS and RESULTS: We have found that none of the forkhead family of proteins, FKHR, is phosphorylated after

  6. Kinetics of Ca2+- and ATP-dependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum

    NARCIS (Netherlands)

    Elzenga, J.T.M.; van Volkenburgh, E.

    Whole-cell patch-clamp techniques were used to measure anion currents through the plasma membrane of protoplasts of mesophyll cells of expanding pea (Pisum sativum L.) leaves. Voltage-induced changes of the currents could be modelled with single exponential activation and deactivation kinetics. The

  7. Nuclear fuel element leak detection system

    International Nuclear Information System (INIS)

    John, C.D. Jr.

    1978-01-01

    Disclosed is a leak detection system integral with a wall of a building used to fabricate nuclear fuel elements for detecting radiation leakage from the nuclear fuel elements as the fuel elements exit the building. The leak detecting system comprises a shielded compartment constructed to withstand environmental hazards extending into a similarly constructed building and having sealed doors on both ends along with leak detecting apparatus connected to the compartment. The leak detecting system provides a system for removing a nuclear fuel element from its fabrication building while testing for radiation leaks in the fuel element

  8. 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

  9. Leak rate measurements and detection systems

    International Nuclear Information System (INIS)

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

    1983-10-01

    A research program is under way to evaluate and develop improve 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 detection by vibrational diagnostic methods

    International Nuclear Information System (INIS)

    Siklossy, P.

    1983-01-01

    The possibilities and methods of leak detection due to mechanical failures in nuclear power plants are reviewed on the basis of the literature. Great importance is attributed to vibrational diagnostic methods for their adventageous characteristics which enable them to become final leak detecting methods. The problems of noise analysis, e.g. leak detection by impact sound measurements, probe characteristics, gain problems, probe selection, off-line analysis and correlation functions, types of leak noises etc. are summarized. Leak detection based on noise analysis can be installed additionally to power plants. Its maintenance and testing is simple. On the other hand, it requires special training and measuring methods. (Sz.J.)

  11. Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes.

    Science.gov (United States)

    Girotti, S; Ferri, E; Cascione, M L; Comuzio, S; Mazzuca, A; Orlandini, A; Breccia, A

    1989-07-01

    Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.

  12. Antihypertrophic Effects of Small Molecules that Maintain Mitochondrial ATP Levels Under Hypoxia

    Directory of Open Access Journals (Sweden)

    Hiroaki Nagai

    2017-10-01

    Full Text Available Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin β, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.

  13. 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

  14. Leaking electricity in domestic appliances

    International Nuclear Information System (INIS)

    Meier, Alan; Rosen, Karen

    1999-01-01

    Many types of home electronic equipment draw electric power when switched off or not performing their principal functions. Standby power use (or ''leaking electricity'') for most appliances ranges from 1 - 20 watts. Even though standby use of each device is small, the combined standby power use of all appliances in a home can easily exceed 50 watts. Leaking electricity is already responsible for 5 to 10 percent of residential electricity use in the United States and over 10 percent in Japan. An increasing number of white goods also have standby power requirements. There is a growing international effort to limit standby power to around one watt per device. New and existing technologies are available to meet this target at little or no extra cost

  15. 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.

  16. 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

  17. Role of connexin 32 hemichannels in the release of ATP from peripheral nerves.

    Science.gov (United States)

    Nualart-Marti, Anna; del Molino, Ezequiel Mas; Grandes, Xènia; Bahima, Laia; Martin-Satué, Mireia; Puchal, Rafel; Fasciani, Ilaria; González-Nieto, Daniel; Ziganshin, Bulat; Llobet, Artur; Barrio, Luis C; Solsona, Carles

    2013-12-01

    Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy. Copyright © 2013 Wiley Periodicals, Inc.

  18. Extracellular ATP acts as a damage associated molecular pattern (DAMP signal in plants

    Directory of Open Access Journals (Sweden)

    Kiwamu eTanaka

    2014-09-01

    Full Text Available 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, 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 Ca2+ 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 the future research of extracellular ATP as a DAMP signal.

  19. Leak testing. Environment and workplaces

    International Nuclear Information System (INIS)

    1984-01-01

    Workplaces specified for leak testing are divided into clean workplaces of the 1st degree, clean workplaces of the second degree, clean workplaces of the third degree and semi-clean workplaces. Clean workplaces are further subdivided into permanent and temporary workplaces. For all said types of workplaces the standard sets the following provisions: basic equipment, machines and instrumentation, permitted and prohibited working activities and principles for maintenance and inspection. (E.S.)

  20. 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.

  1. Repair of EL4 leaks

    International Nuclear Information System (INIS)

    1985-03-01

    The reactor shutdown was decided on the 15th of November 1984, because the evolution of the carbon dioxide quantity in the helium blanket of the heavy water. Leaks have been localized on three different channels. Repairs have been made in hard conditions taking into account the reactor state (materials strongly irradiated). The restart has been authorized on the 24th of January 1985 [fr

  2. Single K ATP channel opening in response to action potential firing in mouse dentate granule neurons.

    Science.gov (United States)

    Tanner, Geoffrey R; Lutas, Andrew; Martínez-François, Juan Ramón; Yellen, Gary

    2011-06-08

    ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

  3. 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 ...

  4. 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

  5. Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

    Science.gov (United States)

    Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

    2016-01-01

    Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the

  6. 3,3'-Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells.

    Science.gov (United States)

    Gong, Yixuan; Sohn, Heesook; Xue, Ling; Firestone, Gary L; Bjeldanes, Leonard F

    2006-05-01

    Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.

  7. 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.

  8. 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...

  9. 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).

  10. Electromotive Potential Distribution and Electronic Leak Currents in Working YSZ Based SOCs

    DEFF Research Database (Denmark)

    Mogensen, Mogens Bjerg; Jacobsen, Torben

    2009-01-01

    The size of electronic leak currents through the YSZ electrolyte of solid oxide cells have been calculated using basic solid state electrochemical relations and literature data. The distribution of the electromotive potential, of Galvani potential, of concentration of electrons, e, and electron...... holes, h, was also calculated as these parameters are the basis for the understanding of the electronic conductivity that causes the electronic leak currents. The results are illustrated with examples. The effects of electrolyte thickness, temperature and cell voltage on the electronic leak current...

  11. Activation of liver X receptor decreases atherosclerosis in Ldlr⁻/⁻ mice in the absence of ATP-binding cassette transporters A1 and G1 in myeloid cells.

    Science.gov (United States)

    Kappus, Mojdeh S; Murphy, Andrew J; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L; Tall, Alan R; Westerterp, Marit

    2014-02-01

    Liver X receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly upregulate genes involved in reverse cholesterol transport and (2) exert anti-inflammatory effects mediated by transrepression of nuclear factor-κB target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate reverse cholesterol transport, would abolish the beneficial effects of LXR activation on atherosclerosis. LXR activator T0901317 substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr(-/-) mice were transplanted with Abca1(-/-)Abcg1(-/-) or wild-type bone marrow (BM) and fed a Western-type diet for 6 weeks with or without T0901317 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0901317 markedly decreased lesion area, complexity, and inflammatory cell infiltration in the Abca1(-/-)Abcg1(-/-) BM-transplanted mice. To investigate whether this was because of macrophage Abca1/g1 deficiency, Ldlr(-/-) mice were transplanted with LysmCreAbca1(fl/fl)Abcg1(fl/fl) or Abca1(fl/fl)Abcg1(fl/fl) BM and fed Western-type diet with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups, and the decrease was more prominent in the LysmCreAbca1(fl/fl)Abcg1(fl/fl) group. The results suggest that anti-inflammatory effects of LXR activators are of key importance to their antiatherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to transrepress inflammatory genes.

  12. 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

  13. 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.

  14. 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)

  15. Paravalvular Leak in Structural Heart Disease.

    Science.gov (United States)

    Goel, Kashish; Eleid, Mackram F

    2018-03-06

    This review will summarize the growing importance of diagnosing and managing paravalvular leak associated with surgical and transcatheter valves. The burden of paravalvular leak is increasing; however, advanced imaging techniques and high degree of clinical suspicion are required for diagnosis and management. The latest data from pivotal clinical trials in the field of transcatheter aortic valve replacement suggest that any paravalvular leak greater than mild was associated with worse clinical outcomes. Percutaneous techniques for paravalvular leak closure are now the preferred approach, and surgical repair is reserved for contraindications and unsuccessful procedures. Recent data from studies evaluating paravalvular leak closure outcomes report a greater than 90% success rate with a significant improvement in patient symptoms. Paravalvular leak is a growing problem in the structural heart disease arena. Percutaneous closure is successful in more than 90% of the procedures with a low complication rate.

  16. 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

  17. Benign Biliary Strictures and Leaks.

    Science.gov (United States)

    Devière, Jacques

    2015-10-01

    The major causes of benign biliary strictures include surgery, chronic pancreatitis, primary sclerosing cholangitis, and autoimmune cholangitis. Biliary leaks mainly occur after surgery and, rarely, abdominal trauma. These conditions may benefit from a nonsurgical approach in which endoscopic retrograde cholangiopancreatography (ERCP) plays a pivotal role in association with other minimally invasive approaches. This approach should be evaluated for any injury before deciding about the method for repair. ERCP, associated with peroral cholangioscopy, plays a growing role in characterizing undeterminate strictures, avoiding both unuseful major surgeries and palliative options that might compromise any further management. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. 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

  19. 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.

  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 molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Acoustic emission leak monitoring system LMS-96

    International Nuclear Information System (INIS)

    Liska, J.; Cvrcek, M.; Mueller, L.

    1997-01-01

    On-line acoustic emission leak monitoring under industrial conditions of nuclear power plants is a problem with specific features setting specific demands on the leak monitoring system. The paper briefly reviews those problems (attenuation pattern of a real structure, acoustic background, alarm system, etc.) and the solution of some of them is discussed. Information is presented on the Acoustic Emission Leak Monitoring System LMS-96 by SKODA NUCLEAR MACHINERY and the system's function is briefly described. (author)

  3. 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.

  4. Hepatobiliary scintigraphy in patients with bile leaks

    International Nuclear Information System (INIS)

    Carichner, S.L.; Nagle, C.E.

    1987-01-01

    Hepatobiliary scintigraphy has been recognized as a useful tool in detecting the presence and sites of bile leaks. The clinical settings in which bile leaks are likely to occur, as well as some of the scintigraphic patterns seen in patients with bile leaks, are reviewed here. Tips for technologists are offered on interventions that might enhanced the quality of information available to the nuclear physician

  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. 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.

  7. ATP-induced changes in rat skeletal muscle contractility.

    Science.gov (United States)

    Gabdrakhmanov, A I; Khayrullin, A E; Grishin, C H; Ziganshin, A U

    2015-01-01

    Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be

  8. 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

  9. 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.

