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

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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.

Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

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Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki

2012-01-01

We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses. PMID

Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate.

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Nilsson, H; Lindgren, D; Mandahl Forsberg, A; Mulder, H; Axelson, H; Johansson, M E

2015-01-08

Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC.

Lactate: link between glycolytic and oxidative metabolism.

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Brooks, George A

2007-01-01

Once thought to be the consequence of oxygen lack in contracting skeletal muscle, the glycolytic product lactate is formed and utilised continuously under fully aerobic conditions. 'Cell-cell' and 'intracellular lactate shuttle' concepts describe the roles of lactate in delivery of oxidative and gluconeogenic substrates as well as in cell signalling. Examples of cell-cell shuttles include lactate exchanges (i) between white-glycolytic and red-oxidative fibres within a working muscle bed; (ii) between working skeletal muscle and heart; and (iii) between tissues of net lactate release and gluconeogenesis. Lactate shuttles exist in diverse tissues including in the brain, where a shuttle between astrocytes and neurons is linked to glutamatergic signalling. Because lactate, the product of glycogenolysis and glycolysis, is disposed of by oxidative metabolism, lactate shuttling unites the two major processes of cellular energy transduction. Lactate disposal is mainly through oxidation, especially during exercise when oxidation accounts for 70-75% of removal and gluconeogenesis the remainder. Lactate flux occurs down proton and concentration gradients that are established by the mitochondrial lactate oxidation complex. Marathon running is a power activity requiring high glycolytic and oxidative fluxes; such activities require lactate shuttling. Knowledge of the lactate shuttle is yet to be imparted to the sport.

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

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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.

Estimation of PMI depends on the changes in ATP and its degradation products.

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Mao, Shiwei; Fu, Gaowen; Seese, Ronald R; Wang, Zhen-Yuan

2013-09-01

Estimating the time since death, or postmortem interval (PMI), has been one of the biggest difficulties in modern forensic investigation. This study tests if the concentrations of breakdown products of adenosine triphosphate (ATP) correlate with PMI in multiple organs from rat. Brains, spleens, and kidneys of rats were harvested at different time points in carcasses maintained at 4°C or 20°C. High Performance Liquid Chromatography (HPLC) was used to quantify concentrations of metabolites related to ATP degradation. A K value (Kv=100×(Hx+HxR)/(ATP+ADP+AMP+IMP+HxR+Hx)) was calculated and correlated with PMI for each organ and temperature. The results indicate that the K value is a robust index for the estimation of PMI based on highly significant linear correlations between PMI and concentrations of ATP breakdown products. Compared with other current research methods, the changing tendency of ATP and its degradation products may be potentially a better way for the estimation of PMI in medico-legal practice. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

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Liu, Jianming; Kandasamy, Vijayalakshmi; Würtz, Anders

2016-01-01

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F1-ATPase into a Lact...

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

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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.

Sodium bicarbonate ingestion increases glycolytic contribution and improves performance during simulated taekwondo combat.

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Lopes-Silva, João Paulo; Da Silva Santos, Jonatas Ferreira; Artioli, Guilherme Giannini; Loturco, Irineu; Abbiss, Chris; Franchini, Emerson

2018-04-01

To investigate the effect of sodium bicarbonate (NaHCO 3 ) on performance and estimated energy system contribution during simulated taekwondo combat. Nine taekwondo athletes completed two experimental sessions separated by at least 48 h. Athletes consumed 300 mg/kg body mass of NaHCO 3 or placebo (CaCO 3 ) 90 min before the combat simulation (three rounds of 2 min separated by 1 min passive recovery), in a double-blind, randomized, repeated-measures crossover design. All simulated combat was filmed to quantify the time spent fighting in each round. Lactate concentration [La - ] and rating of perceived exertion (RPE) were measured before and after each round, whereas heart rate (HR) and the estimated contribution of the oxidative (W OXI ), ATP (adenosine triphosphate)-phosphocreatine (PCr) (W PCR ), and glycolytic (W [ La - ] ) systems were calculated during the combat simulation. [La - ] increased significantly after NaHCO 3 ingestion, when compared with the placebo condition (+14%, P = 0.04, d = 3.70). NaHCO 3 ingestion resulted in greater estimated glycolytic energy contribution in the first round when compared with the placebo condition (+31%, P = 0.01, d = 3.48). Total attack time was significantly greater after NaHCO 3 when compared with placebo (+13%, P = 0.05, d = 1.15). W OXI , W PCR , VO 2 , HR and RPE were not different between conditions (P > 0.05). NaHCO 3 ingestion was able to increase the contribution of glycolytic metabolism and, therefore, improve performance during simulated taekwondo combat.

Hyperthyroidism results in increased glycolytic capacity in the rat heart. A 31P-NMR study.

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Seymour, A M; Eldar, H; Radda, G K

1990-11-12

We have investigated the metabolic adaptations that occur in the thyroxine-treated rat heart. Rats were made hyperthyroid by daily intra-peritoneal injections of thyroxine (35 micrograms/100 g body weight) over seven days. 31P-NMR investigations of isolated glucose-perfused isometric hearts showed that thyroxine treatment caused an increase in Pi (from 4.9 mumols.(g dry wt.)-1 in control hearts to 11.7 mumols.(g dry wt.)-1 in hyperthyroid hearts), a decrease in phosphocreatine (from 36.5 mumols.(g dry wt.)-1 to 21.8 mumols.(g dry wt.)-1) with no change in ATP or ADP concentrations under the same conditions of cardiac work. The unidirectional exchange flux Pi----ATP was measured by saturation transfer NMR in hyperthyroid rat hearts. This exchange (which has been shown to contain a significant glycolytic component) increased by 2.2-fold in thyroxine-treated hearts in comparison to control hearts (to 3.6 mumols.(g dry wt.)-1.s-1, from 1.6 mumols.(g dry wt.)-1.s-1). In parallel experiments, NMR analysis of extracts from hyperthyroid rat hearts showed significantly elevated levels of glucose 6-phosphate, and fructose 6-phosphate. Measurements of enzyme activities isolated from hyperthyroid and control tissue showed a 40% increase in phosphofructokinase activity. These data together with the increased concentration of Pi show that both glycolytic and glycogenolytic fluxes are increased in the hyperthyroid rat heart. This metabolic adaptation may be necessary to cope with the increased number and activity of Na+/K(+)-ATPase pumps that occur in response to thyroxine treatment.

Hyperoxia decreases glycolytic capacity, glycolytic reserve and oxidative phosphorylation in MLE-12 cells and inhibits complex I and II function, but not complex IV in isolated mouse lung mitochondria.

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Kumuda C Das

Full Text Available High levels of oxygen (hyperoxia are frequently used in critical care units and in conditions of respiratory insufficiencies in adults, as well as in infants. However, hyperoxia has been implicated in a number of pulmonary disorders including bronchopulmonary dysplasia (BPD and adult respiratory distress syndrome (ARDS. Hyperoxia increases the generation of reactive oxygen species (ROS in the mitochondria that could impair the function of the mitochondrial electron transport chain. We analyzed lung mitochondrial function in hyperoxia using the XF24 analyzer (extracellular flux and optimized the assay for lung epithelial cells and mitochondria isolated from lungs of mice. Our data show that hyperoxia decreases basal oxygen consumption rate (OCR, spare respiratory capacity, maximal respiration and ATP turnover in MLE-12 cells. There was significant decrease in glycolytic capacity and glycolytic reserve in MLE-12 cells exposed to hyperoxia. Using mitochondria isolated from lungs of mice exposed to hyperoxia or normoxia we have shown that hyperoxia decreased the basal, state 3 and state3 μ (respiration in an uncoupled state respirations. Further, using substrate or inhibitor of a specific complex we show that the OCR via complex I and II, but not complex IV was decreased, demonstrating that complexes I and II are specific targets of hyperoxia. Further, the activities of complex I (NADH dehydrogenase, NADH-DH and complex II (succinate dehydrogenase, SDH were decreased in hyperoxia, but the activity of complex IV (cytochrome oxidase, COX remains unchanged. Taken together, our study show that hyperoxia impairs glycolytic and mitochondrial energy metabolism in in tact cells, as well as in lungs of mice by selectively inactivating components of electron transport system.

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

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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.

Biomimicry enhances sequential reactions of tethered glycolytic enzymes, TPI and GAPDHS.

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Chinatsu Mukai

Full Text Available Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase. We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.

Biomimicry enhances sequential reactions of tethered glycolytic enzymes, TPI and GAPDHS.

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Mukai, Chinatsu; Gao, Lizeng; Bergkvist, Magnus; Nelson, Jacquelyn L; Hinchman, Meleana M; Travis, Alexander J

2013-01-01

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

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Sierra, Ana, E-mail: ana-sierra@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Subbotina, Ekaterina, E-mail: ekaterina-subbotina@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Zhu, Zhiyong, E-mail: zhiyong-zhu@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Gao, Zhan, E-mail: zhan-gao@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Koganti, Siva Rama Krishna, E-mail: sivaramakrishna.koganti@ttuhc.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Coetzee, William A., E-mail: william.coetzee@nyumc.org [Department of Pediatrics, NYU School of Medicine, New York, NY 10016 (United States); Goldhamer, David J., E-mail: david.goldhamer@uconn.edu [Center for Regenerative Biology, Department of Molecular and Cell Biology, Advanced Technology Laboratory, University of Connecticut, 1392 Storrs Road Unit 4243, Storrs, Connecticut 06269 (United States); Hodgson-Zingman, Denice M., E-mail: denice-zingman@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine, Iowa City, IA 52242 (United States); Zingman, Leonid V., E-mail: leonid-zingman@uiowa.edu [Department of Internal Medicine, University of Iowa, Carver College of Medicine, Iowa City, IA 52242 (United States); Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Veterans Affairs, Medical Center, Iowa City, IA 52242 (United States)

2016-02-26

Sarcolemmal ATP-sensitive potassium (K{sub ATP}) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle K{sub ATP} channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle K{sub ATP} channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of K{sub ATP} channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish K{sub ATP} channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities. - Highlights: • ATP-sensitive K{sup +} channels regulate musclin production by skeletal muscles. • Lipolytic ANP signaling is promoted by augmented skeletal muscle musclin production. • Skeletal muscle musclin transcription is promoted by a CaMKII/HDAC/FOXO1 pathway. • Musclin links adipose mobilization to energy use in K{sub ATP} channel deficient skeletal muscle.

Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

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Bosche, Bert, E-mail: bert.bosche@uk-essen.de [Department of Neurology, University of Duisburg-Essen (Germany); Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Schäfer, Matthias, E-mail: matthias.schaefer@sanofi.com [Institute of Physiology, Justus-Liebig-University Giessen (Germany); Graf, Rudolf, E-mail: rudolf.graf@nf.mpg.de [Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Härtel, Frauke V., E-mail: frauke.haertel@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany); Schäfer, Ute, E-mail: ute.schaefer@medunigraz.at [Research Unit for Experimental Neurotraumatology, Medical University of Graz (Austria); Noll, Thomas, E-mail: thomas.noll@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany)

2013-05-03

Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

ATP induces NO production in hippocampal neurons by P2X(7 receptor activation independent of glutamate signaling.

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Juan Francisco Codocedo

Full Text Available To assess the putative role of adenosine triphosphate (ATP upon nitric oxide (NO production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3'-O-(4-Benzoylbenzoyl ATP (Bz-ATP elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG or by N(ω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV, but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

Reexamination of the Physiological Role of PykA in Escherichia coli Revealed that It Negatively Regulates the Intracellular ATP Levels under Anaerobic Conditions.

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Zhao, Chunhua; Lin, Zhao; Dong, Hongjun; Zhang, Yanping; Li, Yin

2017-06-01

Pyruvate kinase is one of the three rate-limiting glycolytic enzymes that catalyze the last step of glycolysis, conversion of phosphoenolpyruvate (PEP) into pyruvate, which is associated with ATP generation. Two isozymes of pyruvate kinase, PykF and PykA, are identified in Escherichia coli PykF is considered important, whereas PykA has a less-defined role. Prior studies inactivated the pykA gene to increase the level of its substrate, PEP, and thereby increased the yield of end products derived from PEP. We were surprised when we found a pykA ::Tn 5 mutant in a screen for increased yield of an end product derived from pyruvate ( n -butanol), suggesting that the role of PykA needs to be reexamined. We show that the pykA mutant exhibited elevated intracellular ATP levels, biomass concentrations, glucose consumption, and n -butanol production. We also discovered that the pykA mutant expresses higher levels of a presumed pyruvate transporter, YhjX, permitting the mutant to recapture and metabolize excreted pyruvate. Furthermore, we demonstrated that the nucleotide diphosphate kinase activity of PykA leads to negative regulation of the intracellular ATP levels. Taking the data together, we propose that inactivation of pykA can be considered a general strategy to enhance the production of pyruvate-derived metabolites under anaerobic conditions. IMPORTANCE This study showed that knocking out pykA significantly increased the intracellular ATP level and thus significantly increased the levels of glucose consumption, biomass formation, and pyruvate-derived product formation under anaerobic conditions. pykA was considered to be encoding a dispensable pyruvate kinase; here we show that pykA negatively regulates the anaerobic glycolysis rate through regulating the energy distribution. Thus, knocking out pykA can be used as a general strategy to increase the level of pyruvate-derived fermentative products. Copyright © 2017 American Society for Microbiology.

How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

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Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

2013-07-01

A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

International Nuclear Information System (INIS)

Baba, Miyako; Inoue, Masahiro; Itoh, Kazuyuki; Nishizawa, Yasuko

2008-01-01

CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells

Assessing glycolytic flux alterations resulting from genetic perturbations in E. coli using a biosensor

DEFF Research Database (Denmark)

Lehning, Christina Eva; Siedler, Solvej; Ellabaan, Mostafa M Hashim

2017-01-01

validated the glycolytic flux dependency of the biosensor in a range of different carbon sources in six different E. coli strains and during mevalonate production. Furthermore, we studied the flux-altering effects of genome-wide single gene knock-outs in E. coli in a multiplex FlowSeq experiment. From...... a library consisting of 2126 knock-out mutants, we identified 3 mutants with high-flux and 95 mutants with low-flux phenotypes that did not have severe growth defects. This approach can improve our understanding of glycolytic flux regulation improving metabolic models and engineering efforts....

Programmed mitophagy is essential for the glycolytic switch during cell differentiation.

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Esteban-Martínez, Lorena; Sierra-Filardi, Elena; McGreal, Rebecca S; Salazar-Roa, María; Mariño, Guillermo; Seco, Esther; Durand, Sylvère; Enot, David; Graña, Osvaldo; Malumbres, Marcos; Cvekl, Ales; Cuervo, Ana María; Kroemer, Guido; Boya, Patricia

2017-06-14

Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts. © 2017 The Authors.

Ketogenic diet metabolites reduce firing in central neurons by opening K(ATP) channels.

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Ma, Weiyuan; Berg, Jim; Yellen, Gary

2007-04-04

A low-carbohydrate ketogenic diet remains one of the most effective (but mysterious) treatments for severe pharmacoresistant epilepsy. We have tested for an acute effect of physiological ketone bodies on neuronal firing rates and excitability, to discover possible therapeutic mechanisms of the ketogenic diet. Physiological concentrations of ketone bodies (beta-hydroxybutyrate or acetoacetate) reduced the spontaneous firing rate of neurons in slices from rat or mouse substantia nigra pars reticulata. This region is thought to act as a "seizure gate," controlling seizure generalization. Consistent with an anticonvulsant role, the ketone body effect is larger for cells that fire more rapidly. The effect of ketone bodies was abolished by eliminating the metabolically sensitive K(ATP) channels pharmacologically or by gene knock-out. We propose that ketone bodies or glycolytic restriction treat epilepsy by augmenting a natural activity-limiting function served by K(ATP) channels in neurons.

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

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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.

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

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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.

Glycolytic Enzymes Coalesce in G Bodies under Hypoxic Stress

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Meiyan Jin

2017-07-01

Full Text Available Glycolysis is upregulated under conditions such as hypoxia and high energy demand to promote cell proliferation, although the mechanism remains poorly understood. We find that hypoxia in Saccharomyces cerevisiae induces concentration of glycolytic enzymes, including the Pfk2p subunit of the rate-limiting phosphofructokinase, into a single, non-membrane-bound granule termed the “glycolytic body” or “G body.” A yeast kinome screen identifies the yeast ortholog of AMP-activated protein kinase, Snf1p, as necessary for G-body formation. Many G-body components identified by proteomics are required for G-body integrity. Cells incapable of forming G bodies in hypoxia display abnormal cell division and produce inviable daughter cells. Conversely, cells with G bodies show increased glucose consumption and decreased levels of glycolytic intermediates. Importantly, G bodies form in human hepatocarcinoma cells in hypoxia. Together, our results suggest that G body formation is a conserved, adaptive response to increase glycolytic output during hypoxia or tumorigenesis.

Glycolytic overload and the genesis of breast cancer.

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Robson, J R

1984-03-01

It is suggested that the development of breast cancer is due to overloading of the glycolytic pathways. An excess of substrates or an excessive delivery rate of substrates to the Krebs Cycle is believed to result in the formation of acetyl CoA. Feedback mechanisms controlling the conversion of acetyl CoA to cholesterol may be overcome; the resulting high concentration of cholesterol induces the formation of pregnenolone which may then be converted into androgens, estrogens and progesterone. These steroids are in addition to those produced by gonads and adrenal glands. Glycolytic overload is also associated with an increase in fat stores which have been shown to be the site of interconversion of sex hormones. Excess sex hormones or abnormal sex hormones are believed to be the cause of breast cancer. The hypothesis presented links glycolytic overload with clinical biochemical phenomena and explains some of the anomalies observed in breast cancer experience in different ethnic groups. Changes in dietary habits during the history of man resulting in " gorging " and the consumption of highly refined sugars are possible causes of glycolytic overload. So, also, is impaired thermogenesis due to Brown Fat deficits in certain ethnic groups.

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

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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

Role of Glycolytic Intermediates in Global Regulation and Signal Transduction. Final Report

Energy Technology Data Exchange (ETDEWEB)

Liao, J.C.

2000-05-08

The goal of this project is to determine the role of glycolytic intermediates in regulation of cell physiology. It is known that many glycolytic intermediates are involved in regulation of enzyme activities at the kinetic level. However, little is known regarding the role of these metabolites in global regulation and signal transduction. This project aims to investigate the role of glycolytic intermediates in the regulation of gene expression.

Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae.

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Elbing, Karin; Ståhlberg, Anders; Hohmann, Stefan; Gustafsson, Lena

2004-12-01

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.

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

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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.

Shikonin, vitamin K3 and vitamin K5 inhibit multiple glycolytic enzymes in MCF-7 cells.

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Chen, Jing; Hu, Xun; Cui, Jingjie

2018-05-01

Glycolysis is the most important source of energy for the production of anabolic building blocks in cancer cells. Therefore, glycolytic enzymes are regarded as potential targets for cancer treatment. Previously, naphthaquinones, including shikonin, vitamin K 3 and vitamin K 5 , have been proven to decrease the rate of glycolysis in cancer cells, which is partly due to suppressed pyruvate kinase activity. In the present study, enzymatic assays were performed using MCF-7 cell lysate in order to screen the profile of glycolytic enzymes in cancer cells inhibited by shikonin, vitamin K 3 and vitamin K 5 , in addition to pyruvate kinase. Results revealed that hexokinase, phosphofructokinase-1, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase produced in the process of glycolysis were inhibited by shikonin, vitamin K 3 and vitamin K 5 . The results indicated that shikonin, vitamin K 3 and vitamin K 5 are chemical inhibitors of glycolytic enzymes in cancer cells and have potential uses in translational medical applications.

A glycolytic metabolon in Saccharomyces cerevisiae is stabilized by F-actin.

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Araiza-Olivera, Daniela; Chiquete-Felix, Natalia; Rosas-Lemus, Mónica; Sampedro, José G; Peña, Antonio; Mujica, Adela; Uribe-Carvajal, Salvador

2013-08-01

In the Saccharomyces cerevisiae glycolytic pathway, 11 enzymes catalyze the stepwise conversion of glucose to two molecules of ethanol plus two CO₂ molecules. In the highly crowded cytoplasm, this pathway would be very inefficient if it were dependent on substrate/enzyme diffusion. Therefore, the existence of a multi-enzymatic glycolytic complex has been suggested. This complex probably uses the cytoskeleton to stabilize the interaction of the various enzymes. Here, the role of filamentous actin (F-actin) in stabilization of a putative glycolytic metabolon is reported. Experiments were performed in isolated enzyme/actin mixtures, cytoplasmic extracts and permeabilized yeast cells. Polymerization of actin was promoted using phalloidin or inhibited using cytochalasin D or latrunculin. The polymeric filamentous F-actin, but not the monomeric globular G-actin, stabilized both the interaction of isolated glycolytic pathway enzyme mixtures and the whole fermentation pathway, leading to higher fermentation activity. The associated complexes were resistant against inhibition as a result of viscosity (promoted by the disaccharide trehalose) or inactivation (using specific enzyme antibodies). In S. cerevisiae, a glycolytic metabolon appear to assemble in association with F-actin. In this complex, fermentation activity is enhanced and enzymes are partially protected against inhibition by trehalose or by antibodies. © 2013 FEBS.

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

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Yi-Hsuan Wu

Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

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Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

Nonlinear correlations in the hydrophobicity and average flexibility along the glycolytic enzymes sequences

Energy Technology Data Exchange (ETDEWEB)

Ciorsac, Alecu, E-mail: aleciorsac@yahoo.co [Politehnica University of Timisoara, Department of Physical Education and Sport, 2 P-ta Victoriei, 300006, Timisoara (Romania); Craciun, Dana, E-mail: craciundana@gmail.co [Teacher Training Department, West University of Timisoara, 4 Boulevard V. Pirvan, Timisoara, 300223 (Romania); Ostafe, Vasile, E-mail: vostafe@cbg.uvt.r [Department of Chemistry, West University of Timisoara, 16 Pestallozi, 300115, Timisoara (Romania); Laboratory of Advanced Researches in Environmental Protection, Nicholas Georgescu-Roegen Interdisciplinary Research and Formation Platform, 4 Oituz, Timisoara, 300086 (Romania); Isvoran, Adriana, E-mail: aisvoran@cbg.uvt.r [Department of Chemistry, West University of Timisoara, 16 Pestallozi, 300115, Timisoara (Romania); Laboratory of Advanced Researches in Environmental Protection, Nicholas Georgescu-Roegen Interdisciplinary Research and Formation Platform, 4 Oituz, Timisoara, 300086 (Romania)

2011-04-15

Research highlights: lights: We focus our study on the glycolytic enzymes. We reveal correlation of hydrophobicity and flexibility along their chains. We also reveal fractal aspects of the glycolytic enzymes structures and surfaces. The glycolytic enzyme sequences are not random. Creation of fractal structures requires the operation of nonlinear dynamics. - Abstract: Nonlinear methods widely used for time series analysis were applied to glycolytic enzyme sequences to derive information concerning the correlation of hydrophobicity and average flexibility along their chains. The 20 sequences of different types of the 10 human glycolytic enzymes were considered as spatial series and were analyzed by spectral analysis, detrended fluctuations analysis and Hurst coefficient calculation. The results agreed that there are both short range and long range correlations of hydrophobicity and average flexibility within investigated sequences, the short range correlations being stronger and indicating that local interactions are the most important for the protein folding. This correlation is also reflected by the fractal nature of the structures of investigated proteins.

Genetic disruption of lactate/H+ symporters (MCTs) and their subunit CD147/BASIGIN sensitizes glycolytic tumor cells to phenformin.

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Marchiq, Ibtissam; Le Floch, Renaud; Roux, Danièle; Simon, Marie-Pierre; Pouyssegur, Jacques

2015-01-01

Rapidly growing glycolytic tumors require energy and intracellular pH (pHi) homeostasis through the activity of two major monocarboxylate transporters, MCT1 and the hypoxia-inducible MCT4, in intimate association with the glycoprotein CD147/BASIGIN (BSG). To further explore and validate the blockade of lactic acid export as an anticancer strategy, we disrupted, via zinc finger nucleases, MCT4 and BASIGIN genes in colon adenocarcinoma (LS174T) and glioblastoma (U87) human cell lines. First, we showed that homozygous loss of MCT4 dramatically sensitized cells to the MCT1 inhibitor AZD3965. Second, we demonstrated that knockout of BSG leads to a decrease in lactate transport activity of MCT1 and MCT4 by 10- and 6-fold, respectively. Consequently, cells accumulated an intracellular pool of lactic and pyruvic acids, magnified by the MCT1 inhibitor decreasing further pHi and glycolysis. As a result, we found that these glycolytic/MCT-deficient cells resumed growth by redirecting their metabolism toward OXPHOS. Third, we showed that in contrast with parental cells, BSG-null cells became highly sensitive to phenformin, an inhibitor of mitochondrial complex I. Phenformin addition to these MCT-disrupted cells in normoxic and hypoxic conditions induced a rapid drop in cellular ATP-inducing cell death by "metabolic catastrophe." Finally, xenograft analysis confirmed the deleterious tumor growth effect of MCT1/MCT4 ablation, an action enhanced by phenformin treatment. Collectively, these findings highlight that inhibition of the MCT/BSG complexes alone or in combination with phenformin provides an acute anticancer strategy to target highly glycolytic tumors. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development. ©2014 American Association for Cancer Research.

Nonlinear correlations in the hydrophobicity and average flexibility along the glycolytic enzymes sequences

International Nuclear Information System (INIS)

Ciorsac, Alecu; Craciun, Dana; Ostafe, Vasile; Isvoran, Adriana

2011-01-01

Research highlights: → We focus our study on the glycolytic enzymes. → We reveal correlation of hydrophobicity and flexibility along their chains. → We also reveal fractal aspects of the glycolytic enzymes structures and surfaces. → The glycolytic enzyme sequences are not random. → Creation of fractal structures requires the operation of nonlinear dynamics. - Abstract: Nonlinear methods widely used for time series analysis were applied to glycolytic enzyme sequences to derive information concerning the correlation of hydrophobicity and average flexibility along their chains. The 20 sequences of different types of the 10 human glycolytic enzymes were considered as spatial series and were analyzed by spectral analysis, detrended fluctuations analysis and Hurst coefficient calculation. The results agreed that there are both short range and long range correlations of hydrophobicity and average flexibility within investigated sequences, the short range correlations being stronger and indicating that local interactions are the most important for the protein folding. This correlation is also reflected by the fractal nature of the structures of investigated proteins.