  10. The P2X7 ATP receptor modulates renal cyst development in vitro

    International Nuclear Information System (INIS)

    Hillman, Kate A.; Woolf, Adrian S.; Johnson, Tanya M.; Wade, Angela; Unwin, Robert J.; Winyard, Paul J.D.

    2004-01-01

    P2X 7 , a piercing receptor, is expressed in renal collecting ducts as they undergo fulminant cysto genesis in the cpk/cpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Dissociated cpk/cpk kidneys generate cysts from cell aggregates within 24 h of suspension culture and we demonstrate that BzATP, a P2X 7 agonist, reduces cystogenesis. This effect is P2X 7 -specific, because: (i) equimolar concentrations of other purinergic agonists, ATP and UTP, had lesser effects and (ii) the P2X 7 inhibitor, oxidized ATP, abrogated the BzATP-mediated reduction in cystogenesis. BzATP did not significantly affect total cell number, proliferation, LDH release or caspase 3 activity, and zVAD-fmk, a caspase blocker, failed to modulate BzATP effects. In addition, this P2X 7 agonist did not significantly alter cyst size, probably excluding altered vectorial transport. In vivo, ATP was detected in cyst fluid from cpk/cpk kidneys; moreover, P2X 7 protein was also upregulated in human fetal ARPKD epithelia versus normal fetal collecting ducts. Thus, ATP may inhibit pathological renal cyst growth through P2X 7 signaling

  11. Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

    Science.gov (United States)

    Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

    2014-12-01

    In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    DEFF Research Database (Denmark)

    Hoffmann, E K

    2001-01-01

    . This had a major influence on my later description of a swelling-activated Cl- conductance. (ii) The pump-leak steady-state concept for cell volume control was introduced by Krogh in 1946, but it was developed in detail by Leaf and Ussing in 1959. This concept was the basis for me and others, when we later...... 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...

  13. 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.

  14. Study on water leak-tightness of small leaks on a 1 inch cylinder valve

    International Nuclear Information System (INIS)

    Miyazawa, T.; Kasai, Y.; Inabe, N.; Aritomi, M.

    2002-01-01

    Practical thresholds for water leak-tightness of small leaks were determined by experimentation. Measurements for small leak samples were taken of air leakage rates and water leakage rates for identical leak samples in order to identify parameters that influence water leak-tightness threshold. Four types of leaks were evaluated: a fine wire inserted in an O-ring seal, a glass capillary tube, a stainless steel orifice, and a scratched valve stem on a 1 inch UF 6 cylinder valve. Experimental results demonstrated that the key parameter for water leak-tightness is the opening size of the leak hole. The maximum allowable hole size to achieve water leak-tightness ranged from 10 to 20 μm in diameter in this study. Experimental results with 1 inch UF 6 cylinder valve samples demonstrated that the acceptance criteria for preshipment leakage test, 1x10 -3 ref-cm 3 .s -1 , as prescribed in ANSI N14.5 is an appropriate value from the point of view of water leak-tightness for enriched UF 6 packages. The mechanism of water leak-tightness is plugging by tiny particles existing in water. The water used in experiments in this study contained far fewer particles than in water assumed to be encountered under accident conditions of transport. Therefore, the water leak-tightness threshold determined in this study is a conservative value in a practical evaluation. (author)

  15. 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

    A planar anode-supported solid oxide fuel cell (SOFC) has been tested to investigate gas tightness of the electrolyte and the applied seals. Gas leaks reduce the efficiency of the SOFC and it is thus important to determine and minimise them. Probe gases (He and Ar) and a Quadrupole Mass Spectrome......A planar anode-supported solid oxide fuel cell (SOFC) has been tested to investigate gas tightness of the electrolyte and the applied seals. Gas leaks reduce the efficiency of the SOFC and it is thus important to determine and minimise them. Probe gases (He and Ar) and a Quadrupole Mass...... 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...

  16. Emergence of flagellar beating from the collective behavior of individual ATP-powered dyneins

    OpenAIRE

    Namdeo, S.; Onck, P. R.

    2016-01-01

    Flagella are hair-like projections from the surface of eukaryotic cells, and they play an important role in many cellular functions, such as cell-motility. The beating of flagella is enabled by their internal architecture, the axoneme, and is powered by a dense distribution of motor proteins, dyneins. The dyneins deliver the required mechanical work through the hydrolysis of ATP. Although the dynein-ATP cycle, the axoneme microstructure, and the flagellar-beating kinematics are well studied, ...

  17. Summary of PWR leak detection studies

    International Nuclear Information System (INIS)

    Cho, J.H.; Elia, F.A. Jr.

    1986-01-01

    Thermal-hydraulic analysis can be used to determine the location and magnitude of leaks inside and location of leaks outside a pressurized water reactor (PWR) containment as required by plant technical specifications. The major advantage of this detection method is that it minimizes radiation exposure of maintenance personnel because most of the leak detection process is performed from the control room outside containment. Plant-specific analyses are utilized to predict change in parameters such as local dew point temperature, relative humidity, dry bulb temperature, and flow rate to sump for various leak rates and enthalpies. These parameter responses are then programmed into the plant computer and instrumentation is provided for area monitoring. The actual inputs are continuously monitored and compared to the predicted plant responses to identify the leak location and quantify the leak. This study concludes that a system that monitors dew point (or relative humidity) and dry bulb temperature changes together with the flow rate to the sump will provide the capability to both locate and quantify a leak inside a containment, while a system that monitors dew point temperature (or relative humidity) changes will provide the capability to locate a leak outside a containment

  18. 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.

  19. 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

  20. Improved method of measurement for outer leak

    International Nuclear Information System (INIS)

    Xu Guang

    2012-01-01

    Pneumatic pipeline is installed for the airborne radioactivity measurement equipment, air tightness and outer leak rate are essential for the testing of the characteristics, both in the national criteria and ISO standards, an improved practical method is available for the measurement of the outer air leak rate based on the engineering experiences for the equipment acceptance and testing procedure. (authors)

  1. 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.

  2. 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

  3. Design of sealing arrangements for development of leak tight articulated manipulator for waste management and reprocessing plants

    International Nuclear Information System (INIS)

    Patil, Satish B.; Mudaiya, Avinash K.

    2016-01-01

    As a part of development of Remote handling equipment and gadgets, Leak tight Articulated Manipulator (ARTM) has been developed for shielded facilities of Nuclear Recycle Plant. The ARTM is a Master Slave Manipulator having 8 Kg Payload capacity and most suitable for shielded sampling cubicles of Waste Management plants and dilution hot cells of Reprocessing plants. All the motions and forces are transmitted from Master to slave arm through the mechanical linkages, which are housed inside the wall tube. The mechanical linkages and wall tube run across the shielded wall. The applications involving seal tightness of the glove boxes and dilution hot cells intend to use all the accessories including MSMs to be leak tight. During the design of leak tight ARTM, all the potential leak paths through the components of existing ARTM were examined critically and static and dynamic seals were designed based on the geometry and requirement of particular leak path. A leak proof testing set up was fabricated for evaluating the leak rates across the manipulator contemplating the actual operating conditions. The manipulator was subjected to the Friction, Rigidity and Leak tests. The results of the tests are satisfactory and as per the International standards available. The leak rates measured for different manipulators were in the range of 0.05 to 0.1 % of air volume/hr @ 200 mm of water columndifferential negative pressure. (author)

  4. Mice Lacking Pannexin 1 Release ATP and Respond Normally to All Taste Qualities.

    Science.gov (United States)

    Vandenbeuch, Aurelie; Anderson, Catherine B; Kinnamon, Sue C

    2015-09-01

    Adenosine triphosphate (ATP) is required for the transmission of all taste qualities from taste cells to afferent nerve fibers. ATP is released from Type II taste cells by a nonvesicular mechanism and activates purinergic receptors containing P2X2 and P2X3 on nerve fibers. Several ATP release channels are expressed in taste cells including CALHM1, Pannexin 1, Connexin 30, and Connexin 43, but whether all are involved in ATP release is not clear. We have used a global Pannexin 1 knock out (Panx1 KO) mouse in a series of in vitro and in vivo experiments. Our results confirm that Panx1 channels are absent in taste buds of the knockout mice and that other known ATP release channels are not upregulated. Using a luciferin/luciferase assay, we show that circumvallate taste buds from Panx1 KO mice normally release ATP upon taste stimulation compared with wild type (WT) mice. Gustatory nerve recordings in response to various tastants applied to the tongue and brief-access behavioral testing with SC45647 also show no difference between Panx1 KO and WT. These results confirm that Panx1 is not required for the taste evoked release of ATP or for neural and behavioral responses to taste stimuli. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Leak detector of liquid sodium

    International Nuclear Information System (INIS)

    Himeno, Yoshiaki.

    1975-01-01

    Object: To arrange a cable core connected to a leakage current detector on the outer wall of piping for liquid sodium, devices or the like and apply a voltage to said core and outer wall to quickly and securely detect the leakage of liquid sodium. Structure: A cable, which is composed of metal coating formed of metal material (copper, steel, stainless, etc.) which is apt to be corroded by reaction products of liquid sodium with water and oxygen in air, and metal oxide (such as magnesium oxide, beryllium oxide, aluminum oxide) as an electric insulator is arranged on the outer wall of pipes or devices. In the event sodium is leaked from the pipes or devices, said metal coating and the insulator are corroded, and the leakage of sodium is sensed by a leakage current detector through the core in the cable. (Kamimura, M.)

  6. On the helium gas leak test

    International Nuclear Information System (INIS)

    Nishikawa, Akira; Ozaki, Susumu

    1975-01-01

    The helium gas leak test (Helium mass spectrometer testing) has a leak detection capacity of the highest level in practical leak tests and is going to be widely applied to high pressure vessels, atomic and vacuum equipments that require high tightness. To establish a standard test procedure several series of experiments were conducted and the results were investigated. The conclusions are summarized as follows: (1) The hood method is quantitatively the most reliable method. The leak rate obtained by tests using 100% helium concentration should be the basis of the other method of test. (2) The integrating method, bell jar method, and vacuum spray method can be considered quantitative when particular conditions are satisfied. (3) The sniffer method is not to be considered quantitive. (4) The leak rate of the hood, integrating, and bell jar methods is approximately proportional to the square of the helium partial pressure. (auth.)

  7. 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

  8. SCTI chemical leak detection test plan

    International Nuclear Information System (INIS)

    1981-01-01

    Tests will be conducted on the CRBRP prototype steam generator at SCTI to determine the effects of steam generator geometry on the response of the CRBRP chemical leak detection system to small water-to-sodium leaks in various regions of the steam generator. Specifically, small injections of hydrogen gas (simulating water leaks) will be made near the two tubesheets, and the effective transport times to the main stream exit and vent line hydrogen meters will be measured. The magnitude and time characteristics of the meters' response will also be measured. This information will be used by the Small Leak Protection Base Program (SG027) for improved predictions of meter response times and leak detection sensitivity

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

    Science.gov (United States)

    Droguett, Karla; Rios, Mariana; Carreño, Daniela V; Navarrete, Camilo; Fuentes, Christian; Villalón, Manuel; Barrera, Nelson P

    2017-07-15

    Extracellular ATP, in association with [Ca 2+ ] i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca 2+ ] i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca 2+ ] i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml -1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an

  14. 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.