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae.

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Elbing, Karin; Larsson, Christer; Bill, Roslyn M; Albers, Eva; Snoep, Jacky L; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena

2004-09-01

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells

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Amemiya, Takashi; Shibata, Kenichi; Itoh, Yoshihiro; Itoh, Kiminori; Watanabe, Masatoshi; Yamaguchi, Tomohiko

2017-10-01

We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

Mutations in AtPS1 (Arabidopsis thaliana parallel spindle 1 lead to the production of diploid pollen grains.

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Isabelle d'Erfurth

2008-11-01

Full Text Available Polyploidy has had a considerable impact on the evolution of many eukaryotes, especially angiosperms. Indeed, most--if not all-angiosperms have experienced at least one round of polyploidy during the course of their evolution, and many important crop plants are current polyploids. The occurrence of 2n gametes (diplogametes in diploid populations is widely recognised as the major source of polyploid formation. However, limited information is available on the genetic control of diplogamete production. Here, we describe the isolation and characterisation of the first gene, AtPS1 (Arabidopsis thaliana Parallel Spindle 1, implicated in the formation of a high frequency of diplogametes in plants. Atps1 mutants produce diploid male spores, diploid pollen grains, and spontaneous triploid plants in the next generation. Female meiosis is not affected in the mutant. We demonstrated that abnormal spindle orientation at male meiosis II leads to diplogamete formation. Most of the parent's heterozygosity is therefore conserved in the Atps1 diploid gametes, which is a key issue for plant breeding. The AtPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. The isolation of a gene involved in diplogamete production opens the way for new strategies in plant breeding programmes and progress in evolutionary studies.

The glycolytic pathway of Trimastix pyriformis is an evolutionary mosaic

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Silberman Jeffrey D

2006-11-01

Full Text Available Abstract Background Glycolysis and subsequent fermentation is the main energy source for many anaerobic organisms. The glycolytic pathway consists of ten enzymatic steps which appear to be universal amongst eukaryotes. However, it has been shown that the origins of these enzymes in specific eukaryote lineages can differ, and sometimes involve lateral gene transfer events. We have conducted an expressed sequence tag (EST survey of the anaerobic flagellate Trimastix pyriformis to investigate the nature of the evolutionary origins of the glycolytic enzymes in this relatively unstudied organism. Results We have found genes in the Trimastix EST data that encode enzymes potentially catalyzing nine of the ten steps of the glycolytic conversion of glucose to pyruvate. Furthermore, we have found two different enzymes that in principle could catalyze the conversion of phosphoenol pyruvate (PEP to pyruvate (or the reverse reaction as part of the last step in glycolysis. Our phylogenetic analyses of all of these enzymes revealed at least four cases where the relationship of the Trimastix genes to homologs from other species is at odds with accepted organismal relationships. Although lateral gene transfer events likely account for these anomalies, with the data at hand we were not able to establish with confidence the bacterial donor lineage that gave rise to the respective Trimastix enzymes. Conclusion A number of the glycolytic enzymes of Trimastix have been transferred laterally from bacteria instead of being inherited from the last common eukaryotic ancestor. Thus, despite widespread conservation of the glycolytic biochemical pathway across eukaryote diversity, in a number of protist lineages the enzymatic components of the pathway have been replaced by lateral gene transfer from disparate evolutionary sources. It remains unclear if these replacements result from selectively advantageous properties of the introduced enzymes or if they are neutral

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium.

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Terrell, Kimberly A; Wildt, David E; Anthony, Nicola M; Bavister, Barry D; Leibo, S P; Penfold, Linda M; Marker, Laurie L; Crosier, Adrienne E

2011-06-01

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.

The unique features of glycolytic pathways in Archaea.

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Verhees, Corné H; Kengen, Servé W M; Tuininga, Judith E; Schut, Gerrit J; Adams, Michael W W; De Vos, Willem M; Van Der Oost, John

2003-10-15

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden-Meyerhof and the Entner-Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

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

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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

A chemometric method to identify enzymatic reactions leading to the transition from glycolytic oscillations to waves

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Zimányi, László; Khoroshyy, Petro; Mair, Thomas

2010-06-01

In the present work we demonstrate that FTIR-spectroscopy is a powerful tool for the time resolved and noninvasive measurement of multi-substrate/product interactions in complex metabolic networks as exemplified by the oscillating glycolysis in a yeast extract. Based on a spectral library constructed from the pure glycolytic intermediates, chemometric analysis of the complex spectra allowed us the identification of many of these intermediates. Singular value decomposition and multiple level wavelet decomposition were used to separate drifting substances from oscillating ones. This enabled us to identify slow and fast variables of glycolytic oscillations. Most importantly, we can attribute a qualitative change in the positive feedback regulation of the autocatalytic reaction to the transition from homogeneous oscillations to travelling waves. During the oscillatory phase the enzyme phosphofructokinase is mainly activated by its own product ADP, whereas the transition to waves is accompanied with a shift of the positive feedback from ADP to AMP. This indicates that the overall energetic state of the yeast extract determines the transition between spatially homogeneous oscillations and travelling waves.

Keragaman Genetik Sekuen Gen ATP Synthase FO Subunit 6 (ATP6 Monyet Hantu (Tarsius Indonesia (GENETIC DIVERSITY STUDY OF ATP6 GENE SEQUENCES OF TARSIERS FROM INDONESIA

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Rini Widayanti

2013-07-01

Full Text Available In a conservation effort, the identification of Tarsier species, on the bases of the morphological andmolecular characteristic is necessary. Up to now, the identification of the animals were based on themorphology and vocalizations, which is extremely difficult to identify each, tarsier species. The objective ofthis research was to study the genetic diversity on ATP6 gene of Tarsius sp. Based on sequencing of PCRproduct using primer ATP6F and ATP6R with 681 nts. PCR product. The sequence of ATP6 fragmentswere aligned with other primates from Gene bank with aid of software Clustal W, and were analyzed usingMEGA program version 4.0. Three different nucleotide sites were found (nucleotide no. 288, 321 and 367.The genetic distance based on nucleotide ATP6 sequence calculated using Kimura 2-parameter modelindicated that the smallest genetic distance 0%, biggest 0.8% and average 0, 2%. The phylogenetic treeusing neighbor joining method based on the sequence of nucleotide ATP6 gene could not be used todifferentiate among T. Dianae (from Central Sulawesi, T. Spectrum (from North Sulawesi, T. bancanus(from lampung, South Sumatera and T.bancanus from West Kalimantan.

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

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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.

Caffeine Ingestion Increases Estimated Glycolytic Metabolism during Taekwondo Combat Simulation but Does Not Improve Performance or Parasympathetic Reactivation.

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João Paulo Lopes-Silva

Full Text Available The aim of this study was to evaluate the effect of caffeine ingestion on performance and estimated energy system contribution during simulated taekwondo combat and on post-exercise parasympathetic reactivation.Ten taekwondo athletes completed two experimental sessions separated by at least 48 hours. Athletes consumed a capsule containing either caffeine (5 mg∙kg-1 or placebo (cellulose one hour before the combat simulation (3 rounds of 2 min separated by 1 min passive recovery, in a double-blind, randomized, repeated-measures crossover design. All simulated combat was filmed to quantify the time spent fighting in each round. Lactate concentration and rating of perceived exertion were measured before and after each round, while heart rate (HR and the estimated contribution of the oxidative (WAER, ATP-PCr (WPCR, and glycolytic (W[La-] systems were calculated during the combat simulation. Furthermore, parasympathetic reactivation after the combat simulation was evaluated through 1 taking absolute difference between the final HR observed at the end of third round and the HR recorded 60-s after (HRR60s, 2 taking the time constant of HR decay obtained by fitting the 6-min post-exercise HRR into a first-order exponential decay curve (HRRτ, or by 3 analyzing the first 30-s via logarithmic regression analysis (T30.Caffeine ingestion increased estimated glycolytic energy contribution in relation to placebo (12.5 ± 1.7 kJ and 8.9 ± 1.2 kJ, P = 0.04. However, caffeine did not improve performance as measured by attack number (CAF: 26. 7 ± 1.9; PLA: 27.3 ± 2.1, P = 0.48 or attack time (CAF: 33.8 ± 1.9 s; PLA: 36.6 ± 4.5 s, P = 0.58. Similarly, RPE (CAF: 11.7 ± 0.4 a.u.; PLA: 11.5 ± 0.3 a.u., P = 0.62, HR (CAF: 170 ± 3.5 bpm; PLA: 174.2 bpm, P = 0.12, oxidative (CAF: 109.3 ± 4.5 kJ; PLA: 107.9 kJ, P = 0.61 and ATP-PCr energy contributions (CAF: 45.3 ± 3.4 kJ; PLA: 46.8 ± 3.6 kJ, P = 0.72 during the combat simulation were unaffected. Furthermore

Caffeine Ingestion Increases Estimated Glycolytic Metabolism during Taekwondo Combat Simulation but Does Not Improve Performance or Parasympathetic Reactivation.

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Lopes-Silva, João Paulo; Silva Santos, Jonatas Ferreira da; Branco, Braulio Henrique Magnani; Abad, César Cavinato Cal; Oliveira, Luana Farias de; Loturco, Irineu; Franchini, Emerson

2015-01-01

The aim of this study was to evaluate the effect of caffeine ingestion on performance and estimated energy system contribution during simulated taekwondo combat and on post-exercise parasympathetic reactivation. Ten taekwondo athletes completed two experimental sessions separated by at least 48 hours. Athletes consumed a capsule containing either caffeine (5 mg∙kg-1) or placebo (cellulose) one hour before the combat simulation (3 rounds of 2 min separated by 1 min passive recovery), in a double-blind, randomized, repeated-measures crossover design. All simulated combat was filmed to quantify the time spent fighting in each round. Lactate concentration and rating of perceived exertion were measured before and after each round, while heart rate (HR) and the estimated contribution of the oxidative (WAER), ATP-PCr (WPCR), and glycolytic (W[La-]) systems were calculated during the combat simulation. Furthermore, parasympathetic reactivation after the combat simulation was evaluated through 1) taking absolute difference between the final HR observed at the end of third round and the HR recorded 60-s after (HRR60s), 2) taking the time constant of HR decay obtained by fitting the 6-min post-exercise HRR into a first-order exponential decay curve (HRRτ), or by 3) analyzing the first 30-s via logarithmic regression analysis (T30). Caffeine ingestion increased estimated glycolytic energy contribution in relation to placebo (12.5 ± 1.7 kJ and 8.9 ± 1.2 kJ, P = 0.04). However, caffeine did not improve performance as measured by attack number (CAF: 26. 7 ± 1.9; PLA: 27.3 ± 2.1, P = 0.48) or attack time (CAF: 33.8 ± 1.9 s; PLA: 36.6 ± 4.5 s, P = 0.58). Similarly, RPE (CAF: 11.7 ± 0.4 a.u.; PLA: 11.5 ± 0.3 a.u., P = 0.62), HR (CAF: 170 ± 3.5 bpm; PLA: 174.2 bpm, P = 0.12), oxidative (CAF: 109.3 ± 4.5 kJ; PLA: 107.9 kJ, P = 0.61) and ATP-PCr energy contributions (CAF: 45.3 ± 3.4 kJ; PLA: 46.8 ± 3.6 kJ, P = 0.72) during the combat simulation were unaffected

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.

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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

L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

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Nakatsu, Daiki; Horiuchi, Yuta; Kano, Fumi; Noguchi, Yoshiyuki; Sugawara, Taichi; Takamoto, Iseki; Kubota, Naoto; Kadowaki, Takashi; Murata, Masayuki

2015-03-10

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

Glycolysis and mitochondrial respiration in mouse LDHC-null sperm.

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Odet, Fanny; Gabel, Scott; London, Robert E; Goldberg, Erwin; Eddy, Edward M

2013-04-01

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

KREBER`S ADVENTURES: COMPUTER GAME ABOUT THE ATP FORMATION

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F. J. Baêta

2015-08-01

Full Text Available Introduction: Biochemistry, as well as other subjects related to molecular area, have several abstract and difficult concepts to be understood, therefore, many educational innovations have been developed, highlighting the digital games. The digital games feature a playful and motivational character that encourages students during the concepts learning, with a different way to learning the concepts studied. Objectives: The objective of this study was the development of a computer game focused on the concepts of ATP formation, including the glycolytic pathway, Krebs cycle and Electron Transport Chain, as well as aspects related to the regulation, and evaluate the usability of it, as well as some evidence of its educational potential. Material and methods: The development of the game followed the following steps: definition of the subject; understanding of game developer (it was chosen the GameMaker; storyboard creation of the game; prototyping, implementation and usability testing. For the evaluation, inspection usability was performed (without involving end users and subsequently the cognitive route and the usability questionnaire (the latter two with students of the discipline of Biochemistry. Results: The game approached the energy metabolism in three phases: the glycolytic pathway, Krebs cycle and electron transport chain. Each phase has a different purpose, with some questions about the ways. To complete the game, you must correctly answer the questions, avoid the obstacles and achieve the goals of each phase. After usability testing, it found that users could, in a playful manner, actively interact with the content addressed and, through the difficulties presented in the game, had the opportunity to expand and review their knowledge. Conclusions: The game was identified as a motivating and innovative proposal for  teaching, and it had good usability for undergraduate students. The ludic worked as a pedagogical practice encourages student

Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

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Gómez-Arreaza, Amaranta; Acosta, Hector; Quiñones, Wilfredo; Concepción, Juan Luis; Michels, Paul A M; Avilán, Luisana

2014-02-01

In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence. Copyright © 2014 Elsevier B.V. All rights reserved.

Non-preferential fuelling of the Na(+)/K(+)-ATPase pump.

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Fernández-Moncada, Ignacio; Barros, L Felipe

2014-06-15

There is abundant evidence that glycolysis and the Na(+)/K(+)-ATPase pump are functionally coupled, and it is thought that the nature of the coupling is energetic, with glycolysis providing the ATP that fuels the pump. This notion has been instrumental to current models of brain energy metabolism. However, structural and biophysical considerations suggest that the pump should also have access to mitochondrial ATP, which is much more abundant. In the present study, we have investigated the source of ATP that fuels the Na(+) pump in astrocytes, taking advantage of the high temporal resolution of recently available FRET nanosensors for glucose, lactate and ATP. The activity of the Na(+) pump was assessed in parallel with the Na(+)-sensitive dye SBFI AM (Na(+)-binding benzofuran isophthalate acetoxymethyl ester). OXPHOS (oxidative phosphorylation) inhibition resulted in bulk ATP depletion and a 5-fold stimulation of glycolytic flux, in spite of which Na(+) pumping was inhibited by 90%. Mathematical modelling of ATP dynamics showed that the observed pump failure is inconsistent with preferential fuelling of the Na(+) pump by glycolytic ATP. We conclude that the nature of the functional coupling between the Na(+) pump and the glycolytic machinery is not energetic and that the pump is mainly fuelled by mitochondrial ATP.

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

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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.

ATP Release Channels

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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.

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

Performance and energy systems contributions during upper-body sprint interval exercise.

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Franchini, Emerson; Takito, Monica Yuri; Dal'Molin Kiss, Maria Augusta Peduti

2016-12-01

The main purpose of this study was to investigate the performance and energy systems contribution during four upper-body Wingate tests interspersed by 3-min intervals. Fourteen well-trained male adult Judo athletes voluntarily took part in the present study. These athletes were from state to national level, were in their competitive period, but not engaged in any weight loss procedure. Energy systems contributions were estimated using oxygen uptake and blood lactate measurements. The main results indicated that there was higher glycolytic contribution compared to oxidative ( P creatine phosphate, ATP-PCr) contribution during bout 3 ( P <0.001), lower glycolytic contribution compared to oxidative and ATP-PCr ( P <0.001 for both comparisons) contributions during bout 4 and lower oxidative compared to ATP-PCr during bout 4 ( P =0.040). For the energy system contribution across Wingate bouts, the ATP-PCr contribution during bout 1 was lower than that observed during bout 4 ( P =0.005), and the glycolytic system presented higher percentage contribution in the first bout compared to the third and fourth bouts ( P <0.001 for both comparisons), and higher percentage participation in the second compared to the fourth bout ( P <0.001). These results suggest that absolute oxidative and ATP-PCr participations were kept constant across Wingate tests, but there was an increase in relative participation of ATP-PCr in bout 4 compared to bout 1, probably due to the partial phosphocreatine resynthesis during intervals and to the decreased glycolytic activity.

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

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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.

Experimental determination of control of glycolysis in Lactococcus lactis

DEFF Research Database (Denmark)

Købmann, Brian Jensen; Andersen, Heidi Winterberg; Solem, Christian

2002-01-01

), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass...... production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis...

Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans.

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Gay, Sean C; Fribourgh, Jennifer L; Donohoue, Paul D; Segel, Irwin H; Fisher, Andrew J

2009-09-01

The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

Preslaughter handling effects on pork quality and glycolytic potential in two muscles differing in fiber type composition

NARCIS (Netherlands)

Hambrecht, E.; Eissen, J.J.; Newman, D.J.; Smits, C.H.M.; Verstegen, M.W.A.; Hartog, den L.A.

2005-01-01

The objective of the present experiment was to investigate the effects of transportation, lairage, and preslaughter stressor treatment on glycolytic potential and pork quality of the glycolytic longissimus and the oxidative supraspinatus (SSP) or serratus ventralis (SV) muscles. In a 2 x 2 x 2

Augmentation of glycolytic metabolism by meclizine is indispensable for protection of dorsal root ganglion neurons from hypoxia-induced mitochondrial compromise.

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Zhuo, Ming; Gorgun, Murat F; Englander, Ella W

2016-10-01

To meet energy demands, dorsal root ganglion (DRG) neurons harbor high mitochondrial content, which renders them acutely vulnerable to disruptions of energy homeostasis. While neurons typically rely on mitochondrial energy production and have not been associated with metabolic plasticity, new studies reveal that meclizine, a drug, recently linked to modulations of energy metabolism, protects neurons from insults that disrupt energy homeostasis. We show that meclizine rapidly enhances glycolysis in DRG neurons and that glycolytic metabolism is indispensable for meclizine-exerted protection of DRG neurons from hypoxic stress. We report that supplementation of meclizine during hypoxic exposure prevents ATP depletion, preserves NADPH and glutathione stores, curbs reactive oxygen species (ROS) and attenuates mitochondrial clustering in DRG neurites. Using extracellular flux analyzer, we show that in cultured DRG neurons meclizine mitigates hypoxia-induced loss of mitochondrial respiratory capacity. Respiratory capacity is a measure of mitochondrial fitness and cell ability to meet fluctuating energy demands and therefore, a key determinant of cellular fate. While meclizine is an 'old' drug with long record of clinical use, its ability to modulate energy metabolism has been uncovered only recently. Our findings documenting neuroprotection by meclizine in a setting of hypoxic stress reveal previously unappreciated metabolic plasticity of DRG neurons as well as potential for pharmacological harnessing of the newly discovered metabolic plasticity for protection of peripheral nervous system under mitochondria compromising conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

MCT1-mediated transport of a toxic molecule is an effective strategy for targeting glycolytic tumors

Science.gov (United States)

Birsoy, Kivanc; Wang, Tim; Possemato, Richard; Yilmaz, Omer H.; Koch, Catherine E.; Chen, Walter W.; Hutchins, Amanda W.; Gultekin, Yetis; Peterson, Tim R.; Carette, Jan E.; Brummelkamp, Thijn R.; Clish, Clary B.; Sabatini, David M.

2012-01-01

SUMMARY There is increasing evidence that oncogenic transformation modifies the metabolic program of cells. A common alteration is the upregulation of glycolysis, and efforts to target glycolytic enzymes for anti-cancer therapy are underway. Here, we performed a genome-wide haploid genetic screen to identify resistance mechanisms to 3-bromopyruvate (3-BrPA), a drug candidate that inhibits glycolysis in a poorly understood fashion. We identified the SLC16A1 gene product, MCT1, as the main determinant of 3-BrPA sensitivity. MCT1 is necessary and sufficient for 3-BrPA uptake by cancer cells. Additionally, MCT1 mRNA levels are the best predictor of 3-BrPA sensitivity and are most elevated in glycolytic cancer cells. Lastly, forced MCT1 expression in 3-BrPA resistant cancer cells sensitizes tumor xenografts to 3-BrPA treatment in vivo. Our results identify a potential biomarker for 3-BrPA sensitivity and provide proof of concept that the selectivity of cancer-expressed transporters can be exploited for delivering toxic molecules to tumors. PMID:23202129

Similar temperature dependencies of glycolytic enzymes: an evolutionary adaptation to temperature dynamics?

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Cruz Luisa Ana B

2012-12-01

Full Text Available Abstract Background Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in Saccharomyces cerevisiae during temperature changes. Results Saccharomyces cerevisiae was grown under different temperature regimes and glucose availability conditions. These included glucose-excess batch cultures at different temperatures and glucose-limited chemostat cultures, subjected to fast linear temperature shifts and circadian sinoidal temperature cycles. An observed temperature-independent relation between intracellular levels of glycolytic metabolites and residual glucose concentration for all experimental conditions revealed that it is the substrate availability rather than temperature that determines intracellular metabolite profiles. This observation corresponded with predictions generated in silico with a kinetic model of yeast glycolysis, when the catalytic capacities of all glycolytic enzymes were set to share the same normalized temperature dependency. Conclusions From an evolutionary perspective, such similar temperature dependencies allow cells to adapt more rapidly to temperature changes, because they result in minimal perturbations of intracellular metabolite levels, thus circumventing the need for extensive modification of enzyme levels.

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.

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...

Co-factor engineering in lactobacilli: Effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

DEFF Research Database (Denmark)

Rud, Ida; Solem, Christian; Jensen, Peter Ruhdal

2008-01-01

The hydrolytic F-1-part of the F1F0-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity...... resulted in a decrease in intracellular energy level (ATP/ADP ratio), biomass yield and growth rate. Interestingly, the glycolytic and ribolytic flux increased in L. plantarum with uncoupled ATPase activity compared to the reference strain by up to 20% and 50%, respectively. The ATP demand was estimated...... to have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity. (C) 2008 Elsevier Inc. All rights reserved....

Course of the anaerobic alcohol fermentation by Saccharomyces cerevisiae simulated through a mathematical model of the glycolysis

Energy Technology Data Exchange (ETDEWEB)

Martiny, S C

1972-01-01

The model presented here attempts to simulate the course of the conversion of glucose to alcohol through a simulation of the glycolytic flux rate. The model is based on dynamic stationarity through the glycolytic reactions, equilibrium in the action of ATPase. The model does not simulate experimental data, mainly because ATPase cannot keep pace with ATP formation. The simulations stress the need for better understanding of the mechanism of ATP removal within the cell.

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...

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

Science.gov (United States)

Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

2015-07-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The unique features of glycolytic pathways in Archaea.

OpenAIRE

Verhees, Corné H; Kengen, Servé W M; Tuininga, Judith E; Schut, Gerrit J; Adams, Michael W W; De Vos, Willem M; Van Der Oost, John

2003-01-01

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a vari...

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

International Nuclear Information System (INIS)

Sierra, Ana; Subbotina, Ekaterina; Zhu, Zhiyong; Gao, Zhan; Koganti, Siva Rama Krishna; Coetzee, William A.; Goldhamer, David J.; Hodgson-Zingman, Denice M.; Zingman, Leonid V.

2016-01-01

Sarcolemmal ATP-sensitive potassium (K_A_T_P) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle K_A_T_P channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle K_A_T_P channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of K_A_T_P channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology to atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish K_A_T_P channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities. - Highlights: • ATP-sensitive K"+ channels regulate musclin production by skeletal muscles. • Lipolytic ANP signaling is promoted by augmented skeletal muscle musclin production. • Skeletal muscle musclin transcription is promoted by a CaMKII/HDAC/FOXO1 pathway. • Musclin links adipose mobilization to energy use in K_A_T_P channel deficient skeletal muscle.

Metal-dependent regulation of ATP7A and ATP7B in fibroblast cultures

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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.

Impact of Autoantibodies against Glycolytic Enzymes on Pathogenicity of Autoimmune Retinopathy and Other Autoimmune Disorders

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Grazyna Adamus

2017-04-01

Full Text Available Autoantibodies (AAbs against glycolytic enzymes: aldolase, α-enolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase are prevalent in sera of patients with blinding retinal diseases, such as paraneoplastic [cancer-associated retinopathy (CAR] and non-paraneoplastic autoimmune retinopathies, as well as in many other autoimmune diseases. CAR is a degenerative disease of the retina characterized by sudden vision loss in patients with cancer and serum anti-retinal AAbs. In this review, we discuss the widespread serum presence of anti-glycolytic enzyme AAbs and their significance in autoimmune diseases. There are multiple mechanisms responsible for antibody generation, including the innate anti-microbial response, anti-tumor response, or autoimmune response against released self-antigens from damaged, inflamed tissue. AAbs against enolase, GADPH, and aldolase exist in a single patient in elevated titers, suggesting their participation in pathogenicity. The lack of restriction of AAbs to one disease may be related to an increased expression of glycolytic enzymes in various metabolically active tissues that triggers an autoimmune response and generation of AAbs with the same specificity in several chronic and autoimmune conditions. In CAR, the importance of serum anti-glycolytic enzyme AAbs had been previously dismissed, but the retina may be without pathological consequence until a failure of the blood–retinal barrier function, which would then allow pathogenic AAbs access to their retinal targets, ultimately leading to damaging effects.

Limits to sustainable muscle performance: interaction between glycolysis and oxidative phosphorylation.