  15. 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.

  16. 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...

  17. Ultrasonic Detectors Safely Identify Dangerous, Costly Leaks

    Science.gov (United States)

    2013-01-01

    In 1990, NASA grounded its space shuttle fleet. The reason: leaks detected in the hydrogen fuel systems of the Space Shuttles Atlantis and Columbia. Unless the sources of the leaks could be identified and fixed, the shuttles would not be safe to fly. To help locate the existing leaks and check for others, Kennedy Space Center engineers used portable ultrasonic detectors to scan the fuel systems. As a gas or liquid escapes from a leak, the resulting turbulence creates ultrasonic noise, explains Gary Mohr, president of Elmsford, New York-based UE Systems Inc., a long-time leader in ultrasonic detector technologies. "In lay terms, the leak is like a dog whistle, and the detector is like the dog ear." Because the ultrasound emissions from a leak are highly localized, they can be used not only to identify the presence of a leak but also to help pinpoint a leak s location. The NASA engineers employed UE s detectors to examine the shuttle fuel tanks and solid rocket boosters, but encountered difficulty with the devices limited range-certain areas of the shuttle proved difficult or unsafe to scan up close. To remedy the problem, the engineers created a long-range attachment for the detectors, similar to "a zoom lens on a camera," Mohr says. "If you are on the ground, and the leak is 50 feet away, the detector would now give you the same impression as if you were only 25 feet away." The enhancement also had the effect of reducing background noise, allowing for a clearer, more precise detection of a leak s location.

  18. 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...

  19. Interaction between neuronal nitric oxide synthase signaling and temperature influences sarcoplasmic reticulum calcium leak: role of nitroso-redox balance.

    Science.gov (United States)

    Dulce, Raul A; Mayo, Vera; Rangel, Erika B; Balkan, Wayne; Hare, Joshua M

    2015-01-02

    Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca(2+)) leak. We measured SR Ca(2+) leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1(-/-)]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR(-/-)) mice. In WT cardiomyocytes, SR Ca(2+) leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1(-/-) cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR(-/-) cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1(-/-) cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1(-/-) cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1(-/-) cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca(2+) content in NOS1(-/-) cells but had no effect in WT or GSNOR(-/-). Ca(2+) leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR

  20. Imaging review of cerebrospinal fluid leaks.

    Science.gov (United States)

    Vemuri, Naga V; Karanam, Lakshmi S P; Manchikanti, Venkatesh; Dandamudi, Srinivas; Puvvada, Sampath K; Vemuri, Vineet K

    2017-01-01

    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. 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.

  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. 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

  4. Vacuum leak test technique of JT-60

    International Nuclear Information System (INIS)

    Kaminaga, Atsushi; Arai, Takashi; Kodama, Kozo; Sasaki, Noboru; Saidoh, Masahiro

    1998-01-01

    Since a vacuum vessel of JT-60 is very large (167 m 3 ) and is combined with many components, such as magnetic coils, neutral beam injection systems and RF heating systems, etc., the position of leak testing exceeds 700. The two kind of techniques for vacuum leak test used in JT-60 has been described. Firstly the probe helium gas can be fed remotely in the three-dimensionally sectioned 54 regions of the JT-60 torus. The leak test was very rapidly performed by using this method. Secondly the helium detector system has been modified by the additional installation of the cryopump, which reduced the background level of the deuterium gas. The sensitivity of vacuum leak test with the cryopump was two orders of magnitude larger than that of without it. The examples of the performed vacuum leak test are stated. The vacuum leaks during experiments were 9 times. They were caused by thermal strain and plasma discharge. The vacuum leaks just after maintenance are 36 times which mainly caused by mis-installation. (author)

  5. Transient leak detection in crude oil pipelines

    Energy Technology Data Exchange (ETDEWEB)

    Beushausen, R.; Tornow, S.; Borchers, H. [Nord-West Oelleitung, Wilhelmshaven (Germany); Murphy, K.; Zhang, J. [Atmos International Ltd., Manchester (United Kingdom)

    2004-07-01

    Nord-West Oelleitung (NWO) operates 2 crude oil pipelines from Wilhemshaven to Koln and Hamburg respectively. German regulations for transporting flammable substances stipulate that 2 independent continuously working procedures be used to detect leaks. Leak detection pigs are used routinely to complement the surveillance system. This paper described the specific issues of transient leak detection in crude oil pipelines. It was noted that traditional methods have failed to detect leaks that occur immediately after pumps are turned on or off because the pressure wave generated by the transient dominates the pressure wave that results from the leak. Frequent operational changes in a pipeline are often accompanied by an increased number of false alarms and failure to detect leaks due to unsteady operations. NWO therefore decided to have the Atmos statistical pipeline leak detection (SPLD) system installed on their pipelines. The key to the SPLD system is the sequential probability ratio test. Comprehensive data validation is performed following reception of pipeline data from the supervisory control and data acquisition (SCADA) system. The validated data is then used to calculate the corrected flow imbalance, which is fed into the SPRT to determine if there is an increase in the flow imbalance. Pattern recognition is then used to distinguish a leak from operational changes. The SPLD is unique because it uses 3 computational pipeline monitoring methods simultaneously, namely modified volume balance, statistical analysis, and pressure and flow monitoring. The successful installation and testing of the SPLD in 2 crude oil pipelines was described along with the main difficulties associated with transient leaks. Field results were presented for both steady-state and transient conditions. 5 refs., 2 tabs., 16 figs.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Means for preventing radioactive fluid leaking

    International Nuclear Information System (INIS)

    Akatsu, Jun-ichi.

    1975-01-01

    Object: To permit prevention of leakage of radioactive fluid from line and valve leak sections while also recovering the liquid by producing a vacuum state in a leak-off line by means of a water ejector. Structure: A portion of the water from a condenser is forced by a condensed water pump through a water ejector tank to a recovery tank while controlling an orifice and valve, whereby a vacuum state is produced in the leak-off line to withdraw the leakage fluid. (Kamimura, M.)

  11. Leak detection system for RBMK coolant circuit

    Energy Technology Data Exchange (ETDEWEB)

    Cherkashov, Ju M; Strelkov, B P; Korolev, Yu V; Eperin, A P; Kozlov, E P; Belyanin, L A; Vanukov, V N [Leningrad Nuclear Power Plant, Leningrad (Russian Federation). Research and Development Inst. of Power Engineering

    1997-12-31

    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.

  12. Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak.

    Science.gov (United States)

    Leon-Aparicio, Daniel; Pacheco, Jonathan; Chavez-Reyes, Jesus; Galindo, Jose M; Valdes, Jesus; Vaca, Luis; Guerrero-Hernandez, Agustin

    2017-07-01

    We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca 2+ leak using synthetic Ca 2+ indicators that report changes in both the cytoplasmic ([Ca 2+ ] i ) and the luminal ER ([Ca 2+ ] ER ) Ca 2+ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca 2+ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca 2+ ] ER but did not decrease the ER Ca 2+ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP 3 R and only Orai3 channel supported the 2-APB-induced ER Ca 2+ leak, while Orai1 and Orai2 inhibited this type of ER Ca 2+ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca 2+ leak but did not modify the ER Ca 2+ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca 2+ ] i response after TG but only when the ER store had been overloaded with Ca 2+ by eliminating the acidic internal Ca 2+ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca 2+ leak but forms an ER Ca 2+ leak channel that is limiting the overloading with Ca 2+ of the ER store. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. 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...

  14. 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

    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 pro...

  15. The leak microstructure, preliminary results

    International Nuclear Information System (INIS)

    Lombardi, M.

    1997-01-01

    The leak microstructure, a new type of element for position-sensitive proportional gas counter, is introduced. For every single detected ionizing radiation it gives a pair of ''induced'' charges of the same quantity (pulses of the same amplitude), of opposite sign, with the same collection time and essentially in time coincidence, that are proportional to the collected primary ionization. A gas multiplication up to 1.5 x 10 5 was achieved. The complete absence of insulating materials in the active volume of this microstructure enables to avoid problems of charging-up and makes its behaviour stable and repeatable. By using the charge-pair generated, it allows the development of a position-sensitive detecting board with a two-dimensional read-out. Between the two external surfaces of this board it is possible to insert an intermediate third conducting layer which reduces (or practically suppresses) the capacitive cross-talk between the X and Y read-out strip systems. Furthermore, this intermediate layer can give a very fast trigger to coordinate the charge-pair and to govern the data acquisition system. By reading every strip separately it is possible to resolve the multi hit problem in two-dimensions. Using isobutane as the gas, an energy resolution of about 8% FWHM was recorded with α-particles from an 241 Am source. In isobutane gas, X-rays from a 55 Fe source and β-particles from a 14 C source were also detected. (orig.)

  16. 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

  17. 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

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

    African Journals Online (AJOL)

    Endoscopic management of bile leaks after laparoscopic cholecystectomy. ... endoscopic management at a median of 12 days (range 2 - 104 days) after surgery. Presenting features included intra-abdominal collections with pain in 58 cases ...

  19. 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)

  20. The new CSA standard for leak detection

    Energy Technology Data Exchange (ETDEWEB)

    Pietsch, Ulli [TAU, Edmonton, Alberta, (Canada); Scott, Don [TransCanada Pipelines, Edmonton, Alberta, (Canada)

    2010-07-01

    Standards need to be updated regularly to reflect current technology and industry practices. This paper describes the new Canadian Standards Association (CSA) for leak detection called Recommended Practice for Liquid Hydrocarbon Pipeline System Leak Detection, Annex E, which can be found in the CSA Z662 Oil and Gas Pipeline Systems standard. The CSA formed a task force of industry experts and regulators for a period of 18 months to draft the new standards. Several comparisons were made with the American Petroleum Institute (API) recommended practice API 1130. This new version introduces and defines the terms, critical instrument, critical process and dependent instrument. The most significant improvement made by the new Annex E is the new requirement that an operating company must develop a leak detection strategy. The writing of a leak detection manual is given high priority. The use of both Annex E and API 1130 is recommended.

  1. Postoperative ascitic leaks: the ongoing challenge.

    Science.gov (United States)

    Rosemurgy, A S; Statman, R C; Murphy, C G; Albrink, M H; McAllister, E W

    1992-06-01

    The leak of ascitic fluid from surgical incisions is thought to be associated with a very high mortality rate. There have been few reports, however, focusing on the clinical characteristics, management, or mortality rates of this condition. During a 10-year period, 18 patients with postoperative ascitic fluid leaks were treated. All patients had ascites before surgery and all had liver disease; in 13 of the 18 patients alcoholic liver disease was the cause of ascites. Ten of the 18 patients died (56%). Midline incisions were more often associated with recalcitrant leaks and fatal complications than were transverse incisions. Early consideration of fascial dehiscence and prompt repair is emphasized. The most effective predictor of survival was cessation of the leak.

  2. Helium leak testing the Westinghouse LCP coil

    International Nuclear Information System (INIS)

    Merritt, P.A.; Attaar, M.H.; Hordubay, T.D.

    1983-01-01

    The tests, equipment, and techniques used to check the Westinghouse LCP coil for coolant flow path integrity and helium leakage are unique in terms of test sensitivity and application. This paper will discuss the various types of helium leak testing done on the LCP coil as it enters different stages of manufacture. The emphasis will be on the degree of test sensitivity achieved under shop conditions, and what equipment, techniques and tooling are required to achieve this sensitivity (5.9 x 10 -8 scc/sec). Other topics that will be discussed are helium flow and pressure drop testing which is used to detect any restrictions in the flow paths, and the LCP final acceptance test which is the final leak test performed on the coil prior to its being sent for testing. The overall allowable leak rate for this coil is 5 x 10 -6 scc/sec. A general evaluation of helium leak testing experience are included

  3. 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.

  4. 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)

  5. Acoustic leak detection in nuclear power plants

    International Nuclear Information System (INIS)

    McElroy, J.W.

    1986-01-01

    For several years now, utilities have been utilizing acoustic leak detection methods as an operating tool in their nuclear power stations. The purpose for using the leak detection system at the various stations vary from safety, ALARA, improved operations, preventive maintenance, or increased plant availability. This paper describes the various acoustic techniques and their application. The techniques are divided into three categories: specific component leakage, intersystem leakage, and pipe through-wall crack leakage. The paper addresses each category in terms of motivation to monitor, method of application and operation, and benefits to be gained. Current requirements are reviewed and analyzed with respect to the acoustic techniques. The paper shows how acoustic leak detection is one of the most effective leak detection tools available. 9 figures, 1 table

  6. 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.

  7. Routine intraoperative leak testing for sleeve gastrectomy: is the leak test full of hot air?

    Science.gov (United States)

    Bingham, Jason; Lallemand, Michael; Barron, Morgan; Kuckelman, John; Carter, Preston; Blair, Kelly; Martin, Matthew

    2016-05-01

    Staple line leak after sleeve gastrectomy (SG) is a rare but dreaded complication with a reported incidence of 0% to 8%. Many surgeons routinely test the staple line with an intraoperative leak test (IOLT), but there is little evidence to validate this practice. In fact, there is a theoretical concern that the leak test may weaken the staple line and increase the risk of a postop leak. Retrospective review of all SGs performed over a 7-year period was conducted. Cases were grouped by whether an IOLT was performed, and compared for the incidence of postop staple line leaks. The ability of the IOLT for identifying a staple line defect and for predicting a postoperative leak was analyzed. Five hundred forty-two SGs were performed between 2007 and 2014. Thirteen patients (2.4%) developed a postop staple line leak. The majority of patients (n = 494, 91%) received an IOLT, including all 13 patients (100%) who developed a subsequent clinical leak. There were no (0%) positive IOLTs and no additional interventions were performed based on the IOLT. The IOLT sensitivity and positive predictive value were both 0%. There was a trend, although not significant, to increase leak rates when a routine IOLT was performed vs no routine IOLT (2.6% vs 0%, P = .6). The performance of routine IOLT after SG provided no actionable information, and was negative in all patients who developed a postoperative leak. The routine use of an IOLT did not reduce the incidence of postop leak, and in fact was associated with a higher leak rate after SG. Published by Elsevier Inc.

  8. 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...

  9. 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.

  10. Evaluation of pipeline leak detection systems

    International Nuclear Information System (INIS)

    Glauz, W.D.; Flora, J.D.; Hennon, G.J.

    1993-01-01

    Leaking underground storage tank system presents an environmental concern and a potential health hazard. It is well known that leaks in the piping associated with these systems account for a sizeable fraction of the leaks. EPA has established performance standards for pipeline leak detection systems, and published a document presenting test protocols for evaluating these systems against the standards. This paper discusses a number of facets and important features of evaluating such systems, and presents results from tests of several systems. The importance of temperature differences between the ground and the product in the line is shown both in theory and with test data. The impact of the amount of soil moisture present is addressed, along with the effect of frozen soil. These features are addressed both for line tightness test systems, which must detect leaks of 0.10 gal/h (0.38 L/h) at 150% of normal line pressure, or 0.20 gal/h (0.76 L/h) at normal line pressure, and for automatic line leak detectors that must detect leaks of 3 gal/h (11 L/h) at 10 psi (69 kPa) within an hour of the occurrence of the leak. This paper also addresses some statistical aspects of the evaluation of these systems. Reasons for keeping the evaluation process ''blind'' to the evaluated company are given, along with methods for assuring that the tests are blind. Most importantly, a test procedure is presented for evaluating systems that report a flow rate (not just a pass/fail decision) that is much more efficient than the procedure presented in the EPA protocol, and is just as stringent

  11. 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

  12. 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)

  13. 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.)

  14. Ca2+ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

    Science.gov (United States)

    Lee, Jaekwang; Chun, Ye-Eun; Han, Kyung-Seok; Lee, Jungmoo; Woo, Dong Ho

    2015-01-01

    Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of Gαq-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte. PMID:25792866

  15. Wireless sensor network for sodium leak detection

    International Nuclear Information System (INIS)

    Satya Murty, S.A.V.; Raj, Baldev; Sivalingam, Krishna M.; Ebenezer, Jemimah; Chandran, T.; Shanmugavel, M.; Rajan, K.K.

    2012-01-01

    Highlights: ► Early detection of sodium leak is mandatory in any reactor handling liquid sodium. ► Wireless sensor networking technology has been introduced for detecting sodium leak. ► We designed and developed a wireless sensor node in-house. ► We deployed a pilot wireless sensor network for handling nine sodium leak signals. - Abstract: To study the mechanical properties of Prototype Fast Breeder Reactor component materials under the influence of sodium, the IN Sodium Test (INSOT) facility has been erected and commissioned at Indira Gandhi Centre for Atomic Research. Sodium reacts violently with air/moisture leading to fire. Hence early detection of sodium leak if any is mandatory for such plants and almost 140 sodium leak detectors are placed throughout the loop. All these detectors are wired to the control room for data collection and monitoring. To reduce the cost, space and maintenance that are involved in cabling, the wireless sensor networking technology has been introduced in the sodium leak detection system of INSOT. This paper describes about the deployment details of the pilot wireless sensor network and the measures taken for the successful deployment.

  16. Helium leak testing of scanning electron microscope

    International Nuclear Information System (INIS)

    Ahmad, Anis; Tripathi, S.K.; Mukherjee, D.

    2015-01-01

    Scanning Electron Microscope (SEM) is a specialized electron-optical device which is used for imaging of miniscule features on topography of material specimens. Conventional SEMs used finely focused high energy (about 30 KeV) electron beam probes of diameter of about 10nm for imaging of solid conducting specimens. Vacuum of the order of 10"-"5 Torr is prerequisite for conventional Tungsten filament type SEMs. One such SEM was received from one of our laboratory in BARC with a major leak owing to persisting poor vacuum condition despite continuous pumping for several hours. He-Leak Detection of the SEM was carried out at AFD using vacuum spray Technique and various potential leak joints numbering more than fifty were helium leak tested. The major leak was detected in the TMP damper bellow. The part was later replaced and the repeat helium leak testing of the system was carried out using vacuum spray technique. The vacuum in SEM is achieved is better than 10"-"5 torr and system is now working satisfactorily. (author)

  17. 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.)

  18. 40 CFR 63.424 - Standards: Equipment leaks.

    Science.gov (United States)

    2010-07-01

    ....424 Standards: Equipment leaks. (a) Each owner or operator of a bulk gasoline terminal or pipeline... location of all equipment in gasoline service at the facility. (c) Each detection of a liquid or vapor leak... replacement of leaking equipment shall be completed within 15 calendar days after detection of each leak...

  19. Small leak shutdown, location, and behavior in LMFBR steam generators

    International Nuclear Information System (INIS)

    Sandusky, D.W.

    1976-01-01

    The paper summarizes an experimental study of small leaks tested under LMFBR steam generator conditions. Defected tubes were exposed to flowing sodium and steam. The observed behavior of the defected tubes is reported along with test results of shutdown methods. Leak location methods were investigated. Methods were identified to open plugged defects for helium leak testing and detect plugged leaks by nondestructive testing

  20. 40 CFR 86.328-79 - Leak checks.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 18 2010-07-01 2010-07-01 false Leak checks. 86.328-79 Section 86.328... Gasoline-Fueled and Diesel-Fueled Heavy-Duty Engines; Gaseous Exhaust Test Procedures § 86.328-79 Leak checks. (a) Vacuum side leak check. (1) Any location within the analysis system where a vacuum leak could...

  1. 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...

  2. 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

  3. Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.

    Directory of Open Access Journals (Sweden)

    Patrizia Pellegatti

    2008-07-01

    Full Text Available There is growing awareness that tumour cells build up a "self-advantageous" microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP.Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours.Our results show that ATP in the tumour interstitium is in the hundreds micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

  4. Field tests and commercialization of natural gas leak detectors

    Energy Technology Data Exchange (ETDEWEB)

    Choi, D S; Jeon, J S; Kim, K D; Cho, Y A [R and D Center, Korea Gas Corporation, Ansan (Korea)

    1999-09-01

    Objectives - (1) fields test of industrial gas leak detection monitoring system. (2) commericialization of residential gas leak detector. Contents - (1) five sets of gas leak detection monitoring system were installed at natural gas transmition facilities and tested long term stability and their performance. (2) improved residential gas leak detector was commercialised. Expected benefits and application fields - (1) contribution to the improvement of domestic gas sensor technology. (2) localization of fabrication technology for gas leak detectors. 23 refs., 126 figs., 37 tabs.

  5. 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.

  6. 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

  7. 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

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

    DEFF Research Database (Denmark)

    Hübschmann, Martin; Skladchikova, Galina

    2008-01-01

    overlaps with the site of NCAM-FGFR interaction, and ATP is capable of disrupting NCAM-FGFR binding. This implies that NCAM signaling through FGFR can be regulated by ATP, which is supported by the observation that ATP can abrogate NCAM-induced neurite outgrowth. Finally, ATP can induce NCAM ectodomain...... shedding, possibly affecting the structural plasticity associated with learning and memory....