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Conley, K E; Kemper, W F; Crowther, G J

2001-09-01

This paper proposes a mechanism responsible for setting the sustainable level of muscle performance. Our contentions are that the sustainable work rate is determined (i) at the muscle level, (ii) by the ability to maintain ATP supply and (iii) by the products of glycolysis that may inhibit the signal for oxidative phosphorylation. We argue below that no single factor 'limits' sustainable performance, but rather that the flux through and the interaction between glycolysis and oxidative phosphorylation set the level of sustainable ATP supply. This argument is based on magnetic resonance spectroscopy measurements of the sources and sinks for energy in vivo in human muscle and rattlesnake tailshaker muscle during sustained contractions. These measurements show that glycolysis provides between 20% (human muscle) and 40% (tailshaker muscle) of the ATP supply during sustained contractions in these muscles. We cite evidence showing that this high glycolytic flux does not reflect an O(2) limitation or mitochondria operating at their capacity. Instead, this flux reflects a pathway independent of oxidative phosphorylation for ATP supply during aerobic exercise. The consequence of this high glycolytic flux is accumulation of H(+), which we argue inhibits the rise in the signal activating oxidative phosphorylation, thereby restricting oxidative ATP supply to below the oxidative capacity. Thus, both glycolysis and oxidative phosphorylation play important roles in setting the highest steady-state ATP synthesis flux and thereby determine the sustainable level of work by exercising muscle.

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...

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

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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

Contributions of citrate in redox potential maintenance and ATP production: metabolic pathways and their regulation in Lactobacillus panis PM1.

Science.gov (United States)

Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji

2013-10-01

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli and can utilize various NADH-reoxidizing routes (e.g., citrate, glycerol, and oxygen) according to environmental conditions. In this study, we investigated the ability of L. panis PM1 to produce succinate, acetate, and lactate via citrate utilization. Possible pathways, as well as regulation, for citrate metabolism were examined on the basis of the genome sequence data and metabolic profiles of L. panis PM1. The presence of citrate led to the up-regulation, at the transcriptional level, of the genes encoding for citrate lyase, malate dehydrogenase, and malic enzyme of the citrate pathways by 10- to 120-fold. The transcriptional regulator of the dha operon coding for glycerol dehydratase of L. panis PM1 repressed the expression of the citrate lyase gene (10-fold). Metabolite analyses indicated that the transcriptional enhancement by citrate stimulated succinate yield. Citrate metabolism contributed to energy production by providing a major alternate pathway for NAD(+) regeneration and allowed acetyl phosphate to yield acetate/ATP instead of ethanol/NAD(+). Additionally, a branching pathway from oxaloacetate to pyruvate increased the pool of lactate, which was then used to produce ATP during stationary phase. However, the redirection of NADH-to-citrate utilization resulted in stress caused by end-products (i.e., succinate and acetate). This stress reduced succinate production by up to 50 % but did not cause significant changes at transcriptional level. Overall, citrate utilization was beneficial for the growth of L. panis PM1 by providing a NAD(+) regeneration route and producing extra ATP.

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

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.

Similar temperature dependencies of glycolytic enzymes : An evolutionary adaptation to temperature dynamics?

NARCIS (Netherlands)

Cruz, L.A.B.; Hebly, M.; Duong, G.H.; Wahl, S.A.; Pronk, J.T.; Heijnen, J.J.; Daran-Lapujade, P.; Van Gulik, W.M.

2012-01-01

Background Temperature strongly affects microbial growth, and many microorganisms have to deal with temperature fluctuations in their natural environment. To understand regulation strategies that underlie microbial temperature responses and adaptation, we studied glycolytic pathway kinetics in

Switch between life history strategies due to changes in glycolytic enzyme gene dosage in Saccharomyces cerevisiae.

Science.gov (United States)

Wang, Shaoxiao; Spor, Aymé; Nidelet, Thibault; Montalent, Pierre; Dillmann, Christine; de Vienne, Dominique; Sicard, Delphine

2011-01-01

Adaptation is the process whereby a population or species becomes better fitted to its habitat through modifications of various life history traits which can be positively or negatively correlated. The molecular factors underlying these covariations remain to be elucidated. Using Saccharomyces cerevisiae as a model system, we have investigated the effects on life history traits of varying the dosage of genes involved in the transformation of resources into energy. Changing gene dosage for each of three glycolytic enzyme genes (hexokinase 2, phosphoglucose isomerase, and fructose-1,6-bisphosphate aldolase) resulted in variation in enzyme activities, glucose consumption rate, and life history traits (growth rate, carrying capacity, and cell size). However, the range of effects depended on which enzyme was expressed differently. Most interestingly, these changes revealed a genetic trade-off between carrying capacity and cell size, supporting the discovery of two extreme life history strategies already described in yeast populations: the "ants," which have lower glycolytic gene dosage, take up glucose slowly, and have a small cell size but reach a high carrying capacity, and the "grasshoppers," which have higher glycolytic gene dosage, consume glucose more rapidly, and allocate it to a larger cell size but reach a lower carrying capacity. These results demonstrate antagonist pleiotropy for glycolytic genes and show that altered dosage of a single gene drives a switch between two life history strategies in yeast.

Loss of the gene for the alpha subunit of ATP synthase (ATP5A1) from the W chromosome in the African grey parrot (Psittacus erithacus).

Science.gov (United States)

de Kloet, S R

2001-08-01

This study describes the results of an analysis using Southern blotting, the polymerase chain reaction, and sequencing which shows that the African grey parrot (Psittacus erithacus) lacks the W-chromosomal gene for the alpha subunit of mitochondrial ATP synthase (ATP5A1W). Additional evidence shows that in other psittacines a fragment of the ATP5A1W gene contains five times as many nonsynonymous nucleotide replacements as the homologous fragment of the Z gene. Therefore, whereas in these other psittacines the corresponding ATP5A1Z protein fragment is highly conserved and varies by only a few, moderately conservative amino acid substitutions, the homologous ATP5A1W fragments contain a considerable number of, sometimes highly nonconservative, amino acid replacements. In one of these species, the ringneck parakeet (Psittacula krameri), the ATP5A1W gene is present in an inactive form because of the presence of a nonsense codon. Other changes, possibly leading to an inactive ATP5A1W gene product, involve the substitution of arginine residues by cysteine in the ATP5A1W protein of the mitred conure (Aratinga mitrata) and the blue and gold macaw (Ara ararauna). The data suggest also that although the divergence of the psittacine ATP5A1W and ATP5A1Z genes preceded the origin of the psittacidae, this divergence occurred independently of a similar process in the myna (Gracula religiosa), the outgroup used in this study.

Respiratory ATP cost and benefit of arbuscular mycorrhizal symbiosis with Nicotiana tabacum at different growth stages and under salinity.

Science.gov (United States)

Del-Saz, Néstor Fernández; Romero-Munar, Antonia; Alonso, David; Aroca, Ricardo; Baraza, Elena; Flexas, Jaume; Ribas-Carbo, Miquel

2017-11-01

Growth and maintenance partly depend on both respiration and ATP production during oxidative phosphorylation in leaves. Under stress, ATP is needed to maintain the accumulated biomass. ATP production mostly proceeds from the cytochrome oxidase pathway (COP), while respiration via the alternative oxidase pathway (AOP) may decrease the production of ATP per oxygen consumed, especially under phosphorus (P) limitation and salinity conditions. Symbiosis with arbuscular mycorrhizal (AM) fungi is reputed by their positive effect on plant growth under stress at mature stages of colonization; however, fungal colonization may decrease plant growth at early stages. Thus, the present research is based on the hypothesis that AM fungus colonization will increase both foliar respiration and ATP production at mature stages of plant growth while decreasing them both at early stages. We used the oxygen-isotope-fractionation technique to study the in vivo respiratory activities and ATP production of the COP and AOP in AM and non-AM (NM) tobacco plants grown under P-limiting and saline conditions in sand at different growth stages (14, 28 and 49days). Our results suggest that AM symbiosis represents an ATP cost detrimental for shoot growth at early stages, whilst it represents a benefit on ATP allowing for faster rates of growth at mature stages, even under salinity conditions. Copyright © 2017 Elsevier GmbH. All rights reserved.

Mitochondrial Dysfunction Contributes to Impaired Insulin Secretion in INS-1 Cells with Dominant-negative Mutations of HNF-1α and in HNF-1α-deficient Islets*

Science.gov (United States)

Pongratz, Rebecca L.; Kibbey, Richard G.; Kirkpatrick, Clare L.; Zhao, Xiaojian; Pontoglio, Marco; Yaniv, Moshe; Wollheim, Claes B.; Shulman, Gerald I.; Cline, Gary W.

2009-01-01

Maturity Onset Diabetes of the Young-type 3 (MODY-3) has been linked to mutations in the transcription factor hepatic nuclear factor (HNF)-1α, resulting in deficiency in glucose-stimulated insulin secretion. In INS-1 cells overexpressing doxycycline-inducible HNF-1α dominant-negative (DN-) gene mutations, and islets from Hnf-1α knock-out mice, insulin secretion was impaired in response to glucose (15 mm) and other nutrient secretagogues. Decreased rates of insulin secretion in response to glutamine plus leucine and to methyl pyruvate, but not potassium depolarization, indicate defects specific to mitochondrial metabolism. To identify the biochemical mechanisms responsible for impaired insulin secretion, we used 31P NMR measured mitochondrial ATP synthesis (distinct from glycolytic ATP synthesis) together with oxygen consumption measurements to determine the efficiency of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling was significantly higher in DN-HNF-1α cells, such that rates of ATP synthesis were decreased by approximately one-half in response to the secretagogues glucose, glutamine plus leucine, or pyruvate. In addition to closure of the ATP-sensitive K+ channels with mitochondrial ATP synthesis, mitochondrial production of second messengers through increased anaplerotic flux has been shown to be critical for coupling metabolism to insulin secretion. 13C-Isotopomer analysis and tandem mass spectrometry measurement of Krebs cycle intermediates revealed a negative impact of DN-HNF-1α and Hnf-1α knock-out on mitochondrial second messenger production with glucose but not amino acids. Taken together, these results indicate that, in addition to reduced glycolytic flux, uncoupling of mitochondrial oxidative phosphorylation contributes to impaired nutrient-stimulated insulin secretion with either mutations or loss of HNF-1α. PMID:19376774

MCT4 Defines a Glycolytic Subtype of Pancreatic Cancer with Poor Prognosis and Unique Metabolic Dependencies

Directory of Open Access Journals (Sweden)

GuemHee Baek

2014-12-01

Full Text Available KRAS mutation, which occurs in ∼95% of pancreatic ductal adenocarcinoma (PDA, has been shown to program tumor metabolism. MCT4 is highly upregulated in a subset of PDA with a glycolytic gene expression program and poor survival. Models with high levels of MCT4 preferentially employ glycolytic metabolism. Selectively in such “addicted” models, MCT4 attenuation compromised glycolytic flux with compensatory induction of oxidative phosphorylation and scavenging of metabolites by macropinocytosis and autophagy. In spite of these adaptations, MCT4 depletion induced cell death characterized by elevated reactive oxygen species and metabolic crisis. Cell death induced by MCT4-depletion was augmented by inhibition of compensatory pathways. In xenograft models, MCT4 had a significant impact on tumor metabolism and was required for rapid tumor growth. Together, these findings illustrate the metabolic diversity of PDA described by MCT4, delineate pathways through which this lactate transporter supports cancer growth, and demonstrate that PDA can be rationally targeted based on metabolic addictions.

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.

Science.gov (United States)

Jiang, Yu; Tao, Rongsheng; Shen, Zhengquan; Sun, Liangdong; Zhu, Fuyun; Yang, Sheng

2016-12-01

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l -1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol -1 added cysteine and a productivity of 6.1 ± 0.0 g l -1  h -1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.

Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger

DEFF Research Database (Denmark)

Meijer, Susan Lisette; Nielsen, Michael Lynge; Olsson, Lisbeth

2009-01-01

With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell...... factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which...... the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic model predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger....

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.

Carbohydrate metabolism in ripening banana and its alteration on gamma irradiation in relation to delay in ripening

International Nuclear Information System (INIS)

Surendranathan, K.K.; Nair, P.M.

1980-01-01

Ripening, of climacteric class of fruits like banana, is accompanied with an upsurge in respiration, indicating a change in metabolism from hexose monophosphate (HMP) shunt pathway to glycolytic pathway. The key enzyme in glycolytic pathway, namely, phosphofructokinase, is activated and this activation paralleled with the increase in respiration rate. The enhancement in the activity of enzymes of glycolytic and Kreb's cycle help the fruit to assimilate energy as ATP produced from the breakdown and oxidation of storage starch. The demand for energy supply is great for the different ripening processes. Gamma irradiation of the fruit at the preclimacteric stage delayed the onset of climacteric to about 7 to 8 days, thereby extending the ripening to 15-20 days. This delay was brought about by the alterations in the metabolism of carbohydrate. There is a predominance of HMP pathway in irradiated banana. This along with the activation of phosphatases like FDPase and F-6-Pase restricted the entrance of sugar phosphate esters to Kreb's cycle for oxidation. The functioning of Kreb's cycle is also affected by the inhibition of succinic dehydrogenase. But activation of glyoxylate shunt pathway helped to maintain the levels of Kreb's cycle intermediates, like citrate and malate, although energy production is reduced. Finally the activation of gluconeogenic pathway helps in channelling the metabolites back to sugars. All these metabolic changes cause a considerable depletion in the production of ATP. (auth.)

Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria.

Science.gov (United States)

Sánchez-Pascuala, Alberto; Nikel, Pablo I; de Lorenzo, Víctor

2018-01-01

The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (i.e., GlucoBrick) that facilitates their grouping in the form of functional modules that can be combined at the user's will. This novel genetic tool allows for the à la carte implementation or boosting of EMP pathway activities into different Gram-negative bacteria. The potential of the GlucoBrick platform is further illustrated by engineering novel glycolytic activities in the most representative members of the Pseudomonas genus (Pseudomonas putida and Pseudomonas aeruginosa).

uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells.

Science.gov (United States)

Laurenzana, Anna; Chillà, Anastasia; Luciani, Cristina; Peppicelli, Silvia; Biagioni, Alessio; Bianchini, Francesca; Tenedini, Elena; Torre, Eugenio; Mocali, Alessandra; Calorini, Lido; Margheri, Francesca; Fibbi, Gabriella; Del Rosso, Mario

2017-09-15

In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma. © 2017 UICC.

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.

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...

Catalytic strategy used by the myosin motor to hydrolyze ATP.

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Kiani, Farooq Ahmad; Fischer, Stefan

2014-07-22

Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

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

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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.

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

Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study.

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Harshini Sarojini

Full Text Available We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery-extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold

Metabolomic Analysis of Oxidative and Glycolytic Skeletal Muscles by Matrix-Assisted Laser Desorption/IonizationMass Spectrometric Imaging (MALDI MSI)

Science.gov (United States)

Tsai, Yu-Hsuan; Garrett, Timothy J.; Carter, Christy S.; Yost, Richard A.

2015-06-01

Skeletal muscles are composed of heterogeneous muscle fibers that have different physiological, morphological, biochemical, and histological characteristics. In this work, skeletal muscles extensor digitorum longus, soleus, and whole gastrocnemius were analyzed by matrix-assisted laser desorption/ionization mass spectrometry to characterize small molecule metabolites of oxidative and glycolytic muscle fiber types as well as to visualize biomarker localization. Multivariate data analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to extract significant features. Different metabolic fingerprints were observed from oxidative and glycolytic fibers. Higher abundances of biomolecules such as antioxidant anserine as well as acylcarnitines were observed in the glycolytic fibers, whereas taurine and some nucleotides were found to be localized in the oxidative fibers.

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.

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

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.

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.

Modeling diauxic glycolytic oscillations in yeast

DEFF Research Database (Denmark)

Hald, Bjørn Olav; Sørensen, Preben Graae

2010-01-01

for investigations of central metabolism dynamics of yeast cells. We have previously proposed a model for the open system comprised of the primary fermentative reactions in yeast that quantitatively describes the oscillatory dynamics. However, this model fails to describe the transient behavior of metabolic......Glycolytic oscillations in a stirred suspension of starved yeast cells is an excellent model system for studying the dynamics of metabolic switching in living systems. In an open-flow system the oscillations can be maintained indefinitely at a constant operating point where they can....... Experimental and computational results strongly suggest that regulation of acetaldehyde explains the observed behavior. We have extended the original model with regulation of pyruvate decarboxylase, a reversible alcohol dehydrogenase, and drainage of pyruvate. Using the method of time rescaling in the extended...

Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria

DEFF Research Database (Denmark)

Sánchez-Pascuala, Alberto; Nikel, Pablo I.; de Lorenzo, Víctor

2018-01-01

the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals......The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved...... and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account...

Metabolic engineering of an ATP-neutral Embden-Meyerhof-Parnas pathway in Corynebacterium glutamicum: growth restoration by an adaptive point mutation in NADH dehydrogenase.

Science.gov (United States)

Komati Reddy, Gajendar; Lindner, Steffen N; Wendisch, Volker F

2015-03-01

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH

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

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Daniela eTrono

2015-12-01

Full Text Available In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about fifteen years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP. Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous and tissues/organs (etiolated and green have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane towards the matrix, so collapsing membrane potential (ΔΨ, the main component of the protonmotive force (Δp in plant mitochondria; moreover, cooperation between PmitoKATP and the K+/H+ antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate

Assembly of the membrane domain of ATP synthase in human mitochondria.

Science.gov (United States)

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

2018-03-20

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

Acceleration of the glycolytic flux by steroid receptor coactivator-2 is essential for endometrial decidualization.

Directory of Open Access Journals (Sweden)

Ramakrishna Kommagani

2013-10-01

Full Text Available Early embryo miscarriage is linked to inadequate endometrial decidualization, a cellular transformation process that enables deep blastocyst invasion into the maternal compartment. Although much of the cellular events that underpin endometrial stromal cell (ESC decidualization are well recognized, the individual gene(s and molecular pathways that drive the initiation and progression of this process remain elusive. Using a genetic mouse model and a primary human ESC culture model, we demonstrate that steroid receptor coactivator-2 (SRC-2 is indispensable for rapid steroid hormone-dependent proliferation of ESCs, a critical cell-division step which precedes ESC terminal differentiation into decidual cells. We reveal that SRC-2 is required for increasing the glycolytic flux in human ESCs, which enables rapid proliferation to occur during the early stages of the decidualization program. Specifically, SRC-2 increases the glycolytic flux through induction of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3, a major rate-limiting glycolytic enzyme. Similarly, acute treatment of mice with a small molecule inhibitor of PFKFB3 significantly suppressed the ability of these animals to exhibit an endometrial decidual response. Together, these data strongly support a conserved mechanism of action by which SRC-2 accelerates the glycolytic flux through PFKFB3 induction to provide the necessary bioenergy and biomass to meet the demands of a high proliferation rate observed in ESCs prior to their differentiation into decidual cells. Because deregulation of endometrial SRC-2 expression has been associated with common gynecological disorders of reproductive-age women, this signaling pathway, involving SRC-2 and PFKFB3, promises to offer new clinical approaches in the diagnosis and/or treatment of a non-receptive uterus in patients presenting idiopathic infertility, recurrent early pregnancy loss, or increased time to pregnancy.

Journey in guidelines for lipid management: From adult treatment panel (ATP-I to ATP-III and what to expect in ATP-IV

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P G Talwalkar

2013-01-01

Full Text Available Adult Treatment Panel (ATP, an expert panel to supervise cholesterol management was set up under the aegis of National Cholesterol Education Program (NCEP in 1985. Since then NCEP-ATP has been revising and framing guidelines to enable clinician to deliver better treatment to cardiovascular patients and to educate general people. As a result, considerable reduction in cardiovascular related deaths has been observed in recent times. All three ATP guidelines viz. ATP-I, ATP-II and ATP-III have targeted low density lipoprotein as their primary goal. The ATP-III guideline was updated in the light of evidences from 5-major clinical trials and was released in 2004. It added therapeutic lifestyle changes, concept of risk equivalents, Framingham CHD-risk score non-high density lipoprotein cholesterol (non-HDL-C as secondary target and gave strong emphasis on metabolic risk factors. The earlier treat-to-target paradigm faced fierce criticism from clinicians across the globe because of insufficient proof of safety and benefits of treating patients with respect to an individual′s low density lipoprotein (LDL level. Further, demonstration of non-HDL-C and total cholesterol/HDL-C ratio as strong predictors of overall cardiovascular risk foresees new guidelines. A tailored-treatment approach was suggested instead of LDL-C target based treatment approach which was soundly based on direct clinical trials evidences and proposes treatment based on individual′s overall 5- to 10-year cardiovascular risk irrespective of LDL-C level, leading to lower number of people on high dose/s of statins. Recent report of the Cholesterol Treatment Trialist′s Collaborators meta-analysis strongly supported primary prevention of LDL with statins in low risk individuals and showed that its benefits completely outweighed its known hazards. Markers other than LDL-C like apolipoprotein B, non-HDL-C and total cholesterol/HDL-C ratio would take precedence in the risk assessment and

ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects

DEFF Research Database (Denmark)

Minet, Ariane D; Gaster, Michael

2010-01-01

To date, it is unknown whether mitochondrial dysfunction in skeletal muscle from subjects with type 2 diabetes is based on primarily reduced mitochondrial mass and/or a primarily decreased mitochondrial ATP synthesis. Mitochondrial mass were determined in myotubes established from eight lean, eight...... mass and the ATP synthesis rate, neither at baseline nor during acute insulin stimulation, were not different between groups. The ratio of ATP synthesis rate at hexokinase versus ATP synthesis rate at baseline was lower in diabetic mitochondria compared to lean mitochondria. Thus the lower content...... obese and eight subjects with type 2 diabetes precultured under normophysiological conditions. Furthermore, mitochondria were isolated and ATP production was measured by luminescence at baseline and during acute insulin stimulation with or without concomitant ATP utilization by hexokinase. Mitochondrial...

The Structural and Functional Coordination of Glycolytic Enzymes in Muscle: Evidence of a Metabolon?

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Lynda Menard

2014-09-01

Full Text Available Metabolism sustains life through enzyme-catalyzed chemical reactions within the cells of all organisms. The coupling of catalytic function to the structural organization of enzymes contributes to the kinetic optimization important to tissue-specific and whole-body function. This coupling is of paramount importance in the role that muscle plays in the success of Animalia. The structure and function of glycolytic enzyme complexes in anaerobic metabolism have long been regarded as a major regulatory element necessary for muscle activity and whole-body homeostasis. While the details of this complex remain to be elucidated through in vivo studies, this review will touch on recent studies that suggest the existence of such a complex and its structure. A potential model for glycolytic complexes and related subcomplexes is introduced.

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

3-bromopyruvate and sodium citrate target glycolysis, suppress survivin, and induce mitochondrial-mediated apoptosis in gastric cancer cells and inhibit gastric orthotopic transplantation tumor growth.

Science.gov (United States)

Wang, Ting-An; Zhang, Xiao-Dong; Guo, Xing-Yu; Xian, Shu-Lin; Lu, Yun-Fei

2016-03-01

Glycolysis is the primary method utilized by cancer cells to produce the energy (adenosine triphosphate, ATP) required for cell proliferation. Therefore, inhibition of glycolysis may inhibit tumor growth. We previously found that both 3-bromopyruvate (3-BrPA) and sodium citrate (SCT) can inhibit glycolysis in vitro; however, the underlying inhibitory mechanisms remain unclear. In the present study, we used a human gastric cancer cell line (SGC-7901) and an orthotopic transplantation tumor model in nude mice to explore the specific mechanisms of 3-BrPA and SCT. We found that both 3-BrPA and SCT effectively suppressed cancer cell proliferation, arrested the cell cycle, induced apoptosis, and decreased the production of lactate and ATP. 3-BrPA significantly reduced the glycolytic enzyme hexokinase activity, while SCT selectively inhibited phosphofructokinase-1 activity. Furthermore, 3-BrPA and SCT upregulated the expression of pro-apoptotic proteins (Bax, cytochrome c, and cleaved caspase-3) and downregulated the expression of anti-apoptotic proteins (Bcl-2 and survivin). Finally, our animal model of gastric cancer indicated that intraperitoneal injection of 3-BrPA and SCT suppressed orthotopic transplantation tumor growth and induced tumor apoptosis. Taken together, these results suggest that 3-BrPA and SCT selectively suppress glycolytic enzymes, decrease ATP production, induce mitochondrial-mediated apoptosis, downregulate survivin, and inhibit tumor growth. Moreover, an intraperitoneal injection is an effective form of administration of 3-BrPA and SCT.

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

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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.

Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

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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.

Symbiotic Association with Mycoplasma hominis Can Influence Growth Rate, ATP Production, Cytolysis and Inflammatory Response of Trichomonas vaginalis

Science.gov (United States)

Margarita, Valentina; Rappelli, Paola; Dessì, Daniele; Pintus, Gianfranco; Hirt, Robert P.; Fiori, Pier L.

2016-01-01

The symbiosis between the parasitic protist Trichomonas vaginalis and the opportunistic bacterium Mycoplasma hominis is the only one currently described involving two obligate human mucosal symbionts with pathogenic capabilities that can cause independent diseases in the same anatomical site: the lower urogenital tract. Although several aspects of this intriguing microbial partnership have been investigated, many questions on the influence of this symbiosis on the parasite pathobiology still remain unanswered. Here, we examined with in vitro cultures how M. hominis could influence the pathobiology of T. vaginalis by investigating the influence of M. hominis on parasite replication rate, haemolytic activity and ATP production. By comparing isogenic mycoplasma-free T. vaginalis and parasites stably associated with M. hominis we could demonstrate that the latter show a higher replication rate, increased haemolytic activity and are able to produce larger amounts of ATP. In addition, we demonstrated in a T. vaginalis-macrophage co-culture system that M. hominis could modulate an aspect of the innate immuno-response to T. vaginalis infections by influencing the production of nitric oxide (NO) by human macrophages, with the parasite-bacteria symbiosis outcompeting the human cells for the key substrate arginine. These results support a model in which the symbiosis between T. vaginalis and M. hominis influences host-microbes interactions to the benefit of both microbial partners during infections and to the detriment of their host. PMID:27379081

Scaling of oxidative and glycolytic enzymes in mammals.

Science.gov (United States)

Emmett, B; Hochachka, P W

1981-09-01

The catalytic activities of several oxidative and glycolytic enzymes were determined in the gastrocnemius muscle of 10 mammalian species differing in body weight by nearly 6 orders of magnitude. When expressed in terms of units gm-1, the activities of enzymes functioning in oxidative metabolism (citrate synthase, beta-hydroxybutyrylCoA dehydrogenase, and malate dehydrogenase) decrease as body weight increases. Log-log plots (activity gm-1 vs body mass) yield straight lines with negative slopes that are less than the allometric exponent (-0.25) typically observed for basal metabolic rates. Since the amount of power a muscle can generate depends upon the catalytic potential of its enzyme machinery (the higher the catalytic potential the higher the maximum rate of energy generation), these data predict that the scope for aerobic activity in large mammals should be greater than in small mammals if nothing else becomes limiting, a result in fact recently obtained by Taylor et al. (Respir. Physiol., 1981). In contrast to the scaling of oxidative enzymes, the activities of enzymes functioning in anaerobic glycogenolysis (glycogen phosphorylase, pyruvate kinase, and lactate dehydrogenase) increase as body size increases. Log-log plots (activity gm-1 vs body mass) display a positive slope indicating that the larger the animal the higher the glycolytic potential of its skeletal muscles. This unexpected result may indicate higher relative power costs for burst type locomotion in larger mammals, which is in fact observed in within-species studies of man. However, the scaling of anaerobic muscle power has not been closely assessed in between-species comparisons of mammals varying greatly in body size.

Functional mitochondrial ATP synthase proteolipid gene produced by recombination of parental genes in a petunia somatic hybrid

International Nuclear Information System (INIS)

Rothenberg, M.; Hanson, M.R.

1988-01-01

A novel ATP synthase subunit 9 gene (atp9) was identified in the mitochondrial genome of a Petunia somatic hybrid line (13-133) which was produced from a fusion between Petunia lines 3688 and 3704. The novel gene was generated by intergenomic recombination between atp9 genes from the two parental plant lines. The entire atp9 coding region is represented on the recombinant gene. Comparison of gene sequences using electrophoresis and autoradiography, indicate that the 5' transcribed region is contributed by an atp9 gene from 3704 and the 3' transcribed region is contributed by an atp9 gene from 3688. The recombinant atp9 gene is transcriptionally active. The location of the 5' and 3' transcript termini are conserved with respect to the parental genes, resulting in the production of hybrid transcripts

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...

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

NARCIS (Netherlands)

Milne, N.; Luttik, M.A.H.; Cueto Rojas, H.F.; Wahl, A.; Van Maris, A.J.A.; Pronk, J.T.; Daran, J.G.

2015-01-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential

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

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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.

Tomatidine Is a Lead Antibiotic Molecule That Targets Staphylococcus aureus ATP Synthase Subunit C.

Science.gov (United States)

Lamontagne Boulet, Maxime; Isabelle, Charles; Guay, Isabelle; Brouillette, Eric; Langlois, Jean-Philippe; Jacques, Pierre-Étienne; Rodrigue, Sébastien; Brzezinski, Ryszard; Beauregard, Pascale B; Bouarab, Kamal; Boyapelly, Kumaraswamy; Boudreault, Pierre-Luc; Marsault, Éric; Malouin, François

2018-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro -generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >10 5 -fold for FC04-100. Copyright © 2018 American Society for Microbiology.

Processing mechanics of alternate twist ply (ATP) yarn technology

Science.gov (United States)

Elkhamy, Donia Said

Ply yarns are important in many textile manufacturing processes and various applications. The primary process used for producing ply yarns is cabling. The speed of cabling is limited to about 35m/min. With the world's increasing demands of ply yarn supply, cabling is incompatible with today's demand activated manufacturing strategies. The Alternate Twist Ply (ATP) yarn technology is a relatively new process for producing ply yarns with improved productivity and flexibility. This technology involves self plying of twisted singles yarn to produce ply yarn. The ATP process can run more than ten times faster than cabling. To implement the ATP process to produce ply yarns there are major quality issues; uniform Twist Profile and yarn Twist Efficiency. The goal of this thesis is to improve these issues through process modeling based on understanding the physics and processing mechanics of the ATP yarn system. In our study we determine the main parameters that control the yarn twist profile. Process modeling of the yarn twist across different process zones was done. A computational model was designed to predict the process parameters required to achieve a square wave twist profile. Twist efficiency, a measure of yarn torsional stability and bulk, is determined by the ratio of ply yarn twist to singles yarn twist. Response Surface Methodology was used to develop the processing window that can reproduce ATP yarns with high twist efficiency. Equilibrium conditions of tensions and torques acting on the yarns at the self ply point were analyzed and determined the pathway for achieving higher twist efficiency. Mechanistic modeling relating equilibrium conditions to the twist efficiency was developed. A static tester was designed to zoom into the self ply zone of the ATP yarn. A computer controlled, prototypic ATP machine was constructed and confirmed the mechanistic model results. Optimum parameters achieving maximum twist efficiency were determined in this study. The

Glycolytic inhibitors 2-deoxyglucose and 3-bromopyruvate synergize with photodynamic therapy respectively to inhibit cell migration.

Science.gov (United States)

Feng, Xiaolan; Wang, Pan; Liu, Quanhong; Zhang, Ting; Mai, Bingjie; Wang, Xiaobing

2015-06-01

Most cancer cells have the specially increased glycolytic phenotype, which makes this pathway become an attractive therapeutic target. Although glycolytic inhibitor 2-deoxyglucose (2-DG) has been demonstrated to potentiate the cytotoxicity of photodynamic therapy (PDT), the impacts on cell migration after the combined treatment has never been reported yet. The present study aimed to analyze the influence of glycolytic inhibitors 2-DG and 3-bromopyruvate (3-BP) combined with Ce6-PDT on cell motility of Triple Negative Breast Cancer MDA-MB-231 cells. As determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium-bromide-Tetraz-olium (MTT) assay, more decreased cell viability was observed in 2-DG + PDT and 3-BP + PDT groups when compared with either monotherapy. Under optimal conditions, synergistic potentiation on cell membrane destruction and the decline of cell adhesion and cells migratory ability were observed in both 2-DG + PDT and 3-BP + PDT by electron microscope observation (SEM), wound healing and trans-well assays. Besides, serious microfilament network collapses as well as impairment of matrix metalloproteinases-9 (MMP-9) were notably improved after the combined treatments by immunofluorescent staining. These results suggest that 2-DG and 3-BP can both significantly potentiated Ce6-PDT efficacy of cell migration inhibition.

Capillary network in slow and fast muscles and in oxidative and glycolytic muscle fibres

Czech Academy of Sciences Publication Activity Database

Čebašek, V.; Kubínová, Lucie; Ribarič, S.; Eržen, I.

2005-01-01

Roč. 24, March (2005), s. 51-58 ISSN 1580-3139 Grant - others:SI-CZ(CZ) KONTAKT 19/2005 Institutional research plan: CEZ:AV0Z50110509 Keywords : capillaries * skeletal muscle fibre s-oxidative and glycolytic * stereology Subject RIV: EA - Cell Biology

Exploiting Unique Structural and Functional Properties of Malarial Glycolytic Enzymes for Antimalarial Drug Development

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Asrar Alam

2014-01-01

Full Text Available Metabolic enzymes have been known to carry out a variety of functions besides their normal housekeeping roles known as “moonlighting functions.” These functionalities arise from structural changes induced by posttranslational modifications and/or binding of interacting proteins. Glycolysis is the sole source of energy generation for malaria parasite Plasmodium falciparum, hence a potential pathway for therapeutic intervention. Crystal structures of several P. falciparum glycolytic enzymes have been solved, revealing that they exhibit unique structural differences from the respective host enzymes, which could be exploited for their selective targeting. In addition, these enzymes carry out many parasite-specific functions, which could be of potential interest to control parasite development and transmission. This review focuses on the moonlighting functions of P. falciparum glycolytic enzymes and unique structural differences and functional features of the parasite enzymes, which could be exploited for therapeutic and transmission blocking interventions against malaria.

Structural models of the human copper P-type ATPases ATP7A and ATP7B

DEFF Research Database (Denmark)

Gourdon, P.; Sitsel, Oleg; Karlsen, J.L.

2012-01-01

The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B......, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type...

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

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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

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

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Olga B. Vadzyuk

2018-03-01

Full Text Available The objective of this study was to detect ATP-sensitive K+ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K+-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of KATP-channel (ATP-sensitive K+-channel – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate. Our data demonstrates that K+ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of KATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of KATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane. Keywords: ATP-sensitive potassium channel, Diazoxide, 5-hydroxydecanoate, Myometrium, Mitochondria, Mitochondrial swelling, Mitochondrial membrane potential, ROS

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.

Optimization time synthesis of nucleotide labelled [γ-32P]-ATP

International Nuclear Information System (INIS)

Rahman, Wira Y; Sarmini, Endang; Herlina; Lubis, Hotman; Triyanto; Hambali

2013-01-01

Adenosine triphosphate-labelled with γ- 32 P([γ- 32 p]-ATP) has been widely used in the biotechnology research, usually as a tracer to study aspects of physiological and pathological processes. In order to support biotechnology research in Indonesia, a process for production of [γ- 32 P]-ATP with enzymatic reaction was used as precursors DL-glyceraldehydde 3-phosphate, Adenosine Diphosphate (ADP) and H 3 32 PO 4 , and enzyme glyceraldehid 3-phosphate dehydrogenase, 3-phosphoglyceryc phosphokinase and lactate dehydrogenase. Optimization of incubation time labeled nucleotide synthesis process is performed to find the optimum conditions, in terms of the most advantageous time in the synthesis process. With the success of the synthesis and optimization is done incubation time of synthesis labeled nucleotide, the result suggested can be used for producing [γ- 32 P] -ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia. (author)

Energy Metabolism in the Liver

OpenAIRE

Rui, Liangyou

2014-01-01

The liver is an essential metabolic organ, and its metabolic activity is tightly controlled by insulin and other metabolic hormones. Glucose is metabolized into pyruvate through glycolysis in the cytoplasm, and pyruvate is completely oxidized to generate ATP through the TCA cycle and oxidative phosphorylation in the mitochondria. In the fed state, glycolytic products are used to synthesize fatty acids through de novo lipogenesis. Long-chain fatty acids are incorporated into triacylglycerol, p...

Defects in mitochondrial ATP synthesis in dystrophin-deficient mdx skeletal muscles may be caused by complex I insufficiency.

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Emma Rybalka

Full Text Available Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca2+ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca2+ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca2+ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb's cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca2+ load in comparison to controls, and 400 nM extramitochondrial Ca2+ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.

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

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

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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.

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

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Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

2011-10-01

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

The danger signal extracellular ATP is an inducer of Fusobacterium nucleatum biofilm dispersal

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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

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.

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress.

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Vassilopoulos, Athanassios; Pennington, J Daniel; Andresson, Thorkell; Rees, David M; Bosley, Allen D; Fearnley, Ian M; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Deng, Chu-Xia; Walker, John E; Gius, David

2014-08-01

Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

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

Serdemetan antagonizes the Mdm2-HIF1α axis leading to decreased levels of glycolytic enzymes.

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Jason A Lehman

Full Text Available Serdemetan (JNJ-26854165, an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1, were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2.

An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts.

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Pateraki, Irini; Renato, Marta; Azcón-Bieto, Joaquín; Boronat, Albert

2013-04-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report the involvement of a plastidial ATP synthase harboring an atypical γ-subunit induced during ripening, which lacks the regulatory dithiol domain present in plant and algae chloroplast γ-subunits. Silencing of this atypical γ-subunit during fruit ripening impairs the capacity of isolated chromoplast to synthesize ATP de novo. We propose that the replacement of the γ-subunit present in tomato leaf and green fruit chloroplasts by the atypical γ-subunit lacking the dithiol domain during fruit ripening reflects evolutionary changes, which allow the operation of chromoplast ATP synthase under the particular physiological conditions found in this organelle. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

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

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.

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.

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...

Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

African Journals Online (AJOL)

Yomi

2012-01-24

Jan 24, 2012 ... protein involved in temperature challenge in S. constricta. Key words: Sinonovacula constricta, ATP synthase, ... MATERIALS AND METHODS. Experimental animals. Sinonovacula constricta (7 to 8 g ... Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to ...

Optimization of L: -methionine feeding strategy for improving S-adenosyl-L: -methionine production by methionine adenosyltransferase overexpressed Pichia pastoris.

Science.gov (United States)

Hu, Hui; Qian, Jiangchao; Chu, Ju; Wang, Yonghong; Zhuang, Yingping; Zhang, Siliang

2009-07-01

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-L: -methionine (SAM). Two L: -methionine (L: -Met) addition strategies were used to supply the precursor: the batch addition strategy (L: -Met was added separately at three time points) and the continuous feeding strategies (L: -Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l(-1) h(-1), respectively). SAM accumulation, L: -Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 +/- 0.31 g l(-1), 41.7 +/- 1.4%, and 0.18 +/- 0.01 g l(-1) h(-1) with the best continuous feeding strategy (0.2 g l(-1) h(-1)), respectively. The bottleneck for SAM production with the low L: -Met feeding rate (0.1 g L(-1) h(-1)) was the insufficient L: -Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing L: -Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the L: -Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the L: -Met feeding rate reached 0.5 g l(-1) h(-1).

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

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Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K; Dean, Dennis R; Hoffman, Brian M; Antony, Edwin; Seefeldt, Lance C

2013-10-08

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 °C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 °C), (iii) Phosphate release (kPi = 16 s(-1), 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein.

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

Science.gov (United States)

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

International Nuclear Information System (INIS)

Penefsky, H.S.

1985-01-01

Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

Science.gov (United States)

Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

2015-01-01

We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

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

An ATP synthase harboring an atypical γ-subunit is involved in ATP synthesis in tomato fruit chromoplasts

DEFF Research Database (Denmark)

Pateraki, Irini; Renato, Marta; Azcõn-Bieto, Joaquín

2013-01-01

Chromoplasts are non-photosynthetic plastids specialized in the synthesis and accumulation of carotenoids. During fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts in a process characterized by the degradation of the thylakoid membranes, and by the active...... synthesis and accumulation of carotenoids. This transition renders chromoplasts unable to photochemically synthesize ATP, and therefore these organelles need to obtain the ATP required for anabolic processes through alternative sources. It is widely accepted that the ATP used for biosynthetic processes...... in non-photosynthetic plastids is imported from the cytosol or is obtained through glycolysis. In this work, however, we show that isolated tomato (Solanum lycopersicum) fruit chromoplasts are able to synthesize ATP de novo through a respiratory pathway using NADPH as an electron donor. We also report...

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

Studies on luciferin-luciferase ATP assay in plants (etiolated wheat germs, and bean leaves

Directory of Open Access Journals (Sweden)

A. Barbaro

2015-01-01

Full Text Available For ATP determination by the method of bioluminescence apparatus of home production was adapted from the equipment available in any isotope laboratory. The measurement error did not exceed 1.5 per cent. Methodical experiments concerned the choice of the extraction, fixation and storage methods of plant material for determination at the given moment of the amount of ATP in the tissues, unchanged by the analytical procedure. The highest ATP amounts were recovered by extraction with perchloric acid at high (25% concentrations of the tissue in the homogenate. The best way of fixation of the material for later analyses was found to be freezing of ready extracts. Lyophilization and freezing of the plant material caused a several-fold decrease of the ATP level in the tissues. These results suggest the necessity of working in conditions of low temperature during the entire analytical procedure and strict observation of time limitation.

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.

A balanced ATP driving force module for enhancing photosynthetic biosynthesis of 3-hydroxybutyrate from CO2.

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Ku, Jason T; Lan, Ethan I

2018-03-01

Using engineered photoautotrophic microorganisms for the direct chemical synthesis from CO 2 is an attractive direction for both sustainability and CO 2 mitigation. However, the behaviors of non-native metabolic pathways may be difficult to control due to the different intracellular contexts between natural and heterologous hosts. While most metabolic engineering efforts focus on strengthening driving forces in pathway design to favor biochemical production in these organisms, excessive driving force may be detrimental to product biosynthesis due to imbalanced cellular intermediate distribution. In this study, an ATP-hydrolysis based driving force module was engineered into cyanobacterium Synechococcus elongatus PCC 7942 to produce 3-hydroxybutyrate (3HB), a valuable chemical feedstock for the synthesis of biodegradable plastics and antibiotics. However, while the ATP driving force module is effective for increasing product formation, uncontrolled accumulation of intermediate metabolites likely led to metabolic imbalance and thus to cell growth inhibition. Therefore, the ATP driving force module was reengineered by providing a reversible outlet for excessive carbon flux. Upon expression of this balanced ATP driving force module with 3HB biosynthesis, engineered strain produced 3HB with a cumulative titer of 1.2 g/L, a significant increase over the initial strain. This result highlighted the importance of pathway reversibility as an effective design strategy for balancing driving force and intermediate accumulation, thereby achieving a self-regulated control for increased net flux towards product biosynthesis. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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...

Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa.

Science.gov (United States)

Yi, Young-Joo; Sutovsky, Miriam; Kennedy, Chelsey; Sutovsky, Peter

2012-01-01

Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.

Identification of the inorganic pyrophosphate metabolizing, ATP substituting pathway in mammalian spermatozoa.

Directory of Open Access Journals (Sweden)

Young-Joo Yi

Full Text Available Inorganic pyrophosphate (PPi is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1 in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.

New insights into the complex regulation of the glycolytic pathway in Lactococcus lactis. I. Construction and diagnosis of a comprehensive dynamic model.

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Dolatshahi, Sepideh; Fonseca, Luis L; Voit, Eberhard O

2016-01-01

This article and the companion paper use computational systems modeling to decipher the complex coordination of regulatory signals controlling the glycolytic pathway in the dairy bacterium Lactococcus lactis. In this first article, the development of a comprehensive kinetic dynamic model is described. The model is based on in vivo NMR data that consist of concentration trends in key glycolytic metabolites and cofactors. The model structure and parameter values are identified with a customized optimization strategy that uses as its core the method of dynamic flux estimation. For the first time, a dynamic model with a single parameter set fits all available glycolytic time course data under anaerobic operation. The model captures observations that had not been addressed so far and suggests the existence of regulatory effects that had been observed in other species, but not in L. lactis. The companion paper uses this model to analyze details of the dynamic control of glycolysis under aerobic and anaerobic conditions.

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

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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.

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway.

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Montaño, Luis M; Carbajal, Verónica; Vargas, Mario H; García-Hernández, Luz M; Díaz-Hernández, Verónica; Checa, Marco; Barajas-López, Carlos

2013-08-01

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

KAUST Repository

Jourdain, P.

2016-02-19

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade

KAUST Repository

Jourdain, P.; Allaman, I.; Rothenfusser, K.; Fiumelli, Hubert; Marquet, P.; Magistretti, Pierre J.

2016-01-01

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.

CE-TOF MS-based metabolomic profiling revealed characteristic metabolic pathways in postmortem porcine fast and slow type muscles.

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Muroya, Susumu; Oe, Mika; Nakajima, Ikuyo; Ojima, Koichi; Chikuni, Koichi

2014-12-01

To determine key compounds and metabolic pathways associated with meat quality, we profiled metabolites in postmortem porcine longissimus lumborum (LL) and vastus intermedius (VI) muscles with different aging times by global metabolomics using capillary electrophoresis-time of flight mass spectrometry. Loading analyses of the principal component analysis showed that hydrophilic amino acids and β-alanine-related compounds contributed to the muscle type positively and negatively, respectively, whereas glycolytic and ATP degradation products contributed to aging time. At 168h postmortem, LL samples were characterized by abundance of combinations of amino acids, dipeptides, and glycolytic products, whereas the VI samples were characterized by abundance of both sulfur-containing compounds and amino acids. The AMP and inosine contents in the VI were approx. 10 times higher than those in the LL at 4h postmortem, suggesting different rates of inosine 5'-monophosphate (IMP) accumulation by adenylate kinase 7 and 5'-nucleotidase, and subsequent different production levels of IMP and hypoxanthine between these two porcine muscles. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biotin enhances ATP synthesis in pancreatic islets of the rat, resulting in reinforcement of glucose-induced insulin secretion.

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Sone, Hideyuki; Sasaki, Yuka; Komai, Michio; Toyomizu, Masaaki; Kagawa, Yasuo; Furukawa, Yuji

2004-02-13

Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.

Molecular characterization of insulin resistance and glycolytic metabolism in the rat uterus

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Zhang, Yuehui; Sun, Xue; Sun, Xiaoyan; Meng, Fanci; Hu, Min; Li, Xin; Li, Wei; Wu, Xiao-Ke; Brännström, Mats; Shao, Ruijin; Billig, Håkan

2016-01-01

Peripheral insulin resistance and hyperandrogenism are the primary features of polycystic ovary syndrome (PCOS). However, how insulin resistance and hyperandrogenism affect uterine function and contribute to the pathogenesis of PCOS are open questions. We treated rats with insulin alone or in combination with human chorionic gonadotropin (hCG) and showed that peripheral insulin resistance and hyperandrogenism alter uterine morphology, cell phenotype, and cell function, especially in glandular epithelial cells. These defects are associated with an aberration in the PI3K/Akt signaling pathway that is used as an indicator for the onset of insulin resistance in classical metabolic tissues. Concomitantly, increased GSK3β (Ser-9) phosphorylation and decreased ERK1/2 phosphorylation in rats treated with insulin and hCG were also observed. We also profiled the expression of glucose transporter (Glut) isoform genes in the uterus under conditions of insulin resistance and/or hyperandrogenism. Finally, we determined the expression pattern of glycolytic enzymes and intermediates during insulin resistance and hyperandrogenism in the uterus. These findings suggest that the PI3K/Akt and MAPK/ERK signaling pathways play a role in the onset of uterine insulin resistance, and they also suggest that changes in specific Glut isoform expression and alterations to glycolytic metabolism contribute to the endometrial dysfunction observed in PCOS patients. PMID:27461373

Energetics of cellular repair processes in a respiratory-deficient mutant of yeast

International Nuclear Information System (INIS)

Jain, V.K.; Gupta, I.; Lata, K.

1982-01-01

Repair of potentially lethal damage induced by cytoxic agents like UV irradiation (254 nm), psorelen-plus-UVA (365 mn), and methyl methanesulfonate has been studied in the presence of a glucose analog, 2-deoxy-D-glucose, in yeast cells. Simultaneously, effects of 2-deoxy-D-glucose were also investigated on parameters of energy metabolism like glucose utilization, rate of ATP production, and ATP content of cells. The following results were obtained. (i) 2-Deoxy-D-glucose is able to inhibit repair of potentially lethal damage induced by all the cytotoxic agents tested. The 2-deoxy-D-glucose-induced inhibition of repair depends upon the type of lesion and the pattern of cellular energy metabolism, the inhibition being greater in respiratory-deficient mutants than in the wild type. (ii) A continuous energy flow is necessary for repair of potentially lethal damage in yeast cells. Energy may be supplied by the glycolytic and/or the respiratory pathway; respiratory metabolism is not essential for this purpose. (iii) The magnitude of repair correlates with the rate of ATP production in a sigmoid manner

Tritium labelled nucleotides: Heterogeneous metal catalyzed exchange labelling of ATP with tritium gas

International Nuclear Information System (INIS)

Jaiswal, D.K.; Morimoto, H.; Williams, P.G.; Wemmer, D.E.

1991-09-01

Adenosine 5' triphosphate (ATP) in aqueous solution has been labeled by exchange with tritium gas in the presence of palladium oxide catalyst. Comparison with our experiments using Pd/BaSO 4 as the catalyst shows that we have obtained product with higher specific activity and improved chemical purity. 3 H NMR spectroscopy of the tritiated ATP shows labelling in both the C-8 and C-2 positions, and the integral ratio of these positions was found to vary from 3:1 to 1:1 under different reaction conditions. 5 refs., 1 fig., 2 tabs

Mitochondrial PKA mediates sperm motility.

Science.gov (United States)

Mizrahi, Rashel; Breitbart, Haim

2014-12-01

Mitochondria are the major source of ATP to power sperm motility. Phosphorylation of mitochondrial proteins has been proposed as a major regulatory mechanism for mitochondrial bioenergetics. Sperm motility was measured by a computer-assisted analyzer, protein detection by western blotting, membrane potential by tetramethylrhodamine, cellular ATP by luciferase assay and localization of PKA by immuno-electron microscopy. Bicarbonate is essential for the creation of mitochondrial electro-chemical gradient, ATP synthesis and sperm motility. Bicarbonate stimulates PKA-dependent phosphorylation of two 60kDa proteins identified as Tektin and glucose-6-phosphate isomerase. This phosphorylation was inhibited by respiration inhibition and phosphorylation could be restored by glucose in the presence of bicarbonate. However, this effect of glucose cannot be seen when the mitochondrial ATP/ADP exchanger was inhibited indicating that glycolytic-produced ATP is transported into the mitochondria and allows PKA-dependent protein phosphorylation inside the mitochondria. Bicarbonate activates mitochondrial soluble adenylyl cyclase (sAC) which catalyzes cAMP production leading to the activation of mitochondrial PKA. Glucose can overcome the lack of ATP in the absence of bicarbonate but it cannot affect the mitochondrial sAC/PKA system, therefore the PKA-dependent phosphorylation of the 60kDa proteins does not occur in the absence of bicarbonate. Production of CO2 in Krebs cycle, which is converted to bicarbonate is essential for sAC/PKA activation leading to mitochondrial membrane potential creation and ATP synthesis. Copyright © 2014 Elsevier B.V. All rights reserved.

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.)

A Petiveria alliacea standardized fraction induces breast adenocarcinoma cell death by modulating glycolytic metabolism.