  9. 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

  10. Single-Shell Tanks Leak Integrity Elements/ SX Farm Leak Causes and Locations - 12127

    Energy Technology Data Exchange (ETDEWEB)

    Girardot, Crystal [URS- Safety Management Solutions, Richland, Washington 99352 (United States); Harlow, Don [ELR Consulting Richland, Washington 99352 (United States); Venetz, Theodore; Washenfelder, Dennis [Washington River Protection Solutions, LLC Richland, Washington 99352 (United States); Johnson, Jeremy [U.S. Department of Energy, Office of River Protection Richland, Washington 99352 (United States)

    2012-07-01

    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-045-91F 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 1-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 dry-wells 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

  11. Capillary leak syndrome: etiologies, pathophysiology, and management.

    Science.gov (United States)

    Siddall, Eric; Khatri, Minesh; Radhakrishnan, Jai

    2017-07-01

    In various human diseases, an increase in capillary permeability to proteins leads to the loss of protein-rich fluid from the intravascular to the interstitial space. Although sepsis is the disease most commonly associated with this phenomenon, many other diseases can lead to a "sepsis-like" syndrome with manifestations of diffuse pitting edema, exudative serous cavity effusions, noncardiogenic pulmonary edema, hypotension, and, in some cases, hypovolemic shock with multiple-organ failure. The term capillary leak syndrome has been used to describe this constellation of disease manifestations associated with an increased capillary permeability to proteins. Diseases other than sepsis that can result in capillary leak syndrome include the idiopathic systemic capillary leak syndrome or Clarkson's disease, engraftment syndrome, differentiation syndrome, the ovarian hyperstimulation syndrome, hemophagocytic lymphohistiocytosis, viral hemorrhagic fevers, autoimmune diseases, snakebite envenomation, and ricin poisoning. Drugs including some interleukins, some monoclonal antibodies, and gemcitabine can also cause capillary leak syndrome. Acute kidney injury is commonly seen in all of these diseases. In addition to hypotension, cytokines are likely to be important in the pathophysiology of acute kidney injury in capillary leak syndrome. Fluid management is a critical part of the treatment of capillary leak syndrome; hypovolemia and hypotension can cause organ injury, whereas capillary leakage of administered fluid can worsen organ edema leading to progressive organ injury. The purpose of this article is to discuss the diseases other than sepsis that produce capillary leak and review their collective pathophysiology and treatment. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  12. 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....

  13. 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.

  14. Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

    Science.gov (United States)

    Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

    2014-01-01

    Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

  15. 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 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.

  16. 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.

  17. Novel cellular bouton structure activated by ATP in the vascular wall of porcine retinal arterioles.

    Science.gov (United States)

    Misfeldt, Mikkel Wölck; Aalkjaer, Christian; Simonsen, Ulf; Bek, Toke

    2010-12-01

    The retinal blood flow is regulated by the tone of resistance arterioles, which is influenced by purinergic compounds such as adenosine and adenosine 5'-triphosphate (ATP) released from the retinal tissue. However, it is unknown what cellular elements in the perivascular retina are responsible for the effect of purines on the tone of retinal arterioles. Porcine retinal arterioles were loaded with the calcium-sensitive fluorophore Oregon green. The vessels were mounted in a confocal myograph for simultaneous recordings of tone and calcium activity in cells of the vascular wall during stimulation with ATP and adenosine, with and without modifiers of these compounds. Additionally, immunohistochemistry was used to localize elements with calcium activity in the vascular wall. Hyperfluorescence indicating calcium activity was recorded in a population of abundant round boutons interspersed in a network of vimentin-positive processes located immediately external to the smooth muscle cell layer but internal to the perivascular glial cells. These structures showed calcium activity when the vessel was relaxed with ATP but not when it was relaxed with adenosine. Ryanodine reduced calcium activity in the boutons, whereas the ATP antagonist adenosine-5'-O-(α, β- methylene diphosphate) reduced calcium activity in both the boutons and vascular tone. The vasodilating effect of purines in porcine retinal tissue involves ATP-dependent calcium activity in a layer of cellular boutons located external to the vascular smooth muscle cells and internal to the perivascular glial cells.

  18. ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain

    Energy Technology Data Exchange (ETDEWEB)

    Celikel, Reha; Veldore, Vidya Harini [University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Mathews, Irimpan [Stanford Synchrotron Radiation Lightsource, 2575 Sand Hill Road, Menlo Park, CA 94025 (United States); Devine, Kevin M., E-mail: kdevine@tcd.ie [Trinity College Dublin, Dublin 2 (Ireland); Varughese, Kottayil I., E-mail: kdevine@tcd.ie [University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

    2012-07-01

    The histidine WalK (YycG) plays a crucial role in coordinating murein synthesis with cell division and the crystal structure of its ATP binding domain has been determined. Interestingly the bound ATP was not hydrolyzed during crystallization and remains intact in the crystal lattice. In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferring the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg{sup 2+} ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3′ of the sugar ring and O2B of the β-phosphate, implying an internal hydrogen bond. The stability of the WalK–ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.

  19. 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

  20. 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...

  1. 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.)

  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. Process for detecting leak faults using a helium mass spectrometer

    International Nuclear Information System (INIS)

    Divet, Claude; Morin, Claude.

    1977-01-01

    The description is given of a process for detecting very small leak faults putting into communication the outer and inner sides of the wall of a containment, one of these wall sides being in contact with gaseous helium under a pressure of around one torr, the other side being one of the limits of a space pumped down to a residual gas pressure under 10 -3 torr. This space is in communication with the measuring cell of a helium mass spectrometer. This process may be applied to the detection of faults in metal claddings of the fuel rods used in nuclear reactors [fr

  4. 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.

  5. Location of leaks in pressurized underground pipelines

    International Nuclear Information System (INIS)

    Eckert, E.G.; Maresca, J.W. Jr.

    1993-01-01

    Millions of underground storage tanks (UST) are used to store petroleum and other chemicals. The pressurized underground pipelines associated with USTs containing petroleum motor fuels are typically 2 in. in diameter and 50 to 200 ft in length. These pipelines typically operate at pressures of 20 to 30 psi. Longer lines, with diameters up to 4 in., are found in some high-volume facilities. There are many systems that can be used to detect leaks in pressurized underground pipelines. When a leak is detected, the first step in the remediation process is to find its location. Passive-acoustic measurements, combined with advanced signal-processing techniques, provide a nondestructive method of leak location that is accurate and relatively simple, and that can be applied to a wide variety of pipelines and pipeline products

  6. Enhanced leak detection risk model development

    Energy Technology Data Exchange (ETDEWEB)

    Harron, Lorna; Barlow, Rick; Farquhar, Ted [Enbridge Pipelines Inc., Edmonton, Alberta (Canada)

    2010-07-01

    Increasing concerns and attention to pipeline safety have engaged pipeline companies and regulatory agencies to extend their approaches to pipeline integrity. The implementation of High Consequence Areas (HCAs) has especially had an impact on the development of integrity management protocols (IMPs) for pipelines. These IMPs can require that a risk based assessment of integrity issues be applied to specific HCA risk factors. This paper addresses the development of an operational risk assessment approach for pipeline leak detection requirements for HCAs. A detailed risk assessment algorithm that includes 25 risk variables and 28 consequence variables was developed for application to all HCA areas. This paper describes the consultative process that was used to workshop the development of this algorithm. Included in this description is how the process addressed various methods of leak detection across a wide variety of pipelines. The paper also looks at development challenges and future steps in applying operation risk assessment techniques to mainline leak detection risk management.

  7. 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

  8. 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.)

  9. Cold Leak Tests of LHC Beam Screens

    CERN Document Server

    Collomb-Patton, C; Jenninger, B; Kos, N

    2009-01-01

    In order to guide the high energy proton beams inside its two 27 km long vacuum rings, the Large Hadron Collider (LHC) at CERN, Geneva, makes use of superconducting technology to create the required magnetic fields. More than 4000 beam screens, cooled at 7 20 K, are inserted inside the 1.9 K beam vacuum tubes to intercept beam induced heat loads and to provide dynamic vacuum stability. As extremely high helium leak tightness is required, all beam screens have been leak tested under cold conditions in a dedicated test stand prior to their installation. After describing the beam screen design and its functions, this report focuses on the cold leak test sequence and discusses the results.

  10. Nuclear genetic defects of mitochondrial ATP synthase

    Czech Academy of Sciences Publication Activity Database

    Hejzlarová, Kateřina; Mráček, Tomáš; Vrbacký, Marek; Kaplanová, Vilma; Karbanová, Vendula; Nůsková, Hana; Pecina, Petr; Houštěk, Josef

    2014-01-01

    Roč. 63, Suppl.1 (2014), S57-S71 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/11/0970; GA ČR GAP303/12/1363; GA MZd(CZ) NT12370; GA MZd(CZ) NT14050 Grant - others:Univerzita Karlova(CZ) 370411 Institutional support: RVO:67985823 Keywords : mitochondrial diseases * TMEM70 * ATPAF1 * ATP5A1 * ATP5E Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.293, year: 2014

  11. 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

  12. CT assessment of anastomotic bowel leak

    Energy Technology Data Exchange (ETDEWEB)

    Power, N. [Department of Radiology, Sunnybrook Hospital, Toronto, Ontario M4N 3M5 (Canada); Atri, M. [Department of Radiology, Sunnybrook Hospital, Toronto, Ontario M4N 3M5 (Canada)]. E-mail: mostafa.atri@sw.ca; Ryan, S. [Department of Radiology, Sunnybrook Hospital, Toronto, Ontario M4N 3M5 (Canada); Haddad, R. [Department of Surgery, Sunnybrook Hospital, Toronto, Ontario M4N 3M5 (Canada); Smith, A. [Department of Surgery, Sunnybrook Hospital, Toronto, Ontario M4N 3M5 (Canada)

    2007-01-15

    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.

  13. LMFBR steam generator leak detection development in the United States

    Energy Technology Data Exchange (ETDEWEB)

    Magee, P M; Gerrels, E E; Greene, D A [General Electric Company, Sunnyvale, CA (United States); McKee, J [Argonne National Laboratory, Argonne, IL (United States)

    1978-10-01

    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{sub 2} and O{sub 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)

  14. 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)

  15. Fuel leak detection on large transport airplanes

    OpenAIRE

    Behbahani-Pour, M.J.; Radice, G.

    2016-01-01

    Fuel leakage has the risk of being ignited by external ignition sources, and therefore it is important to detect\\ud any fuel leakage before the departure of the aircraft. Currently, there are no fuel leak detection systems installed\\ud on commercial aircrafts, to detect fuel tank leakage, while only a small number of more recent aircraft, have a fuel\\ud monitoring system, that generates a fuel leak-warning message in cockpit in the case of fuel imbalance between the\\ud tanks. The approach pro...

  16. 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

  17. 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

  18. 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)

  19. Transanal Drainage of Coloanal Anastomotic Leaks

    Directory of Open Access Journals (Sweden)

    Bradley Sherman

    2018-01-01

    Full Text Available The conventional operative intervention for leaks following coloanal anastomoses has been proximal fecal diversion with or without take-down of anastomosis. A few of these cases are also amenable to percutaneous drainage. Ostomies created in this situation are often permanent, specifically in cases where coloanal anastomoses are taken down at the time of reoperation. We present two patients who developed perianastomotic pelvic abscesses that were treated with transanal large bore catheter drainage resulting in successful salvage of coloanal anastomoses without the need for a laparotomy or ostomy creation. We propose this to be an effective therapeutic approach to leaks involving low coloanal anastomoses in the absence of generalized peritonitis.