Science.gov (United States)

Hernández, John Fredy; Urueña, Claudia Patricia; Cifuentes, Maria Claudia; Sandoval, Tito Alejandro; Pombo, Luis Miguel; Castañeda, Diana; Asea, Alexzander; Fiorentino, Susana

2014-05-14

Folk medicine uses aqueous and alcoholic extracts from Petiveria alliacea (Phytolaccaceae) in leukemia and breast cancer treatment in the Caribbean, Central and South America. Herein, we validated the biological activity of a Petiveria alliacea fraction using a metastatic breast adenocarcinoma model (4T1). Petiveria alliacea fraction biological activity was determined estimating cell proliferation, cell colony growth capacity and apoptosis (caspase-3 activity, DNA fragmentation and mitochondrial membrane potential) in 4T1 cells. Petiveria alliacea was used at IC₅₀ concentration (29 µg/mL) and 2 dilutions below, doxorubicin at 0.27 µg/mL (positive control) and dibenzyl disulfide at 2.93 µg/mL (IC50 fraction marker compound). Proteomic estimations were analyzed by LC-MS-MS. Protein level expression was confirmed by RT-PCR. Glucose and lactate levels were measured by enzymatic assays. LD50 was established in BALB/c mice and antitumoral activity evaluated in mice transplanted with GFP-tagged 4T1 cells. Mice were treated with Petiveria alliacea fraction via I.P (182 mg/kg corresponding to 1/8 of LD₅₀ and 2 dilutions below). Petiveria alliacea fraction in vitro induces 4T1 cells apoptosis, caspase-3 activation, DNA fragmentation without mitochondria membrane depolarization, and decreases cell colony growth capacity. Also, changes in glycolytic enzymes expression cause a decrease in glucose uptake and lactate production. Fraction also promotes breast primary tumor regression in BALB/c mice transplanted with GFP-tagged 4T1 cells. A fraction of Petiveria alliacea leaves and stems induces in vitro cell death and in vivo tumor regression in a murine breast cancer model. Our results validate in partly, the traditional use of Petiveria alliacea in breast cancer treatment, revealing a new way of envisioning Petiveria alliacea biological activity. The fraction effect on the glycolytic pathway enzymes contributes to explain the antiproliferative and antitumor activities

Effects of Interval Training-Based Glycolytic Capacity on Physical Fitness in Recreational Long-Distance Runners

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Zatoń Marek

2015-06-01

Full Text Available Purpose. The aim of this study was to investigate the influence of 8-week-long interval training (targeting glycolytic capacity on selected markers of physical fitness in amateur long-distance runners. Methods. The study involved 17 amateur long-distance runners randomly divided into an experimental (n = 8 and control (n = 9 group. The control group performed three or four continuous training sessions per week whereas the experimental group performed two interval running training sessions and one continuous running training session. A graded treadmill exercise test and the 12-min Cooper test were performed pre- and post-training. Results. O2max and the rate of recovery increased in the experimental group. Relative oxygen uptake, minute ventilation, and heart rate speed decreased in low- (6 km/h and medium-intensity (12 km/h running. Conclusions. Both training modalities showed similar results. However, the significant differences in training volume (4-8 min interval training vs. 40-150 min continuous training indicates that the modalities targeting glycolytic capacity may be more efficient for amateur runners prepare for long-distance events.

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.

NARCIS (Netherlands)

Visch, H.J.; Koopman, W.J.H.; Zeegers, D.; Emst-de Vries, S.E. van; Kuppeveld, F.J.M. van; Heuvel, L.W. van den; Smeitink, J.A.M.; Willems, P.H.G.M.

2006-01-01

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

Science.gov (United States)

Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

2016-12-01

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Genetic variation in ATP5O is associated with skeletal muscle ATP50 mRNA expression and glucose uptake in young twins.

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Tina Rönn

Full Text Available BACKGROUND: Impaired oxidative capacity of skeletal muscle mitochondria contribute to insulin resistance and type 2 diabetes (T2D. Furthermore, mRNA expression of genes involved in oxidative phosphorylation, including ATP5O, is reduced in skeletal muscle from T2D patients. Our aims were to investigate mechanisms regulating ATP5O expression in skeletal muscle and association with glucose metabolism, and the relationship between ATP5O single nucleotide polymorphisms (SNPs and risk of T2D. METHODOLOGY/PRINCIPAL FINDINGS: ATP5O mRNA expression was analyzed in skeletal muscle from young (n = 86 and elderly (n = 68 non-diabetic twins before and after a hyperinsulinemic euglycemic clamp. 11 SNPs from the ATP5O locus were genotyped in the twins and a T2D case-control cohort (n = 1466. DNA methylation of the ATP5O promoter was analyzed in twins (n = 22 using bisulfite sequencing. The mRNA level of ATP5O in skeletal muscle was reduced in elderly compared with young twins, both during basal and insulin-stimulated conditions (p<0.0005. The degree of DNA methylation around the transcription start of ATP5O was <1% in both young and elderly twins and not associated with mRNA expression (p = 0.32. The mRNA level of ATP5O in skeletal muscle was positively related to insulin-stimulated glucose uptake (regression coefficient = 6.6; p = 0.02. Furthermore, two SNPs were associated with both ATP5O mRNA expression (rs12482697: T/T versus T/G; p = 0.02 and rs11088262: A/A versus A/G; p = 0.004 and glucose uptake (rs11088262: A/A versus A/G; p = 0.002 and rs12482697: T/T versus T/G; p = 0.005 in the young twins. However, we could not detect any genetic association with T2D. CONCLUSIONS/SIGNIFICANCE: Genetic variation and age are associated with skeletal muscle ATP5O mRNA expression and glucose disposal rate, suggesting that combinations of genetic and non-genetic factors may cause the reduced expression of ATP5O in T2D muscle. These findings propose a role for ATP5O, in

Comparative proteomics analysis of engineered Saccharomyces cerevisiae with enhanced biofuel precursor production.

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Xiaoling Tang

Full Text Available The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.

Identification of a new Mpl-interacting protein, Atp5d.

Science.gov (United States)

Liu, Hongyan; Zhao, Zhenhu; Zhong, Yuxu; Shan, Yajun; Sun, Xiaohong; Mao, Bingzhi; Cong, Yuwen

2014-06-01

Thrombopoietin (TPO) can regulate hematopoiesis and megakaryopoiesis via activation of its receptor, c-Mpl, and multiple downstream signal transduction pathways. Using the cytoplasmic domain of Mpl as bait, we performed yeast two-hybrid screening, and found that the protein Atp5d might associate with Mpl. Atp5d is known as the δ subunit of mitochondrial ATP synthase, but little is known about the function of dissociative Atp5d. The interaction between Mpl and Atp5d was confirmed by the yeast two-hybrid system, mammalian two-hybrid assay, pull-down experiment, and co-immunoprecipitation study in vivo and in vitro. An additional immunofluorescence assay showed that the two proteins can colocalize along the plasma membrane in the cytoplasm. Using the yeast two-hybrid system, we tested a series of cytoplasmic truncated mutations for their ability to bind Atp5d and found an association between Atp5d and the Aa98-113 domain of Mpl. The dissociation of Atp5d from Mpl after TPO stimulation suggests that Atp5d may be a new component of TPO signaling.

Binding of ATP by pertussis toxin and isolated toxin subunits

International Nuclear Information System (INIS)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-01-01

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [ 3 H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of [ 3 H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [ 3 H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site

Binding of ATP by pertussis toxin and isolated toxin subunits

Energy Technology Data Exchange (ETDEWEB)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

1990-07-03

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

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.

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...

Tin-Containing Silicates: Identification of a Glycolytic Pathway via 3-Deoxyglucosone

DEFF Research Database (Denmark)

Tolborg, Søren; Meier, Sebastian; Sádaba, I.

2016-01-01

a cascade of four to five sequential steps. Currently, there is a limited understanding of the competing glycolytic pathways within these systems. Here we identify dehydration of glucose to 3-deoxyglucosone as an important pathway that occurs in addition to retro-aldol reaction of hexoses when using tin......-containing silicates. It is possible to influence the relative carbon flux through these pathways by controlling the amount of alkali metal salts present in the reaction mixture. In the absence of added potassium carbonate, at least 15–30% carbon flux via 3-deoxyglucosone is observed. Addition of just a few ppm...

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

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Philip Serwer

2017-05-01

Full Text Available Adenosine triphosphate (ATP cleavage powers packaging of a double-stranded DNA (dsDNA molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

Metabolic Trade-offs in Yeast are Caused by F1F0-ATP synthase

DEFF Research Database (Denmark)

Nilsson, Avlant; Nielsen, Jens

2016-01-01

of intermediary metabolism and consequently metabolic trade-offs may take place. One such trade-off, aerobic fermentation, occurs in both yeast (the Crabtree effect) and cancer cells (the Warburg effect) and has been a scientific challenge for decades. Here we show, using flux balance analysis combined...... with in vitro measured enzyme specific activities, that fermentation is more catalytically efficient than respiration, i.e. it produces more ATP per protein mass. And that the switch to fermentation at high growth rates therefore is a consequence of a high ATP production rate, provided by a limited pool...

Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

Science.gov (United States)

González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

2007-11-01

The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

Mitochondrial complex III defects contribute to inefficient respiration and ATP synthesis in the myocardium of Trypanosoma cruzi-infected mice.

Science.gov (United States)

Wen, Jian-Jun; Garg, Nisha Jain

2010-01-01

In this study, we conducted a thorough analysis of mitochondrial bioenergetic function as well as the biochemical and molecular factors that are deregulated and contribute to compromised adenosine triphosphate (ATP) production in the myocardium during Trypanosoma cruzi infection. We show that ADP-stimulated state 3 respiration and ATP synthesis supported by pyruvate/malate (provides electrons to complex I) and succinate (provides electrons to complex II) substrates were significantly decreased in left ventricular tissue and isolated cardiac mitochondria of infected mice. The decreased mitochondrial ATP synthesis in infected murine hearts was not a result of uncoupling between the electron-transport chain and oxidative phosphorylation and decreased availability of the intermediary metabolites (e.g., NADH). The observed decline in the activities of complex-I, -IV, and -V was not physiologically relevant and did not contribute to compromised respiration and ATP synthesis in infected myocardium. Instead, complex III activity was decreased above the threshold level and contributed to respiratory-chain inefficiency and the resulting decline in mitochondrial ATP synthesis in infected myocardium. The loss in complex III activity occurred as a consequence of cytochrome b depletion. Treatment of infected mice with phenyl-alpha-tert-butyl nitrone (PBN, antioxidant) was beneficial in preserving the mtDNA-encoded cytochrome b expression, and subsequently resulted in improved complex III activity, mitochondrial respiration, and ATP production in infected myocardium. Overall, we provide novel data on the mechanism(s) involved in cardiac bioenergetic inefficiency during T. cruzi infection.

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

OpenAIRE

Santangelo, G M; Tornow, J

1990-01-01

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

Astrocytes protect neurons against methylmercury via ATP/P2Y(1) receptor-mediated pathways in astrocytes.

Science.gov (United States)

Noguchi, Yusuke; Shinozaki, Youichi; Fujishita, Kayoko; Shibata, Keisuke; Imura, Yoshio; Morizawa, Yosuke; Gachet, Christian; Koizumi, Schuichi

2013-01-01

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

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.

Comparison of effects of ATP-MgCl2 and adenosine-MgCl2 on renal function following ischemia

International Nuclear Information System (INIS)

Sumpio, B.E.; Hull, M.J.; Baue, A.E.; Chaudry, I.H.

1987-01-01

ATO-MgCl 2 administration had been shown to accelerate the recovery of renal function following warm ischemia. However, since the major breakdown product of ATP is adenosine, the relative contribution of ATP vs. adenosine in improving renal function following ischemia remains to be determined. To study this, kidneys were subjected to 45 min of normothermic ischemia and then perfused at 100 mmHg with oxygenated Krebs-HCO 3 buffer containing albumin, [ 3 H]inulin, substrates, and either 0.3 mM ATP-MgCl 2 or adenosine-MgCl 2 for 110 min. Perfusate and timed urine samples were collected and analyzed for radioactivity and [Na + ]. The functional parameters indicated that although adenosine-MgCl 2 treatment provided a transient improvement, it failed to provided a sustained improvement in renal function or attain control valued compared with ATP-MgCl 2 treatment. Thus, the salutary effects of ATP-MgCl 2 following warm ischemia in the kidney are not mediated by adenosine

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

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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.

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

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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

The Role of ATP in Sleep Regulation

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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.

Altered expression profile of glycolytic enzymes during testicular ischemia reperfusion injury is associated with the p53/TIGAR pathway: effect of fructose 1,6-diphosphate

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May Al-Maghrebi

2016-07-01

Full Text Available Background. Testicular ischemia reperfusion injury (tIRI is considered the mechanism underlying the pathology of testicular torsion and detorsion. Left untreated, tIRI can induce testis dysfunction, damage to spermatogenesis and possible infertility. In this study, we aimed to assess the activities and expression of glycolytic enzymes (GEs in the testis and their possible modulation during tIRI. The effect of fructose 1,6-diphosphate (FDP, a glycolytic intermediate, on tIRI was also investigated. Methods. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + FDP (2 mg/kg. tIRI was induced by occlusion of the testicular artery for 1 h followed by 4 h of reperfusion. FDP was injected peritoneally 30 min prior to reperfusion. Histological and biochemical analyses were used to assess damage to spermatogenesis, activities of major GEs, and energy and oxidative stress markers. The relative mRNA expression of GEs was evaluated by real-time PCR. ELISA and immunohistochemistry were used to evaluate the expression of p53 and TP53-induced glycolysis and apoptosis regulator (TIGAR. Results. Histological analysis revealed tIRI-induced spermatogenic damage as represented by a significant decrease in the Johnsen biopsy score. In addition, tIRI reduced the activities of hexokinase 1, phosphofructokinase-1, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase C. However, mRNA expression downregulation was detected only for hexokinase 1, phosphoglycerate kinase 2, and lactate dehydrogenase C. ATP and NADPH depletion was also induced by tIRI and was accompanied by an increased Malondialdehyde concentration, reduced glutathione level, and reduced superoxide dismutase and catalase enzyme activities. The immunoexpression of p53 and TIGAR was markedly increased after tIRI. The above tIRI-induced alterations were attenuated by FDP treatment. Discussion. Our findings indicate that tIRI-induced spermatogenic damage is

Modification of synthesis nucleotides [γ-32P] ATP

International Nuclear Information System (INIS)

Wira Y Rahman; Endang Sarmini; Herlina; Triyanto; Hambali; Abdul Mutalib; Santi Nurbaiti

2013-01-01

In molecular biology, radionuclides in the form of radiolabeled compounds have been widely used as deoxyribonucleic acid (DNA) / ribonucleic acid (RNA) tracer in order to explore a wide range of physiological and pathological processes. One of such compounds is [γ- 32 P]-adenosine triphosphate {[γ- 32 P]-ATP} [γ- 32 P]-ATP which has been widely used in the biotechnology research. In order to support the biotechnology research in Indonesia in this project, [γ- 32 P]- ATP had been synthesized by enzymatic reactions with modifying the method of synthesis using the precursor DL-glyceraldehyde 3-phosphate, nucleotides Adenosine Diphosphate (ADP) and H 3 32 PO 4 and enzymes glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoroglyceric phosphokinase and lactate dehydrogenase. The purification of the synthesized [γ- 32 P]-ATP, by using DEAE Sephadex column chromatography. The synthesis and purification process that had been performed were able in producing of [γ- 32 P]-ATP with radioactivity of 1,175 mCi and radiochemical purity of 99,49%.. Having successfully prepared the [γ- 32 P]-ATP and application, in the near future the Radioisotopes and Radiopharmaceuticals Centre is expected to be able in providing the above-mentioned radiolabeled nucleotide for biotechnology research in Indonesia. (author)

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....

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.

Kidney outer medulla mitochondria are more efficient compared to cortex mitochondria as a strategy to sustain ATP production in a suboptimal environment.

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Schiffer, Tomas A; Gustafsson, Håkan; Palm, Fredrik

2018-05-30

The kidneys receive approximately 25% of cardiac output, which is a prerequisite in order to maintain sufficient glomerular filtration rate. However, both intrarenal regional renal blood flow and tissue oxygen levels are heterogeneous with decreasing levels in the inner part of the medulla. These differences in combination with the heterogeneous metabolic activity of the different nephron segment located in the different parts of the kidney may constitute a functional problem when challenged. The proximal tubule and the medullary thick ascending limb of Henle are considered to have the highest metabolic rate, which is relating to the high mitochondria content needed to sustain sufficient ATP production from oxidative phosphorylation in order to support high electrolyte transport activity in these nephron segments. Interestingly, the cells located in kidney medulla functions at the verge of hypoxia and the mitochondria may have adapted to the surrounding environment. However, little is known about intrarenal differences in mitochondria function. We therefore investigated functional differences between mitochondria isolated from kidney cortex and medulla of healthy normoglycemic rats were estimated using high-resolution respirometry. The results demonstrate that medullary mitochondria had a higher degree of coupling, are more efficient and have higher oxygen affinity, which would make them more suitable to function in an environment with limited oxygen supply. Furthermore, these results support the hypothesis that mitochondria of medullary cells have adapted to the normal hypoxic in vivo situation as a strategy of sustaining ATP production in a suboptimal environment.

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...

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites.

Science.gov (United States)

Santangelo, G M; Tornow, J

1990-01-01

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity. Images PMID:2405258

Formation of hydrotalcite in aqueous solutions and intercalation of ATP by anion exchange.

Science.gov (United States)

Tamura, Hiroki; Chiba, Jun; Ito, Masahiro; Takeda, Takashi; Kikkawa, Shinichi; Mawatari, Yasuteru; Tabata, Masayoshi

2006-08-15

The formation reaction and the intercalation of adenosine triphosphate (ATP) were studied for hydrotalcite (HT), a layered double hydroxide (LDH) of magnesium and aluminum. Hydrotalcite with nitrate ions in the interlayer (HT-NO(3)) was formed (A) by dropwise addition of a solution of magnesium and aluminum nitrates (pH ca. 3) to a sodium hydroxide solution (pH ca. 14) until the pH decreased from 14 to 10 and (B) by dropwise addition of the NaOH solution to the solution of magnesium and aluminum nitrates with pH increasing from 3 to 10. The precipitate obtained with method B was contaminated with aluminum hydroxide and the crystallinity of the product was low, possibly because aluminum hydroxide precipitates at pH 4 or 5 and remains even after HT-NO(3) forms at pH above 8. With method A, however, the precipitate was pure HT-NO(3) with increased crystallinity, since the solubility of aluminum hydroxide at pH above and around 10 is high as dissolved aluminate anions are stable in this high pH region, and there was no aluminum hydroxide contamination. The formed HT-NO(3) had a composition of [Mg(0.71)Al(0.29)(OH)(2)](NO(3))(0.29).0.58H(2)O. To intercalate ATP anions into the HT-NO(3), HT-NO(3) was dispersed in an ATP solution at pH 7. It was found that the interlayer nitrate ions were completely exchanged with ATP anions by ion exchange, and the interlayer distance expanded almost twice with a free space distance of 1.2 nm. The composition of HT-ATP was established as [Mg(0.68)Al(0.32)(OH)(2)](ATP)(0.080)0.88H(2)O. The increased distance could be explained with a calculated molecular configuration of the ATP as follows: An ATP molecule is bound to an interlayer surface with the triphosphate group, the adenosine group bends owing to its bond angles and projects into the interlayer to a height of 1 nm, and the adenosine groups aligned in the interlayer support the interlayer distance.

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

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Lushchak V.I.

1998-01-01

Full Text Available The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the (V600E)BRAF oncogene

DEFF Research Database (Denmark)

Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS......). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes...

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the V600EBRAF oncogene

DEFF Research Database (Denmark)

Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to V600EBRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS). Notably......, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that V600EBRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes an addiction to V...

Competitive cation binding computations of proton balance for reactions of the phosphagen and glycolytic energy systems within skeletal muscle

Science.gov (United States)

2017-01-01

Limited research and data has been published for the H+ coefficients for the metabolites and reactions involved in non-mitochondrial energy metabolism. The purpose of this investigation was to compute the fractional binding of H+, K+, Na+ and Mg2+ to 21 metabolites of skeletal muscle non-mitochondrial energy metabolism, resulting in 104 different metabolite-cation complexes. Fractional binding of H+ to these metabolite-cation complexes were applied to 17 reactions of skeletal muscle non-mitochondrial energy metabolism, and 8 conditions of the glycolytic pathway based on the source of substrate (glycogen vs. glucose), completeness of glycolytic flux, and the end-point of pyruvate vs. lactate. For pH conditions of 6.0 and 7.0, respectively, H+ coefficients (-‘ve values = H+ release) for the creatine kinase, adenylate kinase, AMP deaminase and ATPase reactions were 0.8 and 0.97, -0.13 and -0.02, 1.2 and 1.09, and -0.01 and -0.66, respectively. The glycolytic pathway is net H+ releasing, regardless of lactate production, which consumes 1 H+. For glycolysis fueled by glycogen and ending in either pyruvate or lactate, H+ coefficients for pH 6.0 and 7.0 were -3.97 and -2.01 (pyruvate), and -1.96 and -0.01 (lactate), respectively. When starting with glucose, the same conditions result in H+ coefficients of -3.98 and -2.67, and -1.97 and –0.67, respectively. The most H+ releasing reaction of glycolysis is the glyceraldehyde-3-phosphate dehydrogenase reaction, with H+ coefficients for pH 6.0 and 7.0 of -1.58 and -0.76, respectively. Incomplete flux of substrate through glycolysis would increase net H+ release due to the absence of the pyruvate kinase and lactate dehydrogenase reactions, which collectively result in H+ coefficients for pH 6.0 and 7.0 of 1.35 and 1.88, respectively. The data presented provide an extensive reference source for academics and researchers to accurately profile the balance of protons for all metabolites and reactions of non-mitochondrial energy

Competitive cation binding computations of proton balance for reactions of the phosphagen and glycolytic energy systems within skeletal muscle.

Science.gov (United States)

Robergs, Robert Andrew

2017-01-01

Limited research and data has been published for the H+ coefficients for the metabolites and reactions involved in non-mitochondrial energy metabolism. The purpose of this investigation was to compute the fractional binding of H+, K+, Na+ and Mg2+ to 21 metabolites of skeletal muscle non-mitochondrial energy metabolism, resulting in 104 different metabolite-cation complexes. Fractional binding of H+ to these metabolite-cation complexes were applied to 17 reactions of skeletal muscle non-mitochondrial energy metabolism, and 8 conditions of the glycolytic pathway based on the source of substrate (glycogen vs. glucose), completeness of glycolytic flux, and the end-point of pyruvate vs. lactate. For pH conditions of 6.0 and 7.0, respectively, H+ coefficients (-'ve values = H+ release) for the creatine kinase, adenylate kinase, AMP deaminase and ATPase reactions were 0.8 and 0.97, -0.13 and -0.02, 1.2 and 1.09, and -0.01 and -0.66, respectively. The glycolytic pathway is net H+ releasing, regardless of lactate production, which consumes 1 H+. For glycolysis fueled by glycogen and ending in either pyruvate or lactate, H+ coefficients for pH 6.0 and 7.0 were -3.97 and -2.01 (pyruvate), and -1.96 and -0.01 (lactate), respectively. When starting with glucose, the same conditions result in H+ coefficients of -3.98 and -2.67, and -1.97 and -0.67, respectively. The most H+ releasing reaction of glycolysis is the glyceraldehyde-3-phosphate dehydrogenase reaction, with H+ coefficients for pH 6.0 and 7.0 of -1.58 and -0.76, respectively. Incomplete flux of substrate through glycolysis would increase net H+ release due to the absence of the pyruvate kinase and lactate dehydrogenase reactions, which collectively result in H+ coefficients for pH 6.0 and 7.0 of 1.35 and 1.88, respectively. The data presented provide an extensive reference source for academics and researchers to accurately profile the balance of protons for all metabolites and reactions of non-mitochondrial energy

Competitive cation binding computations of proton balance for reactions of the phosphagen and glycolytic energy systems within skeletal muscle.

Directory of Open Access Journals (Sweden)

Robert Andrew Robergs

Full Text Available Limited research and data has been published for the H+ coefficients for the metabolites and reactions involved in non-mitochondrial energy metabolism. The purpose of this investigation was to compute the fractional binding of H+, K+, Na+ and Mg2+ to 21 metabolites of skeletal muscle non-mitochondrial energy metabolism, resulting in 104 different metabolite-cation complexes. Fractional binding of H+ to these metabolite-cation complexes were applied to 17 reactions of skeletal muscle non-mitochondrial energy metabolism, and 8 conditions of the glycolytic pathway based on the source of substrate (glycogen vs. glucose, completeness of glycolytic flux, and the end-point of pyruvate vs. lactate. For pH conditions of 6.0 and 7.0, respectively, H+ coefficients (-'ve values = H+ release for the creatine kinase, adenylate kinase, AMP deaminase and ATPase reactions were 0.8 and 0.97, -0.13 and -0.02, 1.2 and 1.09, and -0.01 and -0.66, respectively. The glycolytic pathway is net H+ releasing, regardless of lactate production, which consumes 1 H+. For glycolysis fueled by glycogen and ending in either pyruvate or lactate, H+ coefficients for pH 6.0 and 7.0 were -3.97 and -2.01 (pyruvate, and -1.96 and -0.01 (lactate, respectively. When starting with glucose, the same conditions result in H+ coefficients of -3.98 and -2.67, and -1.97 and -0.67, respectively. The most H+ releasing reaction of glycolysis is the glyceraldehyde-3-phosphate dehydrogenase reaction, with H+ coefficients for pH 6.0 and 7.0 of -1.58 and -0.76, respectively. Incomplete flux of substrate through glycolysis would increase net H+ release due to the absence of the pyruvate kinase and lactate dehydrogenase reactions, which collectively result in H+ coefficients for pH 6.0 and 7.0 of 1.35 and 1.88, respectively. The data presented provide an extensive reference source for academics and researchers to accurately profile the balance of protons for all metabolites and reactions of non

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

Carbohydrate metabolism of Xylella fastidiosa: Detection of glycolytic and pentose phosphate pathway enzymes and cloning and expression of the enolase gene

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Facincani Agda Paula

2003-01-01

Full Text Available The objective of this work was to assess the functionality of the glycolytic pathways in the bacterium Xylella fastidiosa. To this effect, the enzymes phosphoglucose isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase of the glycolytic pathway, and glucose 6-phosphate dehydrogenase of the Entner-Doudoroff pathway were studied, followed by cloning and expression studies of the enolase gene and determination of its activity. These studies showed that X. fastidiosa does not use the glycolytic pathway to metabolize carbohydrates, which explains the increased duplication time of this phytopatogen. Recombinant enolase was expressed as inclusion bodies and solubilized with urea (most efficient extractor, Triton X-100, and TCA. Enolase extracted from X. fastidiosa and from chicken muscle and liver is irreversibly inactivated by urea. The purification of enolase was partial and resulted in a low yield. No enzymatic activity was detected for either recombinant and native enolases, aldolase, and glyceraldehyde-3-phosphate dehydrogenase, suggesting that X. fastidiosa uses the Entner-Doudoroff pathway to produce pyruvate. Evidence is presented supporting the idea that the regulation of genes and the presence of isoforms with regulation patterns might make it difficult to understand the metabolism of carbohydrates in X. fastidiosa.

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

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

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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

Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

International Nuclear Information System (INIS)

Lodato, D.T.; Reed, G.H.

1987-01-01

The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EOR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17 O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the γ-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from 17 O regiospecifically incorporated into the γ-phosphate group of ATP. By contrast, 17 O in the α-phosphate or in the β-phosphate groups of ATP does not influence the spectrum. Experiments in 17 O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme-Mn(II)-oxalate-Mg(II)-ATP complex suggests a scheme for the normal reverse reaction of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the γ-phosphate of ATP

Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Poulsen, Allan K.; Andersen, Ann Zahle; Brasen, Jens Christian

2008-01-01

, while mitochondrial membrane potential was measured using the fluorescent dye DiOC(2)(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP...

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

Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

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Praveen Balabaskaran Nina

2010-07-01

Full Text Available 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 highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a

Effect of 808 nm Diode Laser on Swimming Behavior, Food Vacuole Formation and Endogenous ATP Production of Paramecium primaurelia (Protozoa).

Science.gov (United States)

Amaroli, Andrea; Ravera, Silvia; Parker, Steven; Panfoli, Isabella; Benedicenti, Alberico; Benedicenti, Stefano

2015-01-01

Photobiomodulation (PBM) has been used in clinical practice for more than 40 years. To clarify the mechanisms of action of PBM at cellular and organism levels, we investigated its effect on Paramecium primaurelia (Protozoa) irradiated by an 808 nm infrared diode laser with a flat-top handpiece (1 W in CW). Our results led to the conclusion that: (1) the 808 nm laser stimulates the P. primaurelia without a thermal effect, (2) the laser effect is demonstrated by an increase in swimming speed and in food vacuole formation, (3) the laser treatment affects endogenous adenosine triphosphate (ATP) production in a positive way, (4) the effects of irradiation dose suggest an optimum exposure time of 50 s (64 J cm(-2) of fluence) to stimulate the Paramecium cells; irradiation of 25 s shows no effect or only mild effects and irradiation up to 100 s does not increase the effect observed with 50 s of treatment, (5) the increment of endogenous ATP concentration highlights the positive photobiomodulating effect of the 808 nm laser and the optimal irradiation conditions by the flat-top handpiece. © 2015 The American Society of Photobiology.

Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

Science.gov (United States)

Schühle, Karola

2012-01-01

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

Determination of ATP as a fluorescence probe with europium(III)-doxycycline.

Science.gov (United States)

Hou, Faju; Wang, Xiaolei; Jiang, Chongqiu

2005-03-01

A new spectrofluorimetric method has been developed for the determination of adenosine disodium triphosphate (ATP). We studied the interactions between the doxycycline (DC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-visible absorption and fluorescence spectra. Using doxycycline (DC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP could remarkably enhance the fluorescence intensity of the DC-Eu3+ complex at lambda = 612 nm. The enhanced fluorescence intensity of the Eu3+ ion was in proportion to the concentration of ATP. The optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP were 1.00 x 10(-7) - 2.00 x 10(-6) mol L(-1) with detection limits of 4.07 x 10(-8) mol L(-1). This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to the determination of ATP in samples. The mechanism of fluorescence enhancement between the doxycycline (DC)-Eu3+ complex and ATP was also studied.

Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks

Directory of Open Access Journals (Sweden)

Daniel eGoldman

2012-07-01

Full Text Available Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100ms that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2. The model further predicts how insulin, at concentrations found in prediabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

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

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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.

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

The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

DEFF Research Database (Denmark)

Minet, Ariane D; Gaster, Michael

2011-01-01

compared to lean control. The ATP synthesis rate without ATP consumption was not different between groups and there were no significant gender differences. The mitochondrial dysfunction in type 2 diabetes in vivo is partly based on a primarily impaired ATP synthesis....... or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological...... selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects...

Heat production of pig platelets in relation with glycolysis and respiration.

Science.gov (United States)

Nanri, H; Minakami, S

1983-01-01

Heat production of pig platelets was measured on a flow microcalorimeter with simultaneous measurements of the oxygen consumption and metabolite change of the suspension. The heat production associated with the glycolytic reaction was estimated from the decrease in the heat production caused by the addition of sodium fluoride. The glycolytic heat production was about -75 kJ per mol of lactate formed, which is the sum of the enthalpy values for the conversion of glucose to lactic acid and that for the neutralization of the acid. The heat production due to the respiration was estimated from the heat production of the cells at various pH. The respiratory heat production was about -475 kJ per mol oxygen consumed, which agrees with the enthalpy change for the non-phosphorylating respiration of mitochondria or for the complete oxidation of glucose or fatty acids. The heat production of the cells increased by the alkalinization of the medium, and the increase of the heat production was parallel with the increase in the lactate formation.

Quantal release of ATP from clusters of PC12 cells.

Science.gov (United States)

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

2004-10-15

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

Effects of Irradiation on bacterial atp luminous intensity of cooled pork and chicken

International Nuclear Information System (INIS)

Ju Hua

2010-01-01

The effect of irradiation on cooled pork and chicken was detected with ATP luminous intensity method. The influences of other factors to ATP luminous intensity were also discussed. There was positive correlation between ATP standard concentration and ATP luminous intensity, and negative correlation between irradiation dosage and ATP luminous intensity. The trend of ATP luminous intensity of cooled pork and chicken after irradiation was inverse S, and the maximum ATP luminous intensity appeared at 6.0 kGy, and minimum at 4.0 and 8.0 kGy. Sterilized water and sterilized pork had no interference to ATP luminous intensity of the samples. There was significant positive correlation between E. coli 10003 concentration and ATP luminous intensity, the coefficient correlation was 0.9437. (authors)

Interaction between nucleotide binding sites on chloroplast coupling factor 1 during ATP hydrolysis

Energy Technology Data Exchange (ETDEWEB)

Leckband, D.; Hammes, G.G.

1987-04-21

The initial hydrolysis of radioactively-labelled CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rate constant for the first-order process is independent of substrate concentration but increased hyperbolically to a limiting value of 0.43 s/sup -1/ with increasing concentrations of free Ca/sup 2 +/. A mechanism involving a Ca/sup 2 +/-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approx. 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s/sup -1/ as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changes. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.

Glycogen Shunt Activity and Glycolytic Supercompensation in Astrocytes May Be Distinctly Mediated via the Muscle Form of Glycogen Phosphorylase

DEFF Research Database (Denmark)

Jakobsen, Emil; Bak, Lasse K; Walls, Anne B

2017-01-01

Glycogen is the main storage form of glucose in the brain. In contrast with previous beliefs, brain glycogen has recently been shown to play important roles in several brain functions. A fraction of metabolized glucose molecules are being shunted through glycogen before reentering the glycolytic ...

Metabolomic Analysis of Differential Changes in Metabolites during ATP Oscillations in Chondrogenesis

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Hyuck Joon Kwon

2013-01-01

Full Text Available Prechondrogenic condensation is a critical step for skeletal pattern formation. Recent studies reported that ATP oscillations play an essential role in prechondrogenic condensation. However, the molecular mechanism to underlie ATP oscillations remains poorly understood. In the present study, it was investigated how changes in metabolites are implicated in ATP oscillations during chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS. CE-TOF-MS detected 93 cationic and 109 anionic compounds derived from known metabolic pathways. 15 cationic and 18 anionic compounds revealed significant change between peak and trough of ATP oscillations. These results implicate that glycolysis, mitochondrial respiration and uronic acid pathway oscillate in phase with ATP oscillations, while PPRP and nucleotides synthesis pathways oscillate in antiphase with ATP oscillations. This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate in antiphase with the ATP-consuming PPRP/nucleotide synthesis pathway during chondrogenesis.

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.

ATP economy of force maintenance in human tibialis anterior muscle

DEFF Research Database (Denmark)

Nakagawa, Yoshinao; Ratkevicius, Aivaras; Mizuno, Masao

2005-01-01

PURPOSE: The aim of this study was investigate ATP economy of force maintenance in the human tibialis anterior muscle during 60 s of anaerobic voluntary contraction at 50% of maximum voluntary contraction (MVC). METHODS: ATP turnover rate was evaluated using P magnetic resonance spectroscopy (P...... contraction. It averaged at 4.81 +/- 0.42 N.s.micromol-1, and correlated with the relative cross-sectional area of the muscle occupied by Type I fiber (r = 0.73, P contraction, subjects dropping in force showed lower ATP economy compared with those maintaining the force (3.......7 +/- 0.6 vs 5.3 +/- 0.6 N.s.micromol-1; P contraction could be due to an increase in the ATP economy of contracting muscle fibers offsetting the effects of increased temperature and low ATP economy...

Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

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Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

2018-02-01

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential role of the glycolytic oscillator in acute hypoxia in tumors

International Nuclear Information System (INIS)

Fru, Leonard Che; Adamson, Erin B; Campos, David D; Fain, Sean B; Song, Chihwa; Kissick, Michael W; Jacques, Steven L; Van der Kogel, Albert J; Nickel, Kwang P; Kimple, Randall J

2015-01-01

Tumor acute hypoxia has a dynamic component that is also, at least partially, coherent. Using blood oxygen level dependent magnetic resonance imaging, we observed coherent oscillations in hemoglobin saturation dynamics in cell line xenograft models of head and neck squamous cell carcinoma. We posit a well-established biochemical nonlinear oscillatory mechanism called the glycolytic oscillator as a potential cause of the coherent oscillations in tumors. These data suggest that metabolic changes within individual tumor cells may affect the local tumor microenvironment including oxygen availability and therefore radiosensitivity. These individual cells can synchronize the oscillations in patches of similar intermediate glucose levels. These alterations have potentially important implications for radiation therapy and are a potential target for optimizing the cancer response to radiation. (paper)

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

Adenosine Triphosphate (ATP Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway

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Zhengchang Liu

2013-03-01

Full Text Available Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.

CFTR mediates noradrenaline-induced ATP efflux from DRG neurons.

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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.

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...

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...

A genetic polymorphism in the sex-linked ATP5A1 gene is associated with individual fitness in Ovenbirds (Seiurus aurocapilla)

Science.gov (United States)

Judith D. Toms; Lori S. Eggert; Wayne J. Arendt; John Faaborg

2012-01-01

While testing genetic sexing techniques in Ovenbirds (Seiurus aurocapilla),we found a genetic polymorphism in the ATP5A1 gene in 38% of individuals. The Z ' allele included changes in both intronic and exonic portions of the sequenced region, but there was no evidence that this changed the resulting ATP synthase product. Males that had one or more copies of...

Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

International Nuclear Information System (INIS)

Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

1986-01-01

The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes 14 C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg 2+ . Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-γ-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated

Sorbitol Can Fuel Mouse Sperm Motility and Protein Tyrosine Phosphorylation via Sorbitol Dehydrogenase1

OpenAIRE

Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa A.; Gerton, George L.

2009-01-01

Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and t...

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.

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-

A new function for ATP: activating cardiac sympathetic afferents during myocardial ischemia.

Science.gov (United States)

Fu, Liang-Wu; Longhurst, John C

2010-12-01

Myocardial ischemia activates cardiac sympathetic afferents leading to chest pain and reflex cardiovascular responses. Brief myocardial ischemia leads to ATP release in the interstitial space. Furthermore, exogenous ATP and α,β-methylene ATP (α,β-meATP), a P2X receptor agonist, stimulate cutaneous group III and IV sensory nerve fibers. The present study tested the hypothesis that endogenous ATP excites cardiac afferents during ischemia through activation of P2 receptors. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicates (T(2)-T(5)) in anesthetized cats. Single fields of 45 afferents (conduction velocities = 0.25-4.92 m/s) were identified in the left ventricle with a stimulating electrode. Five minutes of myocardial ischemia stimulated 39 of 45 cardiac afferents (8 Aδ, 37 C fibers). Epicardial application of ATP (1-4 μmol) stimulated six ischemically sensitive cardiac afferents in a dose-dependent manner. Additionally, epicardial ATP (2 μmol), ADP (2 μmol), a P2Y agonist, and α,β-meATP (0.5 μmol) significantly activated eight other ischemically sensitive afferents. Third, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist, abolished the responses of six afferents to epicardial ATP (2 μmol) and attenuated the ischemia-related increase in activity of seven other afferents by 37%. In the absence of P2 receptor blockade, cardiac afferents responded consistently to repeated application of ATP (n = 6) and to recurrent myocardial ischemia (n = 6). Finally, six ischemia-insensitive cardiac spinal afferents did not respond to epicardial ATP (2-4 μmol), although these afferents did respond to epicardial bradykinin. Taken together, these data indicate that, during ischemia, endogenously released ATP activates ischemia-sensitive, but not ischemia-insensitive, cardiac spinal afferents through stimulation of P2 receptors likely located on the cardiac sensory

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

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Neal J Dawson

Full Text Available One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05. A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively. Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Characterization of fructose-1,6-bisphosphate aldolase during anoxia in the tolerant turtle, Trachemys scripta elegans: an assessment of enzyme activity, expression and structure.

Science.gov (United States)

Dawson, Neal J; Biggar, Kyle K; Storey, Kenneth B

2013-01-01

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

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

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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.

Melatonin antiproliferative effects require active mitochondrial function in embryonal carcinoma cells

Science.gov (United States)

Loureiro, Rute; Magalhães-Novais, Silvia; Mesquita, Katia A.; Baldeiras, Ines; Sousa, Isabel S.; Tavares, Ludgero C.; Barbosa, Ines A.; Oliveira, Paulo J.; Vega-Naredo, Ignacio

2015-01-01

Although melatonin oncostatic and cytotoxic effects have been described in different types of cancer cells, the specific mechanisms leading to its antitumoral effects and their metabolic context specificity are still not completely understood. Here, we evaluated the effects of melatonin in P19 embryonal carcinoma stem cells (CSCs) and in their differentiated counterparts, cultured in either high glucose medium or in a galactose (glucose-free) medium which leads to glycolytic suppression and increased mitochondrial metabolism. We found that highly glycolytic P19 CSCs were less susceptible to melatonin antitumoral effects while cell populations relying on oxidative metabolism for ATP production were more affected. The observed antiproliferative action of melatonin was associated with an arrest at S-phase, decreased oxygen consumption, down-regulation of BCL-2 expression and an increase in oxidative stress culminating with caspase-3-independent cell death. Interestingly, the combined treatment of melatonin and dichloroacetate had a synergistic effect in cells grown in the galactose medium and resulted in an inhibitory effect in the highly resistant P19 CSCs. Melatonin appears to exert its antiproliferative activity in P19 carcinoma cells through a mitochondrially-mediated action which in turn allows the amplification of the effects of dichloroacetate, even in cells with a more glycolytic phenotype. PMID:26025920

Synthesis and purification of [γP32]-ATP

International Nuclear Information System (INIS)

Kukuh, Ratnawati; Santoso, Daniel; Basri, T. Hasan; Natalia Adventini

1995-01-01

The synthesis of [γP 3 2]-ATP has been carried out using an enzymes procedure. The compound was formed by the phosphorylation of ADP during the enzymatic conversion of L-α-glycerol-phosphate to 3-phosphoglycerate. In the present study, lactatedehydrogenase and sodium pyruvat were used in order to maintain β-NAD + concentration and to push the reaction of glyceralaldehyde-3-phosphate dehydrogenase towards the formation of 1,3-diphosphoglycerate. L-α-glycerolphosphate was used as primary substrate, as it is more stable than DL-glyceraldehyde-3-phosphate. The enzymatic reaction was stopped by immersing the reaction vessel in boiling water for about 10 minutes. The labelled [γP 3 2]-ATP formed was separated by thin layer chromatography using PEI-cellulose and the spots of [γP 3 2]-ATP and inorganic P 3 2 residue located by autoradiography using X-ray film. The optimum time for the reaction at room temperature was 90 minutes with a labeling efficiency of 94.9 %. Purification of the [γP 3 2]-ATP by anion exchange chromatography using DEAE sephadex yielded a purity of more than 95%. The results showed that the labeled compound [γP 3 2]-ATP can be synthesized via an enzymatic process with a satisfactory yield. (author), 4 refs, 2 tabs, 2 figs

New control method of on-board ATP system of Shinkansen trains

Energy Technology Data Exchange (ETDEWEB)

Fukuda, N.; Watanabe, T. [Railway Technical Research Inst. (Japan)

2000-07-01

We studied a new control method of the on-board automatic train protection (ATP) system for Shinkansen trains to shorten the operation time and not to degrade ride comfort at changes in deceleration of the train, while maintaining the safety and reliability of the present ATP signal system. We propose a new on-board pattern brake control system based on the present ATP data without changing the wayside equipment. By simulating the ATP braking of the proposed control method, we succeeded in shortening the operation time by 48 seconds per one station in comparison with the present ATP brake control system. This paper reports the concept of the system and simulation results of the on-board pattern. (orig.)

ADP-dependent Phosphofructokinases in Mesophilic and Thermophilic Methanogenic Archaea

NARCIS (Netherlands)

Verhees, C.H.; Tuininga, J.E.; Kengen, S.W.M.; Stams, A.J.M.; Oost, van der J.; Vos, de W.M.

2001-01-01

Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PPi-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus

L-sorbose but not D-tagatose induces hemolysis of dog erythrocytes in vitro

NARCIS (Netherlands)

Bär, A.; Leeman, W.R.

1999-01-01

Previous investigations have demonstrated that L-sorbose induces hemolysis of dog erythrocytes. This effect is probably the consequence of an ATP depletion of the red blood cells subsequent to inhibition of hexokinase, and thus the glycolytic pathway, by sorbose 1-phosphate. In the present study,

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.

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.

Application of luciferase assay for ATP to antimicrobial drug susceptibility

Science.gov (United States)

Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

1977-01-01

The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

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

Science.gov (United States)

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.

Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

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Martinez Alfredo

2008-05-01

Full Text Available Abstract Background A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14 derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. It is suggested that this behavior might be due to lineage differences between E. coli W and C. Results This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. Conclusion Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates.

atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

Science.gov (United States)

2013-01-01

Background The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. Results Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. Conclusions The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples. PMID:24299240

Deformation-induced release of ATP from erythrocytes in a poly(dimethylsiloxane)-based microchip with channels that mimic resistance vessels.

Science.gov (United States)

Price, Alexander K; Fischer, David J; Martin, R Scott; Spence, Dana M

2004-08-15

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 micron (2.22 +/- 0.50 microM ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 +/- 0.15 microM) vs a microchip (2.59 +/- 0.32 microM) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

ATP Synthase, a Target for Dementia and Aging?

Science.gov (United States)

Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

2018-02-01

Advancing age is the biggest risk factor for development for the major life-threatening diseases in industrialized nations accounting for >90% of deaths. Alzheimer's dementia (AD) is among the most devastating. Currently approved therapies fail to slow progression of the disease, providing only modest improvements in memory. Recently reported work describes mechanistic studies of J147, a promising therapeutic molecule previously shown to rescue the severe cognitive deficits exhibited by aged, transgenic AD mice. Apparently, J147 targets the mitochondrial alpha-F1-ATP synthase (ATP5A). Modest inhibition of the ATP synthase modulates intracellular calcium to activate AMP-activated protein kinase to inhibit mammalian target of rapamycin, a known mechanism of lifespan extension from worms to mammals.

Preparation and configurational analysis of the stereoisomers of β,γ-bidentate Rh(H2O)4ATP and α,β,γ-tridentate Rh(H2O)3ATP. A new class of enzyme active site probes

International Nuclear Information System (INIS)

Lu, Z.; Shorter, A.L.; Lin, I.; Dunaway-Mariano, D.

1988-01-01

Exchange-inert Co(III) and Cr(III) complexes of polyphosphates have proved to be useful probes of the structural and biochemical properties of naturally occurring Mg II (polyphosphate) complexes. However, applications of these complexes are not without limitations. The Cr III (polyphosphate) probes or their enzymatic products cannot be used in NMR methods because of the paramagnetic nature of the Cr(III) metal. The redox properties of the metal in the Co III (polyphosphate) complexes require that they also be coordinated to a nitrogen-containing ligand. This requirement is not always convenient. This work reported herein was undertaken to create a new class of exchange-inert metal polyphosphate complexes that contain a metal that is both diamagnetic and redox stable. The preparation, properties, and configurational analysis of the stereoisomers of β, γ-bidentate Rh(H 2 O) 4 ATP (ATP = adenosine 5'-triphosphate) and α,β,γ-tridentate Rh(H 2 O) 3 ATP are described. 12 refs., 5 figs

Enhanced Formation of Methylglyoxal-Derived Advanced Glycation End Products in Arabidopsis Under Ammonium Nutrition

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Klaudia Borysiuk

2018-05-01

Full Text Available Nitrate (NO3– and ammonium (NH4+ are prevalent nitrogen (N sources for plants. Although NH4+ should be the preferred form of N from the energetic point of view, ammonium nutrition often exhibits adverse effects on plant physiological functions and induces an important growth-limiting stress referred as ammonium syndrome. The effective incorporation of NH4+ into amino acid structures requires high activity of the mitochondrial tricarboxylic acid cycle and the glycolytic pathway. An unavoidable consequence of glycolytic metabolism is the production of methylglyoxal (MG, which is very toxic and inhibits cell growth in all types of organisms. Here, we aimed to investigate MG metabolism in Arabidopsis thaliana plants grown on NH4+ as a sole N source. We found that changes in activities of glycolytic enzymes enhanced MG production and that markedly elevated MG levels superseded the detoxification capability of the glyoxalase pathway. Consequently, the excessive accumulation of MG was directly involved in the induction of dicarbonyl stress by introducing MG-derived advanced glycation end products (MAGEs to proteins. The severe damage to proteins was not within the repair capacity of proteolytic enzymes. Collectively, our results suggest the impact of MG (mediated by MAGEs formation in proteins in the contribution to NH4+ toxicity symptoms in Arabidopsis.

Enhanced Formation of Methylglyoxal-Derived Advanced Glycation End Products in Arabidopsis Under Ammonium Nutrition

Science.gov (United States)

Borysiuk, Klaudia; Ostaszewska-Bugajska, Monika; Vaultier, Marie-Noëlle; Hasenfratz-Sauder, Marie-Paule; Szal, Bożena

2018-01-01

Nitrate (NO3–) and ammonium (NH4+) are prevalent nitrogen (N) sources for plants. Although NH4+ should be the preferred form of N from the energetic point of view, ammonium nutrition often exhibits adverse effects on plant physiological functions and induces an important growth-limiting stress referred as ammonium syndrome. The effective incorporation of NH4+ into amino acid structures requires high activity of the mitochondrial tricarboxylic acid cycle and the glycolytic pathway. An unavoidable consequence of glycolytic metabolism is the production of methylglyoxal (MG), which is very toxic and inhibits cell growth in all types of organisms. Here, we aimed to investigate MG metabolism in Arabidopsis thaliana plants grown on NH4+ as a sole N source. We found that changes in activities of glycolytic enzymes enhanced MG production and that markedly elevated MG levels superseded the detoxification capability of the glyoxalase pathway. Consequently, the excessive accumulation of MG was directly involved in the induction of dicarbonyl stress by introducing MG-derived advanced glycation end products (MAGEs) to proteins. The severe damage to proteins was not within the repair capacity of proteolytic enzymes. Collectively, our results suggest the impact of MG (mediated by MAGEs formation in proteins) in the contribution to NH4+ toxicity symptoms in Arabidopsis. PMID:29881392

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

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans

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Neal J. Dawson

2013-01-01

Full Text Available Lactate dehydrogenase (LDH; E.C. 1.1.1.27 is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47% Km for L-lactate and a higher Vmax value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a stimulation of endogenously present protein phosphatases decreased the Km of L-lactate of control LDH to anoxic levels, whereas (b stimulation of kinases increased the Km of L-lactate of anoxic LDH to normoxic levels, and (c dot blot analysis shows significantly less serine (78% and threonine (58% phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans.

Science.gov (United States)

Dawson, Neal J; Bell, Ryan A V; Storey, Kenneth B

2013-01-01

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD(+). The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

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

Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth disease.

LENUS (Irish Health Repository)

Pitceathly, Robert D S

2012-09-11

Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

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.

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

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

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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.

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

Science.gov (United States)

Park, Yoojin; Nim-Anussornkul, Duangrat; Vilaivan, Tirayut; Morii, Takashi; Kim, Byeang Hyean

2018-01-15

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct measurement of newly synthesized ATP dissociation kinetics in sarcoplasmic reticulum ATPase

International Nuclear Information System (INIS)

Teruel-Puche, J.; Kurzmack, M.; Inesi, G.

1987-01-01

Incubation of SR vesicles with Ca 2+ and ( 32 P)acetylphosphate, yields steady state levels of ( 32 P)phosphorylated enzyme (ATPase) intermediate and high concentrations of Ca 2+ in the lumen of the vesicles. At this time, addition of ADP (and EGTA to lower the Ca 2+ concentration in the medium outside the vesicles) results in single cycle formation of (γ- 32 P)ATP by transfer of ( 32 P)phosphate from the enzyme intermediate to ADP. The phosphoenzyme decay and ATP formation exhibit a fast component within the first 20 msec following addition of ADP, and a slower component reaching an asymptote in approximately 100 msec. They have now measured by a rapid filtration method the fraction of newly synthesized ATP which is bound to the enzyme, as opposed to the fraction dissociated into the medium. They find that nearly all the ATP formed during the initial burst is still bound to the enzyme within the initial 20 msec of reaction. Dissociation of newly synthesized ATP occurs then with approximately 13 sec -1 rate constant, permitting reequilibration of the system and further formation of ATP. The rate limiting effect of ATP dissociation and other partial reactions on the slow component of single cycle ATP synthesis is evaluated by appropriate kinetic simulations

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.

A comparative study of ATPase subunit 9 (Atp9) gene between ...

African Journals Online (AJOL)

ATPase subunit 9 gene (Atp9) is an important functional gene in mitochondria, and is closely related with energy supply. RNA editing of atp9 gene was associated with male sterility in plants. In this study, the atp9 gene in soybeans was cloned from a soybean cytoplasmic male sterile line NJCMS2A and its maintainer line ...

Use of L-Glutamic Acid in a New Enrichment Broth (R-TATP Broth) for Detecting the Presence or Absence of Molds in Raw Ingredients/Personal Care Product Formulations by Using an ATP Bioluminescence Assay.