  20. Apparatus for Leak Testing Pressurized Hoses

    Science.gov (United States)

    Underwood, Steve D. (Inventor); Garrison, Steve G. (Inventor); Gant, Bobby D. (Inventor); Palmer, John R. (Inventor)

    2015-01-01

    A hose-attaching apparatus for leak-testing a pressurized hose may include a hose-attaching member. A bore may extend through the hose-attaching member. An internal annular cavity may extend coaxially around the bore. At least one of a detector probe hole and a detector probe may be connected to the internal annular cavity. At least a portion of the bore may have a diameter which is at least one of substantially equal to and less than a diameter of a hose to be leak-tested.

  1. 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).

  2. 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

  3. ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

    Science.gov (United States)

    Liao, Jielou

    2004-03-01

    Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

  4. Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation.

    Science.gov (United States)

    Ishida, Akihiko; Yamada, Yasuko; Kamidate, Tamio

    2008-11-01

    In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.

  5. Effects and mechanism of acid rain on plant chloroplast ATP synthase.

    Science.gov (United States)

    Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2016-09-01

    Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

  6. Rapid granulation tissue regeneration by intracellular ATP delivery--a comparison with Regranex.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Howard

    Full Text Available This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.

  7. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  8. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    International Nuclear Information System (INIS)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-01-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

  9. Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum.

    Science.gov (United States)

    Melani, Alessia; Corti, Francesca; Stephan, Holger; Müller, Christa E; Donati, Chiara; Bruni, Paola; Vannucchi, Maria Giuliana; Pedata, Felicita

    2012-01-01

    In the central nervous system (CNS) ATP and adenosine act as transmitters and neuromodulators on their own receptors but it is still unknown which part of extracellular adenosine derives per se from cells and which part is formed from the hydrolysis of released ATP. In this study extracellular concentrations of adenosine and ATP from the rat striatum were estimated by the microdialysis technique under in vivo physiological conditions and after focal ischemia induced by medial cerebral artery occlusion. Under physiological conditions, adenosine and ATP concentrations were in the range of 130 nmol/L and 40 nmol/L, respectively. In the presence of the novel ecto-ATPase inhibitor, PV4 (100 nmol/L), the extracellular concentration of ATP increased 12-fold to ~360 nmol/L but the adenosine concentration was not altered. This demonstrates that, under physiological conditions, adenosine is not a product of extracellular ATP. In the first 4h after ischemia, adenosine increased to ~690 nmol/L and ATP to ~50 nmol/L. In the presence of PV4 the extracellular concentration of ATP was in the range of 450 nmol/L and a significant decrease in extracellular adenosine (to ~270 nmol/L) was measured. The contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia onset. Furthermore we demonstrated, by immunoelectron microscopy, the presence of the concentrative nucleoside transporter CNT2 on plasma and vesicle membranes isolated from the rat striatum. These results are in favor that adenosine is transported in vesicles and is released in an excitation-secretion manner under in vivo physiological conditions. Early after ischemia, extracellular ATP is hydrolyzed by ecto-nucleotidases which significantly contribute to the increase in extracellular adenosine. To establish the contribution of extracellular ATP to adenosine might constitute the basis for devising a correct putative purinergic strategy aimed at protection from ischemic damage

  10. Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

    Science.gov (United States)

    Meulemans, Ann; Seneca, Sara; Pribyl, Thomas; Smet, Joel; Alderweirldt, Valerie; Waeytens, Anouk; Lissens, Willy; Van Coster, Rudy; De Meirleir, Linda; di Rago, Jean-Paul; Gatti, Domenico L; Ackerman, Sharon H

    2010-02-05

    Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F(1)) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120-124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the death of a 14-month-old patient. We have investigated the impact of the pathogenic W94R mutation on Atp12p structure/function. Plasmid-borne wild type human Atp12p rescues the respiratory defect of a yeast ATP12 deletion mutant (Deltaatp12). The W94R mutation alters the protein at the most highly conserved position in the Pfam sequence and renders HuAtp12p insoluble in the background of Deltaatp12. In contrast, the yeast protein harboring the corresponding mutation, ScAtp12p(W103R), is soluble in the background of Deltaatp12 but not in the background of Deltaatp12Deltafmc1, a strain that also lacks Fmc1p. Fmc1p is a yeast mitochondrial protein not found in higher eukaryotes. Tryptophan 94 (human) or 103 (yeast) is located in a positively charged region of Atp12p, and hence its mutation to arginine does not alter significantly the electrostatic properties of the protein. Instead, we provide evidence that the primary effect of the substitution is on the dynamic properties of Atp12p.

  11. Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

    Science.gov (United States)

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

    2017-08-22

    The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

  12. Analytical study of sodium combustion phenomena under sodium leak accidents

    International Nuclear Information System (INIS)

    Kim, Byung Ho; Jeong, J. Y.; Jeong, K. C.; Kim, T. J.; Choi, J. H.

    2001-12-01

    The rise of temperature and pressure, the release of aerosol in the buildings as a result of sodium fire must be considered for the safety measures of LMR. Therefore for the safety of the LMR, it is necessary to understand the characteristics of sodium fire, resulting from the various type of leakage. ASSCOPS(Analysis of Simultaneous Sodium Combustion in Pool and Spray) is the computer code for the analysis of the thermal consequence of sodium leak and fire in LMR that has been developed by Japan Nuclear Cycle Development Institute(JNC) in Japan. In this study, a preliminary analysis of sodium leak and fire accidents in S/G building of KALIMER is made by using ASSCOPS code. Various phenomena of interest are spray and pool burning, peak pressure, temperature change, local structure temperature, aerosol behavior, drain system into smothering tank, ventilation characteristics at each cell with the safety venting system and nitrogen injection system. In this calculation, the dimension of the S/G building was chosen in accordance with the selected options of LMR name KALIMER(Korea). As a result of this study, it was shown that subsequent effect of sodium fire depended upon whether the sodium continued to leak from the pipe or not, whether the ventilation system was running, whether the inert gas injection system was provided, whether the sodium on floor was drained into the smothering tank or not, whether the building was sealed or not, etc. Specially the excessive rise of pressure into each cell was prevented by installing the pressure release plates on wall of the building

  13. Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

    DEFF Research Database (Denmark)

    Ostergaard, L; Teilum, K; Mirza, O

    2000-01-01

    Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown...... to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover......-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant...

  14. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

    1988-07-12

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

  15. 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

  16. Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak.

    Science.gov (United States)

    Chetham, P M; Sefton, W D; Bridges, J P; Stevens, T; McMurtry, I F

    1997-04-01

    Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown. Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion. (1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective. Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.

  17. Characterization of cellular protective effects of ATP13A2/PARK9 expression and alterations resulting from pathogenic mutants.

    Science.gov (United States)

    Covy, Jason P; Waxman, Elisa A; Giasson, Benoit I

    2012-12-01

    Mutations in ATP13A2, which encodes a lysosomal P-type ATPase of unknown function, cause an autosomal recessive parkinsonian syndrome. With mammalian cells, we show that ATP13A2 expression protects against manganese and nickel toxicity, in addition to proteasomal, mitochondrial, and oxidative stress. Consistent with a recessive mode of inheritance of gene defects, disease-causing mutations F182L and G504R are prone to misfolding and do not protect against manganese and nickel toxicity because they are unstable as a result of degradation via the endoplasmic reticulum-associated degradation (ERAD)-proteasome system. The protective effects of ATP13A2 expression are not due to inhibition of apoptotic pathways or a reduction in typical stress pathways, insofar as these pathways are still activated in challenged ATP13A2-expressing cells; however, these cells display a dramatic reduction in the accumulation of oxidized and damaged proteins. These data indicate that, contrary to a previous suggestion, ATP13A2 is unlikely to convey cellular resilience simply by acting as a lysosomal manganese transporter. Consistent with the recent identification of an ATP13A2 recessive mutation in Tibetan terriers that develop neurodegeneration with neuronal ceroid lipofucinoses, our data suggest that ATP13A2 may function to import a cofactor required for the function of a lysosome enzyme(s). Copyright © 2012 Wiley Periodicals, Inc.

  18. ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis.

    Science.gov (United States)

    Fois, Giorgio; Winkelmann, Veronika Eva; Bareis, Lara; Staudenmaier, Laura; Hecht, Elena; Ziller, Charlotte; Ehinger, Konstantin; Schymeinsky, Jürgen; Kranz, Christine; Frick, Manfred

    2018-02-05

    Vesicular P2X 4 receptors are known to facilitate secretion and activation of pulmonary surfactant in the alveoli of the lungs. P2X 4 receptors are expressed in the membrane of lamellar bodies (LBs), large secretory lysosomes that store lung surfactant in alveolar type II epithelial cells, and become inserted into the plasma membrane after exocytosis. Subsequent activation of P2X 4 receptors by adenosine triphosphate (ATP) results in local fusion-activated cation entry (FACE), facilitating fusion pore dilation, surfactant secretion, and surfactant activation. Despite the importance of ATP in the alveoli, and hence lung function, the origin of ATP in the alveoli is still elusive. In this study, we demonstrate that ATP is stored within LBs themselves at a concentration of ∼1.9 mM. ATP is loaded into LBs by the vesicular nucleotide transporter but does not activate P2X 4 receptors because of the low intraluminal pH (5.5). However, the rise in intravesicular pH after opening of the exocytic fusion pore results in immediate activation of vesicular P2X 4 by vesicular ATP. Our data suggest a new model in which agonist (ATP) and receptor (P2X 4 ) are located in the same intracellular compartment (LB), protected from premature degradation (ATP) and activation (P2X 4 ), and ideally placed to ensure coordinated and timely receptor activation as soon as fusion occurs to facilitate surfactant secretion. © 2018 Fois et al.

  19. 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

  20. 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

  1. GASFLOW analysis of a tritium leak accident

    International Nuclear Information System (INIS)

    Farman, R.F.; Fujita, R.K.; Travis, J.R.

    1994-01-01

    The consequences of an earthquake-induced fire involving a tritium leak were analyzed using the GASFLOW computer code. Modeling features required by the analysis include ventilation boundary conditions, flow of a gas mixture in an enclosure containing obstacles, thermally induced buoyancy, and combustion phenomena. (author). 2 refs., 6 figs

  2. GASFLOW analysis of a tritium leak accident

    International Nuclear Information System (INIS)

    Farman, R.F.; Fujita, R.K.; Travis, J.R.

    1994-01-01

    The consequences of an earthquake-induced fire involving a tritium leak were analyzed using the GASFLOW computer code. Modeling features required by the analysis include ventilation boundary conditions, flow of a gas mixture in an enclosure containing obstacles, thermally induced buoyancy, and combustion phenomena

  3. 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...