Science.gov (United States)

Yang, Youjun; English, Donald J

The present study reports the effects of adding L-glutamic acid to a new enrichment broth designated as R-TATP broth, to promote the growth of slow-growing mold microorganisms such as Aspergillus brasiliensis and Aspergillus oryzae , without interfering in the growth of other types of microorganisms. This L-glutamic acid containing enrichment broth would be particularly valuable in a rapid microbial detection assay such as an adenosine triphosphate (ATP) bioluminescence assay. By using this new enrichment broth, the amount of ATP (represented as relative light unit ratio after normalized with the negative test control) from mold growth was significantly increased by reducing the time of detection of microbial contamination in a raw ingredient or personal care product formulation from an incubation period of 48-18 h. By using L-glutamic acid in this enrichment broth, the lag phase of the mold growth cycle was shortened. In response to various concentrations of L-glutamic acid in R-TATP broth, there was an increased amount of ATP that had been produced by mold metabolism in an ATP bioluminescence assay. By using L-glutamic acid in R-TATP broth in an ATP bioluminescence assay, the presence of mold could be detected in 18 h as well as other types of microorganisms that may or may not be present in a test sample. By detecting the presence or absence of microbial contamination in 18 h, it is superior in comparison to a 48-96 h incubation period by using either a standard or rapid detection method.

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

'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.

ATP storage and uptake by isolated pancreatic zymogen granules

DEFF Research Database (Denmark)

Haanes, Kristian Agmund; Novak, Ivana

2010-01-01

ATP is released from pancreatic acini in response to cholinergic and hormonal stimulation. The same stimuli cause exocytosis of ZG (zymogen granules) and release of digestive enzymes. The aim of the present study was to determine whether ZG stored ATP and to characterize the uptake mechanism for ...

The Glycolytic Versatility of Bacteroides uniformis CECT 7771 and Its Genome Response to Oligo and Polysaccharides

Directory of Open Access Journals (Sweden)

Alfonso Benítez-Páez

2017-08-01

Full Text Available Bacteroides spp. are dominant components of the phylum Bacteroidetes in the gut microbiota and prosper in glycan enriched environments. However, knowledge of the machinery of specific species isolated from humans (like Bacteroides uniformis contributing to the utilization of dietary and endogenous sources of glycans and their byproducts is limited. We have used the cutting-edge nanopore-based technology to sequence the genome of B. uniformis CECT 7771, a human symbiont with a proven pre-clinical efficacy on metabolic and immune dysfunctions in obesity animal models. We have also used massive sequencing approaches to distinguish the genome expression patterns in response to carbon sources of different complexity during growth. At genome-wide level, our analyses globally demonstrate that B. uniformis strains exhibit an expanded glycolytic capability when compared with other Bacteroides species. Moreover, by studying the growth and whole-genome expression of B. uniformis CECT 7771 in response to different carbon sources, we detected a differential growth fitness and expression patterns across the genome depending on the carbon source of the culture media. The dietary fibers used exerted different effects on B. uniformis CECT 7771 activating different molecular pathways and, therefore, allowing the production of different metabolite types with potential impact on gut health. The genome and transcriptome analysis of B. uniformis CECT 7771, in response to different carbon sources, shows its high versatility to utilize both dietary and endogenous glycans along with the production of potentially beneficial end products for both the bacterium and the host, pointing to a mechanistic basis of a mutualistic relationship.

An EMMPRIN–γ-catenin–Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

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Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S.; Enríquez, José A.; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G.

2014-01-01

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. PMID:24994937

An EMMPRIN-γ-catenin-Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions.

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Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S; Enríquez, José A; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G

2014-09-01

Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. © 2014. Published by The Company of Biologists Ltd.

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo.

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Michael Macia

Full Text Available Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA. Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR imaging and 31-phosphorus MR spectroscopy (31P-MRS. Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator. During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz, mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.

Kinetic Characterisation of Phosphofructokinase Purified from Setaria cervi: A Bovine Filarial Parasite

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Bechan Sharma

2011-01-01

Full Text Available Phosphofructokinase (PFK, a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that Km values for F-6-P (1.05 mM and ATP (3 μM were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM, exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP competitively inhibited the filarial enzyme: Ki and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP also inhibited the enzyme competitively with the Ki value equal to 0.8 mM. The Hill coefficient values (>1.5 for F-6-P (at inhibitory concentration of ATP and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase.

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Semkiv, Marta V; Dmytruk, Kostyantyn V; Abbas, Charles A; Sibirny, Andriy A

2014-05-15

The production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity. Intact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production. Overexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.

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

ATP synthesis in the energy metabolism pathway: a new perspective for manipulating CdSe quantum dots biosynthesized in Saccharomyces cerevisiae

Directory of Open Access Journals (Sweden)

Zhang R

2017-05-01

Full Text Available Rong Zhang,1–3 Ming Shao,1–3 Xu Han,1–3 Chuan Wang,3–4 Yong Li,3–4 Bin Hu,3–4 Daiwen Pang,3–4 Zhixiong Xie1–31Hubei Key Laboratory of Cell Homeostasis, 2College of Life Sciences, Wuhan University, 3Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education, 4College of Chemistry and Molecular Science, Wuhan University, Wuhan, People’s Republic of ChinaAbstract: Due to a growing trend in their biomedical application, biosynthesized nanomaterials are of great interest to researchers nowadays with their biocompatible, low-energy consumption, economic, and tunable characteristics. It is important to understand the mechanism of biosynthesis in order to achieve more efficient applications. Since there are only rare studies on the influences of cellular energy levels on biosynthesis, the influence of energy is often overlooked. Through determination of the intracellular ATP concentrations during the biosynthesis process, significant changes were observed. In addition, ATP synthesis deficiency caused great decreases in quantum dots (QDs biosynthesis in the Δatp1, Δatp2, Δatp14, and Δatp17 strains. With inductively coupled plasma-atomic emission spectrometry and atomic absorption spectroscopy analyses, it was found that ATP affected the accumulation of the seleno-precursor and helped with the uptake of Cd and the formation of QDs. We successfully enhanced the fluorescence intensity 1.5 or 2 times through genetic modification to increase ATP or SeAM (the seleno analog of S-adenosylmethionine, the product that would accumulate when ATP is accrued. This work explains the mechanism for the correlation of the cellular energy level and QDs biosynthesis in living cells, demonstrates control of the biosynthesis using this mechanism, and thus provides a new manipulation strategy for the biosynthesis of other nanomaterials to widen their applications. Keywords: ATP, biosynthesis, Saccharomyces cerevisiae, QDs, CdSe

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

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Bele, Tanja; Fabbretti, Elsa

2016-08-01

P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

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Belcik, J Todd; Davidson, Brian P; Xie, Aris; Wu, Melinda D; Yadava, Mrinal; Qi, Yue; Liang, Sherry; Chon, Chae Ryung; Ammi, Azzdine Y; Field, Joshua; Harmann, Leanne; Chilian, William M; Linden, Joel; Lindner, Jonathan R

2017-03-28

Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×10 8 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K ATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of

Synergistic binding of glucose and aluminium ATP to hexokinase from Saccharomyces cerevisiae.

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Woolfitt, A R; Kellett, G L; Hoggett, J G

1988-08-10

The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence. No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM. In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of [L]0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM. The presence of saturating concentrations of AlATP diminished [L]0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer. The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP. The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction.

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,

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

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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.

Spectrographic study of neodymium complexing with ATP and ADP

International Nuclear Information System (INIS)

Svetlova, I.E.; Dobrynina, N.A.; Martynenko, L.N.

1989-01-01

By spectrographic method neodymium complexing with ATP and ADP in aqueous solutions at different pH values has been studied. The composition of the complexes was determined by the method of isomolar series. On the basis of analysis of absorption spectra it has been ascertained that at equimolar ratio of Nd 3+ and ATP absorption band of L278A corresponds to monocomplex, and the band of 4290 A - to biscomplex. For the complexes with ADP the absorption band of 4288 A is referred to bicomplexes. The character of ATP and ADP coordination by Nd 3+ ion is considered. Stability constants of the complexes are calculated

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

Science.gov (United States)

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.

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

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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

[Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

Science.gov (United States)

Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

2015-08-01

Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals.

Inhibition of the Fe(III)-catalyzed dopamine oxidation by ATP and its relevance to oxidative stress in Parkinson's disease.

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Jiang, Dianlu; Shi, Shuyun; Zhang, Lin; Liu, Lin; Ding, Bingrong; Zhao, Bingqing; Yagnik, Gargey; Zhou, Feimeng

2013-09-18

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic cells, which implicates a role of dopamine (DA) in the etiology of PD. A possible DA degradation pathway is the Fe(III)-catalyzed oxidation of DA by oxygen, which produces neuronal toxins as side products. We investigated how ATP, an abundant and ubiquitous molecule in cellular milieu, affects the catalytic oxidation reaction of dopamine. For the first time, a unique, highly stable DA-Fe(III)-ATP ternary complex was formed and characterized in vitro. ATP as a ligand shifts the catecholate-Fe(III) ligand metal charge transfer (LMCT) band to a longer wavelength and the redox potentials of both DA and the Fe(III) center in the ternary complex. Remarkably, the additional ligation by ATP was found to significantly reverse the catalytic effect of the Fe(III) center on the DA oxidation. The reversal is attributed to the full occupation of the Fe(III) coordination sites by ATP and DA, which blocks O2 from accessing the Fe(III) center and its further reaction with DA. The biological relevance of this complex is strongly implicated by the identification of the ternary complex in the substantia nigra of rat brain and its attenuation of cytotoxicity of the Fe(III)-DA complex. Since ATP deficiency accompanies PD and neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) induced PD, deficiency of ATP and the resultant impairment toward the inhibition of the Fe(III)-catalyzed DA oxidation may contribute to the pathogenesis of PD. Our finding provides new insight into the pathways of DA oxidation and its relationship with synaptic activity.

Tight Coupling of Metabolic Oscillations and Intracellular Water Dynamics in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Thoke, Henrik Seir; Tobiesen, Asger; Brewer, Jonathan R.

2015-01-01

We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute...... conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D...

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

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.

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

Science.gov (United States)

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.

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.

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

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Heinzen, Erin L; Swoboda, Kathryn J; Hitomi, Yuki

2012-01-01

and their unaffected parents to identify de novo nonsynonymous mutations in ATP1A3 in all seven individuals. In a subsequent sequence analysis of ATP1A3 in 98 other patients with AHC, we found that ATP1A3 mutations were likely to be responsible for at least 74% of the cases; we also identified one inherited mutation...... affecting the level of protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in ATP1A3....

Motor pathway excitability in ATP13A2 mutation carriers

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Zittel, S; Kroeger, J; van der Vegt, J P M

2012-01-01

OBJECTIVE: To describe excitability of motor pathways in Kufor-Rakeb syndrome (PARK9), an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration caused by a mutation in the ATP13A2 gene, using transcranial magnetic stimulation (TMS). METHODS: Five members of a Chilean family...... with an ATP13A2 mutation (one affected mutation carrier (MC) with a compound heterozygous mutation, 4 asymptomatic MC with a single heterozygous mutation) and 11 healthy subjects without mutations were studied. We measured motor evoked potentials (MEP), the contralateral silent period (cSP), short interval....... RESULTS: CSP duration was increased in the symptomatic ATP13A2 MC. The iSP measurements revealed increased interhemispheric inhibition in both the compound heterozygous and the heterozygous MC. CONCLUSION: A compound heterozygous mutation in the ATP13A2 gene is associated with increased intracortical...

MRP transporters as membrane machinery in the bradykinin-inducible export of ATP.

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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.

Local release of ATP into the arterial inflow and venous drainage of human skeletal muscle: insight from ATP determination with the intravascular microdialysis technique

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Mortensen, Stefan; Thaning, Pia; Nyberg, Michael Permin

2011-01-01

is released into plasma, we measured plasma [ATP] with the intravascular microdialysis technique at rest and during dynamic exercise (normoxia and hypoxia), passive exercise, thigh compressions and arterial ATP, tyramine and ACh infusion in a total of 16 healthy young men. Femoral arterial and venous...

ATP measurements for monitoring microbial drinking water quality

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Vang, Óluva Karin

Current standard methods for surveillance of microbial drinking water quality are culture based, which are laborious and time-consuming, where results not are available before one to three days after sampling. This means that the water may have been consumed before results on deteriorated water....... The overall aim of this PhD study was to investigate various methodological features of the ATP assay for a potential implementation on a sensor platform as a real-time parameter for continuous on-line monitoring of microbial drinking water quality. Commercial reagents are commonly used to determine ATP......, microbial quality in distributed water, detection of aftergrowth, biofilm formation etc. This PhD project demonstrated that ATP levels are relatively low and fairly stable in drinking water without chlorine residual despite different sampling locations, different drinking water systems and time of year...

Effects of Glycolytic-Based Interval Training on Anaerobic Capacity in Soccer Players

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Polczyk Michał

2015-09-01

Full Text Available Purpose. The aim of this study was to determine the magnitude of changes in anaerobic endurance in response to a training protocol targeting glycolytic capacity. Methods. The study involved 24 soccer players from two U-18 teams. One team served as an experimental (E group the other a control (C. Besides standard soccer practice performed by both groups, an interval training protocol was administered to the experimental group twice a week (15 sessions. One training repetition involved running a soccerspecific course. Repetition time was equal to 15 s interspersed with 45 s passive recovery. Total number of repetitions was determined by the ability to maintain target time (power in subsequent repetitions. A 5% reduction in the distance covered (m compared with the first repetition ended a set. The number of sets was based on the ability of player to maintain target time per repetition. Rest interval between sets was 15 min. Anaerobic performance was assessed before and after the 8-week protocol by the Wingate test in which arterial blood gases, blood lactate concentration, and respiratory variables on a breath-by-breath basis were measured. Results. Distance covered in group E in the first training session was 470.38 ± 77.82 m and 1182.31 ± 164.44 m in the last session. Post-intervention total work (273.63 ± 18.32 to 284.98 ± 15.76 J/kg and maximum power (13.28 ± 1.43 to 14.14 ± 1.25 W/kg significantly increased in the Wingate test. Statistically significant increases in lactate concentration (10.64 ± 1.54 and 12.72 ± 1.59 mmol/l and lower blood pH (7.21 ± 0.03 and 7.19 ± 0.02 were also observed. No significant changes in any of the above variables were observed in group C. Conclusions. Interval training develops glycolytic capacity but with large inter-individual variability.

Electrochemical Investigation of the Interaction between Catecholamines and ATP.

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Taleat, Zahra; Estévez-Herrera, Judith; Machado, José D; Dunevall, Johan; Ewing, Andrew G; Borges, Ricardo

2018-02-06

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

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.

Long term effect of curcumin in regulation of glycolytic pathway and angiogenesis via modulation of stress activated genes in prevention of cancer.

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Laxmidhar Das

Full Text Available Oxidative stress, an important factor in modulation of glycolytic pathway and induction of stress activated genes, is further augmented due to reduced antioxidant defense system, which promotes cancer progression via inducing angiogenesis. Curcumin, a naturally occurring chemopreventive phytochemical, is reported to inhibit carcinogenesis in various experimental animal models. However, the underlying mechanism involved in anticarcinogenic action of curcumin due to its long term effect is still to be reported because of its rapid metabolism, although metabolites are accumulated in tissues and remain for a longer time. Therefore, the long term effect of curcumin needs thorough investigation. The present study aimed to analyze the anticarcinogenic action of curcumin in liver, even after withdrawal of treatment in Dalton's lymphoma bearing mice. Oxidative stress observed during lymphoma progression reduced antioxidant enzyme activities, and induced angiogenesis as well as activation of early stress activated genes and glycolytic pathway. Curcumin treatment resulted in activation of antioxidant enzyme super oxide dismutase and down regulation of ROS level as well as activity of ROS producing enzyme NADPH:oxidase, expression of stress activated genes HIF-1α, cMyc and LDH activity towards normal level. Further, it lead to significant inhibition of angiogenesis, observed via MMPs activity, PKCα and VEGF level, as well as by matrigel plug assay. Thus findings of this study conclude that the long term effect of curcumin shows anticarcinogenic potential via induction of antioxidant defense system and inhibition of angiogenesis via down regulation of stress activated genes and glycolytic pathway in liver of lymphoma bearing mice.

Silencing of Atp2b1 increases blood pressure through vasoconstriction.

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Shin, Young-Bin; Lim, Ji Eun; Ji, Su-Min; Lee, Hyeon-Ju; Park, So-Yon; Hong, Kyung-Won; Lim, Mihwa; McCarthy, Mark I; Lee, Young-Ho; Oh, Bermseok

2013-08-01

Recent genome-wide association studies (GWASs) have identified 30 genetic loci that regulate blood pressure, increasing our understanding of the cause of hypertension. However, it has been difficult to define the causative genes at these loci due to a lack of functional analyses. In this study, we aimed to validate the candidate gene ATP2B1 in 12q21, variants near which have the strongest association with blood pressure in Asians and Europeans. ATP2B1 functions as a calcium pump to fine-tune calcium concentrations - necessary for repolarization following muscular contractions. We silenced Atp2b1 using an siRNA complex, injected into mouse tail veins. In treated mice, blood pressure rose and the mesenteric arteries increased in wall : lumen ratio. Moreover, the arteries showed enhanced myogenic responses to pressure, and contractile responses to phenylephrine increased compared with the control, suggesting that blood pressure is regulated by ATP2B1 through the contraction and dilation of the vessel, likely by controlling calcium concentrations in the resting state. These results support that ATP2B1 is the causative gene in the blood pressure-associated 12q21 locus and demonstrate that ATP2B1 expression in the vessel influences blood pressure.

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

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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.

Modulation of cellular radiation responses by 2-deoxy-D-glucose and other glycolytic inhibitors: Implications for cancer therapy

OpenAIRE

Kalia Vijay; Prabhakara S; Narayanan Vidya

2009-01-01

Background: 2-Deoxy-D-glucose (2-DG), a glycolytic inhibitor, was observed earlier to increase DNA, chromosomal, and cellular damage in tumor cells, by inhibiting energy-dependent repair processes. Lonidamine (LND) selectively inhibits glycolysis in cancer cells. It damages the condensed mitochondria in these cells, impairing thereby the activity of hexokinase (predominantly attached to the outer mitochondrial membranes). It inhibits repair of radiation-induced potentially lethal cellular da...

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

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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.

Local and global bifurcations at infinity in models of glycolytic oscillations

DEFF Research Database (Denmark)

Sturis, Jeppe; Brøns, Morten

1997-01-01

We investigate two models of glycolytic oscillations. Each model consists of two coupled nonlinear ordinary differential equations. Both models are found to have a saddle point at infinity and to exhibit a saddle-node bifurcation at infinity, giving rise to a second saddle and a stable node...... at infinity. Depending on model parameters, a stable limit cycle may blow up to infinite period and amplitude and disappear in the bifurcation, and after the bifurcation, the stable node at infinity then attracts all trajectories. Alternatively, the stable node at infinity may coexist with either a stable...... sink (not at infinity) or a stable limit cycle. This limit cycle may then disappear in a heteroclinic bifurcation at infinity in which the unstable manifold from one saddle at infinity joins the stable manifold of the other saddle at infinity. These results explain prior reports for one of the models...

Development of an intracellular glycolytic flux sensor for high throughput applications in E.coli

DEFF Research Database (Denmark)

Lehning, Christina Eva

The aim of this PhD project was to construct, test and apply an intracellular, growth-­‐ independent and direct measureable glycolytic flux biosensor in E. coli. Studying the metabolic flux of bacterial cells is of growing interest as it is of fundamental importance to bacterial physiology as well...... to study the flux-­‐altering effects of gene knockouts in E. coli at the single cell level in a vastly parallelized and high-­‐throughput manner. After growth for several generations in rich and minimal media, 2126 gene knockouts, mainly outside of the core metabolism, could be screened. 3 gene knockouts...

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

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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.

Novel retinoblastoma treatment avoids chemotherapy: the effect of optimally timed combination therapy with angiogenic and glycolytic inhibitors on LHBETATAG retinoblastoma tumors

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Samuel K Houston

2011-01-01

Full Text Available Samuel K Houston1, Yolanda Piña1, Timothy G Murray1, Hinda Boutrid1, Colleen Cebulla2, Amy C Schefler1, Wei Shi1, Magda Celdran1, William Feuer1, Jaime Merchan3, Ted J Lampidis41Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USA; 2Department of Ophthalmology, The Ohio State University, Columbus, OH, USA; 3Division of Hematology/Oncology, Department of Medicine, 4Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine and Sylvester Comprehensive Cancer Center, Miami, FL, USAPurpose: The purpose of this study was to evaluate the effect of optimally timed combination treatment with angiogenic and glycolytic inhibitors on tumor burden, hypoxia, and angiogenesis in advanced retinoblastoma tumors.Methods: LHBETATAG mice (n = 30 were evaluated. Mice were divided into 5 groups (n = 6 and received injections at 16 weeks of age (advanced tumors with a saline, b anecortave acetate (AA, c 2-deoxyglucose (2-DG, d AA + 2-DG (1 day post-AA treatment, or e AA + 2-DG (1 week post-AA treatment. Eyes were enucleated at 21 weeks and tumor sections were analyzed for hypoxia, angiogenesis, and tumor burden.Results: Eyes treated with 2-DG 1 day post-AA injection showed a 23% (P = 0.03 reduction in tumor burden compared with 2-DG alone and a 61% (P < 0.001 reduction compared with saline-treated eyes. Eyes treated with 2-DG 1 week post-AA injection showed no significant decrease in tumor burden compared with 2-DG alone (P = 0.21 and a 56% (P < 0.001 decrease in comparison with saline-treated eyes. 2-DG significantly reduced the total density of new blood vessels in tumors by 44% compared to saline controls (P < 0.001, but did not affect the density of mature vasculature.Conclusions: Combination therapy with angiogenic and glycolytic inhibitors significantly enhanced tumor control. Synergistic effects were shown to be dependent on the temporal course of treatment

Effect of irradiation on detection of bacteria in dehydrated vegetables with ATP bioluminescence assay

International Nuclear Information System (INIS)

Xiao Huan; Luo Shishi; Wang Zegang; Feng Min; Zhu Jiating; Chen Xiulan; Zhai Jianqing

2011-01-01

ATP bioluminescence intensity of 4 kinds of irradiated dehydrated vegetables was inconsistent with the bacteria number, the reasons were investigated in this paper. Results showed that irradiation had little effect on background luminescence, and there was no effect on luciferase-luminous system. When irradiation killed the bacteria, the ATPase activity also decreased. As a result, the ATP content in bacteria didn't decreased with the killed of bacteria, which contributed to the increase of free ATP in ATP extract and finally led to the disagreement between the bioluminescence intensity and the actual number of bacteria. When the free ATP in the dehydrated vegetable was removed, the bioluminescence intensity of ATP extract was consistent with the actual number of bacteria in irradiated dehydrated vegetable and ATP bioluminescence technology could be used in bacteria detection of irradiated samples. (authors)

Selective decrease of components of the creatine kinase system and ATP synthase complex in chronic Chagas disease cardiomyopathy.

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Priscila Camillo Teixeira

2011-06-01

Full Text Available BACKGROUND: Chronic Chagas disease cardiomyopathy (CCC is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC and ischemic (IC cardiomyopathies. METHODOLOGY/PRINCIPAL FINDINGS: Myocardium homogenates from CCC (N=5, IC (N=5 and IDC (N=5 patients, as well as from heart donors (N=5 were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit and muscular creatine kinase (CKM and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. CONCLUSIONS/SIGNIFICANCE: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

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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.

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

International Nuclear Information System (INIS)

Milgrom, Elena M.; Milgrom, Yakov M.

2012-01-01

Highlights: ► MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. ► V-ATPase activity saturation with MgATP is not sufficient for complete protection. ► The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K m values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

Molecular structure of human KATP in complex with ATP and ADP.

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Lee, Kenneth Pak Kin; Chen, Jue; MacKinnon, Roderick

2017-12-29

In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K + channel Kir6.2, in the presence of Mg 2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg 2+ -ATP in the degenerate site and Mg 2+ -ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP's ability to override ATP inhibition. © 2017, Lee et al.

The Glycolytic Metabolite, Fructose-1,6-bisphosphate, Blocks Epileptiform Bursts by Attenuating Voltage-Activated Calcium Currents in Hippocampal Slices

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Li-Rong Shao

2018-06-01

Full Text Available Manipulation of metabolic pathways (e.g., ketogenic diet (KD, glycolytic inhibition alters neural excitability and represents a novel strategy for treatment of drug-refractory seizures. We have previously shown that inhibition of glycolysis suppresses epileptiform activity in hippocampal slices. In the present study, we aimed to examine the role of a “branching” metabolic pathway stemming off glycolysis (i.e., the pentose-phosphate pathway, PPP in regulating seizure activity, by using a potent PPP stimulator and glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP. Employing electrophysiological approaches, we investigated the action of F1,6BP on epileptiform population bursts, intrinsic neuronal firing, glutamatergic and GABAergic synaptic transmission and voltage-activated calcium currents (ICa in the CA3 area of hippocampal slices. Bath application of F1,6BP (2.5–5 mM blocked epileptiform population bursts induced in Mg2+-free medium containing 4-aminopyridine, in ~2/3 of the slices. The blockade occurred relatively rapidly (~4 min, suggesting an extracellular mechanism. However, F1,6BP did not block spontaneous intrinsic firing of the CA3 neurons (when synaptic transmission was eliminated with DNQX, AP-5 and SR95531, nor did it significantly reduce AMPA or NMDA receptor-mediated excitatory postsynaptic currents (EPSCAMPA and EPSCNMDA. In contrast, F1,6BP caused moderate reduction (~50% in GABAA receptor-mediated current, suggesting it affects excitatory and inhibitory synapses differently. Finally and unexpectedly, F1,6BP consistently attenuated ICa by ~40% without altering channel activation or inactivation kinetics, which may explain its anticonvulsant action, at least in this in vitro seizure model. Consistent with these results, epileptiform population bursts in CA3 were readily blocked by the nonspecific Ca2+ channel blocker, CdCl2 (20 μM, suggesting that these bursts are calcium dependent. Altogether, these data

Effect of extraluminal ATP application on vascular tone and blood flow in skeletal muscle

DEFF Research Database (Denmark)

Nyberg, Michael Permin; Al-Khazraji, Baraa K; Mortensen, Stefan P

2013-01-01

During skeletal muscle contractions, the concentration of ATP increases in muscle interstitial fluid as measured by microdialysis probes. This increase is associated with the magnitude of blood flow, suggesting that interstitial ATP may be important for contraction-induced vasodilation. However...... studied. The rat gluteus maximus skeletal muscle model was used to study changes in local skeletal muscle hemodynamics. Superfused ATP at concentrations found during muscle contractions (1-10 µM) increased blood flow by up to 400%. In this model, the underlying mechanism was also examined by inhibition...... in interstitial ATP concentrations increases muscle blood flow, indicating that the contraction-induced increase in skeletal muscle interstitial [ATP] is important for exercise hyperemia. The vasodilator effect of ATP application is mediated by NO and prostanoid formation....