  4. A Quick Guide to Paravalvular Leak Closure

    Science.gov (United States)

    Gafoor, Sameer; Franke, Jennifer; Bertog, Stefan; Lam, Simon; Vaskelyte, Laura; Hofmann, Ilona; Matic, Predrag

    2015-01-01

    Paravalvular leak (PVL) is a seldomly covered aspect of structural heart disease. However, this is a condition that frequently presents after valvular replacement. This article will cover the diagnosising and treating PVL (i.e. imaging, access, and device selection). In addition, specific aspects of aortic and mitral PVL closure will be covered in this review. PMID:29588686

  5. The concept of leak before failure

    International Nuclear Information System (INIS)

    Pellissier-Tanon, A.

    1985-01-01

    The ability to demonstrate the state of leak before failure introduce a new tangible element in the estimation of safety from which it becomes possible to estimate more rationnally the risks associated to crack formation. This paper presents the international positions on the full section break suppression of steam and primary pipes (Federal Republic of Germany, USA, France) [fr

  6. Glucose is required to maintain high ATP-levels for the energy utilizing steps during PDT-induced apoptosis

    International Nuclear Information System (INIS)

    Oberdanner, C.; Plaetzer, K.; Kiesslich, T.; Krammer, B.

    2003-01-01

    Full text: Photodynamic therapy (PDT) may trigger apoptosis or necrosis in cancer cells. Several steps in the induction and execution of apoptosis require high amounts of adenosine-5'-triphosphate (ATP). Since the mitochondrial membrane potential (ΔΨ) decreases early in apoptosis, we raised the question about the mechanisms of maintaining a sufficiently high ATP-level. We therefore monitored ΔΨ and the intracellular ATP-level of apoptotic human epidermoid carcinoma cells (A431) after photodynamic treatment with aluminium (III) phthalocyanine tetrasulfonate chloride. A maximum of caspase-3 activation and nuclear fragmentation was found at fluences of about 4 J.cm -2 . Under these conditions apoptotic cells reduced ΔΨ rapidly, while the ATP-level remained high for 4 to 6 hours after treatment for cells supplied with glucose. To analyze the contribution of glycolysis to the energy supply during apoptosis experiments were carried out with cells deprivated of glucose. These cells showed a rapid drop of ATP-content and neither caspase-activation nor nuclear fragmentation could be detected. We conclude that the use of glucose as a source of ATP is obligatory for the execution of PDT-induced apoptosis. (author)

  7. Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

    Directory of Open Access Journals (Sweden)

    Milos B. Rokic

    2018-04-01

    Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

  8. Developments in steam generator leak detection at Ontario Hydro

    Energy Technology Data Exchange (ETDEWEB)

    Maynard, K.J.; Singh, V.P. [Ontario Hydro Technologies, Toronto, Ontario (Canada)

    1998-07-01

    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 ({approx}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)

  9. 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)

  10. 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)

  11. Experiments and calculations to leak openings and leak rates on typical piping components and systems

    International Nuclear Information System (INIS)

    Hoefler, A.; Grebner, H.

    1992-01-01

    Calculations of leak opening and leak rate for through cracks in piping components have been performed. The analyses are pre- or mostly post-calculations to experiments performed at the HDR facility under PWR operating conditions. Piping components under consideration are small diameter straight pipes with circumferential cracks, pipe bends with longitudinal or circumferential cracks and pipe branches with weldment cracks. The component are loaded by internal pressure and opening as well as closing bending moment. The finite element method and two-phase flow leak rate programs are 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. 6 refs., 16 figs., 2 tabs

  12. 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

  13. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    -generating enzymes in pancreatic juice, adenylate kinase, and NDP kinase, capable of sequentially phosphorylating AMP via ADP to ATP. Activities of nonspecific phosphatases, nucleotide pyrophosphatase/phosphodiesterases, and adenosine deaminase were negligible. Taken together, CCK-8 stimulation of pancreas causes...

  14. Performance evaluation of PFBR wire type sodium leak detectors

    International Nuclear Information System (INIS)

    Vijayakumar, G.; Rajan, K.K.; Nashine, B.K.; Chandramouli, S.; Madhusoodanan, K.; Kalyanasundaram, P.

    2011-01-01

    Highlights: → Performance evaluation of wire type leak detectors was conducted in LEENA facility by creating sodium leaks. → The lowest leak rate of 214 g/h was detected in 50 min and the highest detection time was 6 h for a leak rate of 222 g/h. → Factors affecting the leak detection time are packing density of thermal insulation, layout of heater, temperature, etc. → Relationship between leak rate and detection time was established and a leak rate of 100 g/h is likely to be detected in 11.1 h. → Contact resistance of leaked sodium increased to 3.5 kilo ohms in 20 h. - Abstract: Wire type leak detectors working on conductivity principle are used for detecting sodium leak in the secondary sodium circuits of fast breeder reactors. It is required to assess the performance of these detectors and confirm that they are meeting the requirements. A test facility by name LEENA was constructed at Indira Gandhi Centre for Atomic Research (IGCAR), Kalpakkam to test the wire type leak detector lay out by simulating different sodium leak rates. This test facility consists of a sodium dump tank, a test vessel, interconnecting pipelines with valves, micro filter and test section with leak simulators. There are three different test sections in the test set up of length 1000 mm each. These test sections simulate piping of Prototype Fast Breeder Reactor (PFBR) secondary circuit and the wire type leak detector layout in full scale. All test sections are provided with leak simulators. A leak simulator consists of a hole of size one mm drilled in the test section and closed with a tapered pin. The tapered pin position in the hole is adjusted by a screw mechanism and there by the annular gap of flow area is varied for getting different leak rates. Various experiments were conducted to evaluate the performance of the leak detectors by creating different sodium leak rates. This paper deals with the details of wire type leak detector layout for the secondary sodium circuit of

  15. 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.

  16. 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.

  17. 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.

  18. 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

  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. Dynamic pressure measures for long pipeline leak detection

    Energy Technology Data Exchange (ETDEWEB)

    Likun Wang; Hongchao Wang; Min Xiong; Bin Xu; Dongjie Tan; Hengzhang Zhou [PetroChina Pipeline Company, Langfang (China). R and D Center

    2009-07-01

    Pipeline leak detection method based on dynamic pressure is studied. The feature of dynamic pressure which is generated by the leakage of pipeline is analyzed. The dynamic pressure method is compared with the static pressure method for the advantages and disadvantages in pipeline leak detection. The dynamic pressure signal is suitable for pipeline leak detection for quick-change of pipeline internal pressure. Field tests show that the dynamic pressure method detects pipeline leak rapidly and precisely. (author)

  1. 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.

  2. Characterization of a Ca2+ response to both UTP and ATP at human P2Y11 receptors: evidence for agonist-specific signaling.

    Science.gov (United States)

    White, Pamela J; Webb, Tania E; Boarder, Michael R

    2003-06-01

    Previous reports on heterologously-expressed human P2Y11 receptors have indicated that ATP, but not UTP, is an agonist stimulating both phosphoinositidase C and adenylyl cyclase. Consistent with these findings, we report that in 1321N1 cells expressing human P2Y11 receptors, UTP stimulation did not lead to accumulation of inositol(poly)phosphates under conditions in which ATP gave a robust, concentration-dependent effect. Unexpectedly, however, both UTP and ATP stimulated increases in cytosolic Ca2+ concentration ([Ca2+]c), with both nucleotides achieving similar EC50 and maximal responses. The responses to maximally effective concentrations of ATP and UTP were not additive. The [Ca2+]c increase in response to UTP was less dependent on extracellular Ca2+ than was the response to ATP. AR-C67085 (2-propylthio-beta,gamma-difluoromethylene-d-ATP, a P2Y11-selective agonist), adenosine 5'-O-(3-thiotriphosphate), and benzoyl ATP were all full agonists with potencies similar to those of ATP and UTP. In desensitization experiments, exposure to ATP resulted in loss of the UTP response; this response was more sensitive to desensitization than that of ATP. Pertussis toxin pretreatment attenuated the response to UTP but left the ATP response unaffected. The presence of 2-aminoethyl diphenylborate differentially affected the responses of ATP and UTP. No mRNA transcripts for P2Y2 or P2Y4 were detectable in the P2Y11-expressing cells. We conclude that UTP is a Ca2+-mobilizing agonist at P2Y11 receptors and that ATP and UTP acting at the same receptor recruit distinct signaling pathways. This example of agonist-specific signaling is discussed in terms of agonist trafficking and differential signal strength.

  3. A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

    Science.gov (United States)

    Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

    2016-07-01

    In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. 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.

  5. 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)

  6. Damage phenomena at target surface by small leak

    International Nuclear Information System (INIS)

    Jeong, Kyung Chai; Jeong, J. Y.; Kim, B. H.; Kim, T. J.; Choi, J. H.

    2001-04-01

    Design of the steam generator should be considered the safety about the sodium-water reaction occurred by water leak in heat transfer tube. Water leak mainly occurred from welding defect at the process of tube connection, the vibration of heat transfer tube bundle in steam generating system, fretting, and pin hole in original tube manufacturing. The classification of water leak divided to two parts, roughly, in case of the water leak studies. One is small leak phenomena analysis, and the other is it of large leak which was mainly treated to the evaluation on pressure increasing from hydrogen gas formed by sodium-water reaction in sodium system. In small water leak, the leak propagation phenomena and the development of leak detecting system at initial stage of small water leak were studied, mainly. In this study, the corrosion phenomena on the target tube surface appeared by sodium-water reaction was analyzed through the small water leak experiments, and, also, the jet phenomena formed by N 2 gas injection through the leak nozzle under water medium was observed

  7. Small leak detection requirements for SNR-300 steam generator operation

    International Nuclear Information System (INIS)

    Dumm, K.

    1975-01-01

    The leak detection philosophy of the SNR-300 steam generators is described. Due to operational demands small leaks have already to be detectable at a stage where secondary tube damage has not yet occurred. The requirements on a leak detection instrument are developed and the instrument itself is described. (author)

  8. 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

  9. 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

  10. Special storage of leaking fuel at Paks NPP

    International Nuclear Information System (INIS)

    Biro, Janos; Szőke, L.; Burján, T.; Lukács, R.; Hózer, H.

    2015-01-01

    In this paper the activities related with spent, hermetic as well as leaking fuel handling and storage, including: Spent fuel pool; Transportation criteria for the spent fuel assemblies and Interim spent fuel dry storage; Short-term storage in the spent fuel pool; Identification of the leaking assemblies by the TS-device; Present conception of Identification, handling of the leaking FAs; Modified transport procedure for the leaking FAs; Calculation of solved activity inside the leaking fuel rod; Solved activity limit values for the leaking FAs; Long-term storage in the interim spent Fuel dry storage are presented. At the end authors’ concluded that: 1) The leaking FA can be transported to the interim dry storage together with the other spent fuel assemblies in the transport container. 2) The transport-documentation of the leaking FA has to contain: isotope inventory, calculated solved activity values of the failed FA and the quantity of failed fuel rods. 3) Performing three leakage tests of the identified leaking FA before the transportation in the 5FP. it is useful to decrease the solved activity concentration inside the leaking FA and give additional information about the extent of the leakage. 4) We can calculate simply the solved activity of the leaking FA. 5) The modified transport procedure will have to be authorized. 6) The radiological effects of the leaking FA are negligible relative to the natural background radiation

  11. Final evaluation of advanced and current leak detection systems

    International Nuclear Information System (INIS)

    Kupperman, D.S.; Carlson, R.; Brewer, W.; Lanham, R.

    1989-01-01

    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

  12. 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

  13. 40 CFR 63.648 - Equipment leak standards.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 10 2010-07-01 2010-07-01 false Equipment leak standards. 63.648...) National Emission Standards for Hazardous Air Pollutants From Petroleum Refineries § 63.648 Equipment leak...) through (c)(10) and (e) through (i) of this section. (1) The instrument readings that define a leak for...

  14. 40 CFR 63.769 - Equipment leak standards.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 10 2010-07-01 2010-07-01 false Equipment leak standards. 63.769... § 63.769 Equipment leak standards. (a) This section applies to equipment subject to this subpart and... release to detect leaks, except under the following conditions. (i) The owner or operator has obtained...

  15. 40 CFR 63.1331 - Equipment leak provisions.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 11 2010-07-01 2010-07-01 true Equipment leak provisions. 63.1331... Standards for Hazardous Air Pollutant Emissions: Group IV Polymers and Resins § 63.1331 Equipment leak... in pumps and agitator seals in light liquid service shall not be considered to be a leak. For...

  16. 10 CFR 39.35 - Leak testing of sealed sources.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Leak testing of sealed sources. 39.35 Section 39.35 Energy....35 Leak testing of sealed sources. (a) Testing and recordkeeping requirements. Each licensee who uses... record of leak test results in units of microcuries and retain the record for inspection by the...

  17. 40 CFR 63.1434 - Equipment leak provisions.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 11 2010-07-01 2010-07-01 true Equipment leak provisions. 63.1434... Standards for Hazardous Air Pollutant Emissions for Polyether Polyols Production § 63.1434 Equipment leak provisions. (a) The owner or operator of each affected source shall comply with the HON equipment leak...

  18. 40 CFR 63.1410 - Equipment leak provisions.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 11 2010-07-01 2010-07-01 true Equipment leak provisions. 63.1410... leak provisions. The owner or operator of each affected source shall comply with the requirements of 40 CFR part 63, subpart UU (national emission standards for equipment leaks (control level 2)) for all...

  19. 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...

  20. 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...

  1. Small leak detection requirements for SNR-300 steam generator operation

    Energy Technology Data Exchange (ETDEWEB)

    Dumm, K

    1975-07-01

    The leak detection philosophy of the SNR-300 steam generators is described. Due to operational demands small leaks have already to be detectable at a stage where secondary tube damage has not yet occurred. The requirements on a leak detection instrument are developed and the instrument itself is described. (author)

  2. Carbon and energy metabolism of atp mutants of Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole

    1992-01-01

    strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming......The membrane-bound H+-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth...... rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion...

  3. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

    Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

  4. 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.

  5. 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.

  6. Leak Detection Modeling and Simulation for Oil Pipeline with Artificial Intelligence Method

    OpenAIRE

    Sukarno, Pudjo; Sidarto, Kuntjoro Adji; Trisnobudi, Amoranto; Setyoadi, Delint Ira; Rohani, Nancy; Darmadi, Darmadi

    2007-01-01

    Leak detection is always interesting research topic, where leak location and leak rate are two pipeline leaking parameters that should be determined accurately to overcome pipe leaking problems. In this research those two parameters are investigated by developing transmission pipeline model and the leak detection model which is developed using Artificial Neural Network. The mathematical approach needs actual leak data to train the leak detection model, however such data could not be obtained ...

  7. Intraoperative air leak measured after lobectomy is associated with postoperative duration of air leak.

    Science.gov (United States)

    Brunelli, Alessandro; Salati, Michele; Pompili, Cecilia; Gentili, Paolo; Sabbatini, Armando

    2017-11-01

    To verify the association between the air leak objectively measured intraoperatively (IAL) using the ventilator and the air leak duration after pulmonary lobectomy. Prospective analysis on 111 patients submitted to pulmonary lobectomy (33 by video-assisted thoracic surgery). After resection, objective assessment of air leak (in milliliter per minute) was performed before closure of the chest by measuring the difference between a fixed inspired and expired volume, using a tidal volume of 8 ml/kg, a respiratory rate of 10 and a positive-end expiratory pressure of 5 cmH2O. A multivariable analysis was performed for identifying factors associated with duration of postoperative air leak. Average IAL was 158 ml/min (range 0-1500 ml/min). The best cut-off (receiver-operating characteristics analysis) associated with air leak longer than 5 days was 500 ml/min. Nine patients had IAL >500 ml/min (8%). They had a longer duration of postoperative air leak compared with those with a lower IAL (mean values, 10.1 days, SD 8.8 vs 1.5 days, SD 4.9 P leak duration after multivariable regression: left side resection (P = 0.018), upper site resection (P = 0.031) and IAL >500 ml/min (P leak duration was generated: 1.7 + 2.4 × left side + 2.2 × upper site + 8.8 × IAL >500. The air leak measurement using the ventilator parameters after lung resection may assist in estimating the risk of postoperative prolonged air leak. An IAL > 500 ml/min may warrant the use of intraoperative preventative measures, particularly after video-assisted thoracic surgery lobectomy where a submersion test is often unreliable. © 2017 European Society of Cardiology and European Atherosclerosis Association. All rights reserved. For permissions please email: journals.permissions@oup.com.

  8. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

    Science.gov (United States)

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

    2014-04-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

  9. 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

  10. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are

  11. He leaks in the CERN LHC beam vacuum chambers operating at cryogenic temperatures

    CERN Document Server

    Baglin, V

    2007-01-01

    The 27 km long large hadron collider (LHC), currently under construction at CERN, will collide protons beam at 14 TeV in the centre of mass. In the 8 arcs, the superconducting dipoles and quadrupoles of the FODO cells operate with superfluid He at 1.9 K. In the 8 long straight sections, the cold bores of the superconducting magnets are held at 1.9 or 4.5 K. Thus, in the LHC, 75% of the beam tube vacuum chamber is cooled with He. In many areas of the machine, He leaks could appear in the beam tube. At cryogenic temperature, the gas condenses onto the cold bores or beam screens, and interacts with the circulating beam. He leaks creates a He front propagating along the vacuum chambers, which might cause magnet quench. We discuss the consequences of He leaks, the possible means of detections, the strategies to localise them and the methods to measure their size.

  12. Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump

    International Nuclear Information System (INIS)

    Rhodes, C.J.; Lucas, C.A.; Mutkoski, R.L.; Orci, L.; Halban, P.A.

    1987-01-01

    Isolated rat pancreatic islets were pulse-labeled for 5 min with [ 3 H]leucine then chased for 25 min, during which time endogenously labeled [ 3 H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [ 3 H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer, proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect

  13. Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

    International Nuclear Information System (INIS)

    Mithieux, G.; Rousset, B.

    1989-01-01

    We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells

  14. ATP-dependent human RISC assembly pathways.

    Science.gov (United States)

    Yoda, Mayuko; Kawamata, Tomoko; Paroo, Zain; Ye, Xuecheng; Iwasaki, Shintaro; Liu, Qinghua; Tomari, Yukihide

    2010-01-01

    The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

  15. 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)

  16. 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.

  17. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

    Science.gov (United States)

    Brand, M D; Lehninger, A L

    1977-01-01

    The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

  18. Evolutionary, kinetic and thermodynamic aspects on the bioenergetics of inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP)

    International Nuclear Information System (INIS)

    Baltscheffsky, H.; Baltscheffsky, M.

    1995-01-01

    Energy barriers for energy carriers are of fundamental significance for the successful operation of the bioenergetic reactions in living cells. PPi and ATP are outstanding ''energy-rich'' examples of molecular ''energy currencies'' in biological systems, with kinetic barriers preventing excessively fast thermodynamically feasible hydrolysis from occurring. The barriers may be considered to facilitate the energy coupling roles of these phosphate compounds, which are to secure growth and maintain numerous other energy requiring functions. The enzymes involved in overcoming the energies of activation of the bioenergetic reactions have evolved to be very well tuned for their roles. Three aspects will be discussed in some detail. The first is the fact that ATP at neutral pH is considerably more energy-rich than PPi, which thus has been called a ''poor man's ATP''. This is exemplified by the kinetic and thermodynamic differences observed between the requirements for the photosynthetic formation of PPi and ATP in certain photobacterial chromatophores by varying levels of energy supply. At lower pH, PPi and ATP are equally energy-rich, which may be of significance for acidophiles. The second concerns the possible evolutionary significance of the finding that, in the dark, a pH gradient suffices to drive extensive PPi synthesis, whereas ATP synthesis requires both a pH gradient and a membrane potential (Strid et al, Biochim. Biophys. Acta 892 (1987) 236-244). Thirdly, PPi as the most plausible predecessor to ATP in the origin and early evolution of life, will be discussed. (author). Abstract only

  19. Experiences in integrated leak rate measurements

    International Nuclear Information System (INIS)

    Shirk, R.E.

    1982-01-01

    During a hypothetical design basis accident for nuclear power plants, the reactor containment system is relied upon to maintain radioactive exposure limits below acceptable limits. Integrated leak rate testing is a means of verifying that the leakage of radioactivity material from the reactor containment will be below allowable limits. Leakage rate computations are based on the ideal gas law. The absolute method of leakage rate testing utilizing mass point method of data analysis is recommended. Integrated leak rate testing data is obtained from pressure, drybulb temperature, dewpoint temperature, and flow measuring systems. Test data does not support the usual leakage (flow) - pressure square root relationship. The major source of potential leakage from the reactor containment is reactor containment isolation valves

  20. Monju secondary heat transport system sodium leak

    International Nuclear Information System (INIS)

    Suzuki, Takeo; Hiroi, Hiroshi; Usami, Shin; Iwata, Koji.

    1996-01-01

    On December 8, 1995, the sodium leakage from the secondary heat transport system (SHTS) occurred in the piping room of the reactor auxiliary building in Monju. The secondary sodium leaked through a temperature sensor, due to the breakaway of the tip of the well tube of the sensor installed near the outlet of the intermediate heat exchanger (IHX) in the C loop of SHTS. The reactor core remained cooled and thus, from the viewpoint of radiological hazards, the safety of the reactor was secured. There were no adverse effects for operating personnel or the surrounding environment. The cause of the well tube failure is considered to result from high cycle fatigue due to flow induced vibrations. Delay in draining the sodium from the leaking loop increased the consequential effects from sodium combustion products. (author)