Changes in dermal interstitial ATP levels during local heating of human skin.

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Gifford, Jayson R; Heal, Cory; Bridges, Jarom; Goldthorpe, Scott; Mack, Gary W

2012-12-15

Heating skin is believed to activate vanilloid type III and IV transient receptor potential ion channels (TRPV3, TRPV4, respectively), resulting in the release of ATP into the interstitial fluid. We examined the hypothesis that local skin heating would result in an accumulation of ATP in the interstitial fluid that would be related with a rise in skin blood flow (SkBF) and temperature sensation. Two microdialysis probes were inserted into the dermis on the dorsal aspect of the forearm in 15 young, healthy subjects. The probed skin was maintained at 31°C, 35°C, 39°C and 43°C for 8 min periods, during which SkBF was monitored as cutaneous vascular conductance (CVC). Dialysate was collected and analysed for ATP ([ATP](d)) using a luciferase-based assay, and ratings of perceived warmth were taken at each temperature. At a skin temperature of 31°C, [ATP](d) averaged 18.93 ± 4.06 nm and CVC averaged 12.57 ± 1.59% peak. Heating skin to 35°C resulted in an increase in CVC (17.63 ± 1.27% peak; P ATP](d). Heating skin to 39°C and 43°C resulted in a decreased [ATP](d) (5.88 ± 1.68 nm and 8.75 ± 3.44 nm, respectively; P ATP does not occur during local heating, and therefore does not have a role in temperature sensation or the dilator response in human skin. Nevertheless, the low threshold of dilatation (35°C) indicates a possible role for the TRPV3, TRPV4 channels or the sensitization of other ion channels in mediating the dilator response.

The mitochondrial phosphate transporters modulate plant responses to salt stress via affecting ATP and gibberellin metabolism in Arabidopsis thaliana.

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Wei Zhu

Full Text Available The mitochondrial phosphate transporter (MPT plays crucial roles in ATP production in plant cells. Three MPT genes have been identified in Arabidopsis thaliana. Here we report that the mRNA accumulations of AtMPTs were up-regulated by high salinity stress in A. thaliana seedlings. And the transgenic lines overexpressing AtMPTs displayed increased sensitivity to salt stress compared with the wild-type plants during seed germination and seedling establishment stages. ATP content and energy charge was higher in overexpressing plants than those in wild-type A. thaliana under salt stress. Accordingly, the salt-sensitive phenotype of overexpressing plants was recovered after the exogenous application of atractyloside due to the change of ATP content. Interestingly, Genevestigator survey and qRT-PCR analysis indicated a large number of genes, including those related to gibberellin synthesis could be regulated by the energy availability change under stress conditions in A. thaliana. Moreover, the exogenous application of uniconazole to overexpressing lines showed that gibberellin homeostasis was disturbed in the overexpressors. Our studies reveal a possible link between the ATP content mediated by AtMPTs and gibberellin metabolism in responses to high salinity stress in A. thaliana.

ATP-loading 201Tl myocardial SPECT for the detection of ischemic heart disease

International Nuclear Information System (INIS)

Fujinaga, Tsuyoshi; Yamazaki, Sayaka; Hara, Masatada; Umezawa, Chiaki; Okamura, Tetsuo; Murata, Hajime; Maruno, Hirotaka; Onoguchi, Masahisa.

1993-01-01

To evaluate the usefulness for the detection of ischemic heart disease, ATP myocrdial SPECT was performed in 35 patients (mean; 59±9.4 years) with angina pectoris or old myocardial infarction. Coronary angiography (CAG) was performed in all patients. The ultra-short half-life of ATP required a continuous infusion for its use. ATP was infused intravenously at a rate of 0.16 mg/kg/min for 5 min, with 201 Tl injection taking place at 3 min. Myocardial SPECT imaging was begun 5 min and 4 hr later after the end of ATP infusion. ATP caused a significant decrease in arterial blood pressure (p 201 Tl myocardial SPECT for the detection of coronary artery disease (CAD) was evaluated using CAG as a golden standard. The sensitivity and specificity for CAD detection were 82% and 90%, respectively. ATP myocardial SPECT is a promising new test for the detection of ischemic heart disease. (author)

Separation of metabolic supply and demand: aerobic glycolysis as a normal physiological response to fluctuating energetic demands in the membrane.

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Epstein, Tamir; Xu, Liping; Gillies, Robert J; Gatenby, Robert A

2014-01-01

Cancer cells, and a variety of normal cells, exhibit aerobic glycolysis, high rates of glucose fermentation in the presence of normal oxygen concentrations, also known as the Warburg effect. This metabolism is considered abnormal because it violates the standard model of cellular energy production that assumes glucose metabolism is predominantly governed by oxygen concentrations and, therefore, fermentative glycolysis is an emergency back-up for periods of hypoxia. Though several hypotheses have been proposed for the origin of aerobic glycolysis, its biological basis in cancer and normal cells is still not well understood. We examined changes in glucose metabolism following perturbations in membrane activity in different normal and tumor cell lines and found that inhibition or activation of pumps on the cell membrane led to reduction or increase in glycolysis, respectively, while oxidative phosphorylation remained unchanged. Computational simulations demonstrated that these findings are consistent with a new model of normal physiological cellular metabolism in which efficient mitochondrial oxidative phosphorylation supplies chronic energy demand primarily for macromolecule synthesis and glycolysis is necessary to supply rapid energy demands primarily to support membrane pumps. A specific model prediction was that the spatial distribution of ATP-producing enzymes in the glycolytic pathway must be primarily localized adjacent to the cell membrane, while mitochondria should be predominantly peri-nuclear. The predictions were confirmed experimentally. Our results show that glycolytic metabolism serves a critical physiological function under normoxic conditions by responding to rapid energetic demand, mainly from membrane transport activities, even in the presence of oxygen. This supports a new model for glucose metabolism in which glycolysis and oxidative phosphorylation supply different types of energy demand. Cells use efficient but slow-responding aerobic metabolism

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.

Ethanologenic Enzymes of Zymomonas mobilis

Energy Technology Data Exchange (ETDEWEB)

Ingram, Lonnie O' Neal

1999-03-01

Zymomonas mobilis is a unique microorganism in being both obligately fermentative and utilizing a Entner-Doudoroff pathway for glycolysis. Glycolytic flux in this organism is readily measured as evolved carbon dioxide, ethanol, or glucose consumed and exceeds 1 {micro}mole glucose/min per mg cell protein. To support this rapid glycolysis, approximately 50% of cytoplasmic protein is devoted to the 13 glycolytic and fermentative enzymes which constitute this central catabolic pathway. Only 1 ATP (net) is produced from each glucose metabolized. During the past grant period, we have completed the characterization of 11 of the 13 glycolytic genes from Z. mobilis together with complementary but separate DOE-fimded research by a former post-dot and collaborator, Dr. Tyrrell Conway. Research funded in my lab by DOE, Division of Energy Biosciences can be divided into three sections: A. Fundamental studies; B. Applied studies and utility; and C. Miscellaneous investigations.

Acidic pH facilitates peripheral αβmeATP-mediated nociception in rats: differential roles of P2X, P2Y, ASIC and TRPV1 receptors in ATP-induced mechanical allodynia and thermal hyperalgesia.

Science.gov (United States)

Seo, Hyoung-Sig; Roh, Dae-Hyun; Kwon, Soon-Gu; Yoon, Seo-Yeon; Kang, Suk-Yun; Moon, Ji-Young; Choi, Sheu-Ran; Beitz, Alvin J; Lee, Jang-Hern

2011-03-01

Peripheral ischemia is commonly associated with an increase in tissue ATP concentration and a decrease in tissue pH. Although in vitro data suggest that low tissue pH can affect ATP-binding affinities to P2 receptors, the mechanistic relationship between ATP and low pH on peripheral nociception has not been fully examined. This study was designed to investigate the potential role of an acidified environment on intraplantar αβmeATP-induced peripheral pain responses in rats. The mechanical allodynia (MA) produced by injection of αβmeATP was significantly increased in animals that received the drug diluted in pH 4.0 saline compared to those that received the drug diluted in pH 7.0 saline. Moreover, animals injected with αβmeATP (100 nmol) in pH 4.0 saline developed thermal hyperalgesia (TH), which did not occur in animals treated with αβmeATP diluted in pH 7.0 saline. To elucidate which receptors were involved in this pH-related facilitation of αβmeATP-induced MA and TH, rats were pretreated with PPADS (P2 antagonist), TNP-ATP (P2X antagonist), MRS2179 (P2Y1 antagonist), AMG9810 (TRPV1 antagonist) or amiloride (ASIC blocker). Both PPADS and TNP-ATP dose-dependently blocked pH-facilitated MA, while TH was significantly reduced by pre-treatment with MRS2179 or AMG9810. Moreover, amiloride injection significantly reduced low pH-induced facilitation of αβmeATP-mediated MA, but not TH. These results demonstrate that low tissue pH facilitates ATP-mediated MA via the activation of P2X receptors and ASICs, whereas TH induced by ATP under low pH conditions is mediated by the P2Y1 receptor and TRPV1, but not ASIC. Thus distinct mechanisms are responsible for the development of MA and TH under conditions of tissue acidosis and increased ATP. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effect of Intramuscular Protons, Lactate, and ATP on Muscle Hyperalgesia in Rats.

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Nicholas S Gregory

Full Text Available Chronic muscle pain is a significant health problem leading to disability[1]. Muscle fatigue can exacerbate muscle pain. Metabolites, including ATP, lactate, and protons, are released during fatiguing exercise and produce pain in humans. These substances directly activate purinergic (P2X and acid sensing ion channels (ASICs on muscle nociceptors, and when combined, produce a greater increase in neuron firing than when given alone. Whether the enhanced effect of combining protons, lactate, and ATP is the sum of individual effects (additive or more than the sum of individual effects (synergistic is unknown. Using a rat model of muscle nociceptive behavior, we tested each of these compounds individually over a range of physiologic and supra-physiologic concentrations. Further, we combined all three compounds in a series of dilutions and tested their effect on muscle nociceptive behavior. We also tested a non-hydrolyzable form of ATP (α,β-meATP alone and in combination with lactate and acidic pH. Surprisingly, we found no dose-dependent effect on muscle nociceptive behavior for protons, lactate, or ATP when given alone. We similarly found no effect after application of each two-metabolite combination. Only pH 4 saline and α,β-meATP produced hyperalgesia when given alone. When all 3 substances were combined, however, ATP (2.4μm, lactate (10mM, and acidic pH (pH 6.0 produced an enhanced effect greater than the sum of the effects of the individual components, i.e. synergism. α,β me ATP (3nmol, on the other hand, showed no enhanced effects when combined with lactate (10mM or acidic pH (pH 6.0, i.e. additive. These data suggest that combining fatigue metabolites in muscle produces a synergistic effect on muscle nociception.

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

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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

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

NARCIS (Netherlands)

Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C. D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

2014-01-01

Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we

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.

Calorimetric study on human erythrocyte glycolysis. Heat production in various metabolic conditions.

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Minakami, S; de Verdier, C H

1976-06-01

The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway.

Label free luminescence strategy for sensitive detection of ATP using aptamer-Ru(II) complexes

Energy Technology Data Exchange (ETDEWEB)

Babu, Eththilu [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Muthu Mareeswaran, Paulpandian [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Department of Industrial Chemistry, Alagappa Univesity, Karaikudi 630003, Tamil Nadu (India); Ramdass, Arumugam [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India); Research Department of Chemistry, Aditanar College of Arts and Science, Tiruchendur 628216, Tamil Nadu (India); Ramesh, Pandian [UCIBIO-REQUIMTE, Departmento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica (Portugal); Rajagopal, Seenivasan, E-mail: rajagopalseenivasan@yahoo.com [Department of Physical Che mistry, School of Chemistry, Madurai Kamaraj University, Madurai 625021, Tamil Nadu (India)

2016-07-15

A simple and sensitive aptamer-based luminescence strategy for ATP detection is developed using Ru(II) complexes as probe molecule. It is based on the fact that Ru(II)-dppz complexes show the light switching behavior with DNA aptamers and found to show significant luminescence spectral change on the addition of ATP molecules. The binding efficiencies of aptamer with ATP, ADP and AMP are calculated and compared. The structural change of aptamer is also studied using circular dichroism (CD) spectral techniques. Moreover, the binding nature of aptamer with ATP, ADP and AMP is demonstrated by computational techniques. The proposed strategy was successfully applied to the detection of ATP.

Label free luminescence strategy for sensitive detection of ATP using aptamer-Ru(II) complexes

International Nuclear Information System (INIS)

Babu, Eththilu; Muthu Mareeswaran, Paulpandian; Ramdass, Arumugam; Ramesh, Pandian; Rajagopal, Seenivasan

2016-01-01

A simple and sensitive aptamer-based luminescence strategy for ATP detection is developed using Ru(II) complexes as probe molecule. It is based on the fact that Ru(II)-dppz complexes show the light switching behavior with DNA aptamers and found to show significant luminescence spectral change on the addition of ATP molecules. The binding efficiencies of aptamer with ATP, ADP and AMP are calculated and compared. The structural change of aptamer is also studied using circular dichroism (CD) spectral techniques. Moreover, the binding nature of aptamer with ATP, ADP and AMP is demonstrated by computational techniques. The proposed strategy was successfully applied to the detection of ATP.

De novo mutations in ATP1A3 cause alternating hemiplegia of childhood

Science.gov (United States)

Heinzen, Erin L.; Swoboda, Kathryn J.; Hitomi, Yuki; Gurrieri, Fiorella; Nicole, Sophie; de Vries, Boukje; Tiziano, F. Danilo; Fontaine, Bertrand; Walley, Nicole M.; Heavin, Sinéad; Panagiotakaki, Eleni; Fiori, Stefania; Abiusi, Emanuela; Di Pietro, Lorena; Sweney, Matthew T.; Newcomb, Tara M.; Viollet, Louis; Huff, Chad; Jorde, Lynn B.; Reyna, Sandra P.; Murphy, Kelley J.; Shianna, Kevin V.; Gumbs, Curtis E.; Little, Latasha; Silver, Kenneth; Ptác̆ek, Louis J.; Haan, Joost; Ferrari, Michel D.; Bye, Ann M.; Herkes, Geoffrey K.; Whitelaw, Charlotte M.; Webb, David; Lynch, Bryan J.; Uldall, Peter; King, Mary D.; Scheffer, Ingrid E.; Neri, Giovanni; Arzimanoglou, Alexis; van den Maagdenberg, Arn M.J.M.; Sisodiya, Sanjay M.; Mikati, Mohamad A.; Goldstein, David B.; Nicole, Sophie; Gurrieri, Fiorella; Neri, Giovanni; de Vries, Boukje; Koelewijn, Stephany; Kamphorst, Jessica; Geilenkirchen, Marije; Pelzer, Nadine; Laan, Laura; Haan, Joost; Ferrari, Michel; van den Maagdenberg, Arn; Zucca, Claudio; Bassi, Maria Teresa; Franchini, Filippo; Vavassori, Rosaria; Giannotta, Melania; Gobbi, Giuseppe; Granata, Tiziana; Nardocci, Nardo; De Grandis, Elisa; Veneselli, Edvige; Stagnaro, Michela; Gurrieri, Fiorella; Neri, Giovanni; Vigevano, Federico; Panagiotakaki, Eleni; Oechsler, Claudia; Arzimanoglou, Alexis; Nicole, Sophie; Giannotta, Melania; Gobbi, Giuseppe; Ninan, Miriam; Neville, Brian; Ebinger, Friedrich; Fons, Carmen; Campistol, Jaume; Kemlink, David; Nevsimalova, Sona; Laan, Laura; Peeters-Scholte, Cacha; van den Maagdenberg, Arn; Casaer, Paul; Casari, Giorgio; Sange, Guenter; Spiel, Georg; Boneschi, Filippo Martinelli; Zucca, Claudio; Bassi, Maria Teresa; Schyns, Tsveta; Crawley, Francis; Poncelin, Dominique; Vavassori, Rosaria

2012-01-01

Alternating hemiplegia of childhood (AHC) is a rare, severe neurodevelopmental syndrome characterized by recurrent hemiplegic episodes and distinct neurologic manifestations. AHC is usually a sporadic disorder with unknown etiology. Using exome sequencing of seven patients with AHC, and their unaffected parents, we identified de novo nonsynonymous mutations in ATP1A3 in all seven AHC patients. Subsequent sequence analysis of ATP1A3 in 98 additional patients revealed that 78% of AHC cases have a likely causal ATP1A3 mutation, including one inherited mutation in a familial case of AHC. Remarkably, six ATP1A3 mutations explain the majority of patients, including one observed in 36 patients. Unlike ATP1A3 mutations that cause rapid-onset-dystonia-parkinsonism, AHC-causing mutations revealed consistent reductions in ATPase activity without effects on protein expression. This work identifies de novo ATP1A3 mutations as the primary cause of AHC, and offers insight into disease pathophysiology by expanding the spectrum of phenotypes associated with mutations in this gene. PMID:22842232

Evaluation of ATP measurements to detect microbial ingress by wastewater and surface water in drinking water.

Science.gov (United States)

Vang, Óluva K; Corfitzen, Charlotte B; Smith, Christian; Albrechtsen, Hans-Jørgen

2014-11-01

Fast and reliable methods are required for monitoring of microbial drinking water quality in order to protect public health. Adenosine triphosphate (ATP) was investigated as a potential real-time parameter for detecting microbial ingress in drinking water contaminated with wastewater or surface water. To investigate the ability of the ATP assay in detecting different contamination types, the contaminant was diluted with non-chlorinated drinking water. Wastewater, diluted at 10(4) in drinking water, was detected with the ATP assay, as well as 10(2) to 10(3) times diluted surface water. To improve the performance of the ATP assay in detecting microbial ingress in drinking water, different approaches were investigated, i.e. quantifying microbial ATP or applying reagents of different sensitivities to reduce measurement variations; however, none of these approaches contributed significantly in this respect. Compared to traditional microbiological methods, the ATP assay could detect wastewater and surface water in drinking water to a higher degree than total direct counts (TDCs), while both heterotrophic plate counts (HPC 22 °C and HPC 37 °C) and Colilert-18 (Escherichia coli and coliforms) were more sensitive than the ATP measurements, though with much longer response times. Continuous sampling combined with ATP measurements displays definite monitoring potential for microbial drinking water quality, since microbial ingress in drinking water can be detected in real-time with ATP measurements. The ability of the ATP assay to detect microbial ingress is influenced by both the ATP load from the contaminant itself and the ATP concentration in the specific drinking water. Consequently, a low ATP concentration of the specific drinking water facilitates a better detection of a potential contamination of the water supply with the ATP assay. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Nisin depletes ATP and proton motive force in mycobacteria.

Science.gov (United States)

Chung, H J; Montville, T J; Chikindas, M L

2000-12-01

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

Science.gov (United States)

Koronowski, Kevin B; Khoury, Nathalie; Saul, Isabel; Loris, Zachary B; Cohan, Charles H; Stradecki-Cohan, Holly M; Dave, Kunjan R; Young, Juan I; Perez-Pinzon, Miguel A

2017-11-01

Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1 , accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders. © 2017 American Heart Association, Inc.

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.

Some processes of energy saving and expenditure occurring during ethanol perfusion in the isolated liver of fed rats; a Nuclear Magnetic Resonance study.

Directory of Open Access Journals (Sweden)

Gin Henri

2004-03-01

Full Text Available Abstract Background In the isolated liver of fed rats, a 10 mM ethanol perfusion rapidly induced a rapid 25% decrease in the total ATP content, the new steady state resulting from both synthesis and consumption. The in situ rate of mitochondrial ATP synthesis without activation of the respiration was increased by 27%, implying an increased energy demand. An attempt to identify the ethanol-induced ATP-consuming pathways was performed using 31P and 13C Nuclear Magnetic Resonance. Results Ethanol (i transiently increased sn-glycerol-3-phosphate formation whereas glycogenolysis was continuously maintained; (ii decreased the glycolytic ATP supply and (iii diminished the intracellular pH in a dose-dependent manner in a slight extend. Although the cytosolic oxidation of ethanol largely generated H+ (and NADH, intracellular pHi was maintained by (i the large and passive excretion of cellular acetic acid arising from ethanol oxidation (evidenced by exogenous acetate administration, without energetic cost or (ii proton extrusion via the Na+-HCO3- symport (implying the indirect activation of the Na+-K+-ATPase pump and thus an energy use, demonstrated during the addition of their specific inhibitors SITS and ouabaïn, respectively. Conclusion Various cellular mechanisms diminish the cytosolic concentration of H+ and NADH produced by ethanol oxidation, such as (i the large but transient contribution of the dihydroxyacetone phosphate / sn-glycerol-3-phosphate shuttle between cytosol and mitochondria, mainly implicated in the redox state and (ii the major participation of acetic acid in passive proton extrusion out of the cell. These processes are not ATP-consuming and the latter is a cellular way to save some energy. Their starting in conjunction with the increase in mitochondrial ATP synthesis in ethanol-perfused whole liver was however insufficient to alleviate either the inhibition of glycolytic ATP synthesis and/or the implication of Na+-HCO3- symport and

Motility, ATP levels and metabolic enzyme activity of sperm from bluegill (Lepomis macrochirus).

Science.gov (United States)

Burness, Gary; Moyes, Christopher D; Montgomerie, Robert

2005-01-01

Male bluegill displays one of two life history tactics. Some males (termed "parentals") delay reproduction until ca. 7 years of age, at which time they build nests and actively courts females. Others mature precociously (sneakers) and obtain fertilizations by cuckolding parental males. In the current study, we studied the relations among sperm motility, ATP levels, and metabolic enzyme activity in parental and sneaker bluegill. In both reproductive tactics, sperm swimming speed and ATP levels declined in parallel over the first 60 s of motility. Although sneaker sperm initially had higher ATP levels than parental sperm, by approximately 30 s postactivation, no differences existed between tactics. No differences were noted between tactics in swimming speed, percent motility, or the activities of key metabolic enzymes, although sperm from parentals had a higher ratio of creatine phosphokinase (CPK) to citrate synthase (CS). In both tactics, with increasing CPK and CS activity, sperm ATP levels increased at 20 s postactivation, suggesting that capacities for phosphocreatine hydrolysis and aerobic metabolism may influence interindividual variation in rates of ATP depletion. Nonetheless, there was no relation between sperm ATP levels and either swimming speed or percent of sperm that were motile. This suggests that interindividual variation in ATP levels may not be the primary determinant of variation in sperm swimming performance in bluegill.

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.

ATP Induces IL-1β Secretion in Neisseria gonorrhoeae-Infected Human Macrophages by a Mechanism Not Related to the NLRP3/ASC/Caspase-1 Axis

Directory of Open Access Journals (Sweden)

Killen García

2016-01-01

Full Text Available Neisseria gonorrhoeae (Ngo has developed multiple immune evasion mechanisms involving the innate and adaptive immune responses. Recent findings have reported that Ngo reduces the IL-1β secretion of infected human monocyte-derived macrophages (MDM. Here, we investigate the role of adenosine triphosphate (ATP in production and release of IL-1β in Ngo-infected MDM. We found that the exposure of Ngo-infected MDM to ATP increases IL-1β levels about ten times compared with unexposed Ngo-infected MDM (P0.05 and caspase-1 (CASP1, P>0.05. In addition, ATP was not able to modify caspase-1 activity in Ngo-infected MDM but was able to increase pyroptosis (P>0.01. Notably ATP treatment defined an increase of positive staining for IL-1β with a distinctive intracellular pattern of distribution. Collectively, these data demonstrate that ATP induces IL-1β secretion by a mechanism not related to the NLRP3/ASC/caspase-1 axis and likely is acting at the level of vesicle trafficking or pore formation.

Energy-producing system of the membrane potential generation in γ-irradiated Streptococcus faecalis

International Nuclear Information System (INIS)

Fomenko, B.S.

1976-01-01

γ-irradiated (20-100 krads) Str. faecalis cells exhibited increased glycolytic and ATPase activity whereas the ATP level remained unaffected by radiation. It is concluded that the radiation-induced reduction of the membrane potential in Str. faecalis, that has been earlier described, is not connected with the impairment of the energy-producing system of the potential generation

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.

Functional importance of the astrocytic glycogen-shunt and glycolysis for maintenance of an intact intra/extracellular glutamate gradient

DEFF Research Database (Denmark)

Schousboe, Arne; Sickmann, Helle M; Walls, Anne B

2010-01-01

It has been proposed that a considerable fraction of glucose metabolism proceeds via the glycogen-shunt consisting of conversion of glucose units to glycogen residues and subsequent production of glucose-1-phosphate to be metabolized in glycolysis after conversion to glucose-6-phosphate. The impo......It has been proposed that a considerable fraction of glucose metabolism proceeds via the glycogen-shunt consisting of conversion of glucose units to glycogen residues and subsequent production of glucose-1-phosphate to be metabolized in glycolysis after conversion to glucose-6-phosphate....... The importance of this as well as the significance of ATP formed in glycolysis versus that formed by the concerted action of the tricarboxylic acid (TCA) cycle processes and oxidative phosphorylation for maintenance of glutamate transport capacity in astrocytes is discussed. It is argued that glycolytically...

A novel two-nucleotide deletion in the ATP7A gene associated with delayed infantile onset of Menkes disease.

Science.gov (United States)

Wada, Takahito; Haddad, Marie Reine; Yi, Ling; Murakami, Tomomi; Sasaki, Akiko; Shimbo, Hiroko; Kodama, Hiroko; Osaka, Hitoshi; Kaler, Stephen G

2014-04-01

Determining the relationship between clinical phenotype and genotype in genetic diseases is important in clinical practice. In general, frameshift mutations are expected to produce premature termination codons, leading to production of mutant transcripts destined for degradation by nonsense-mediated decay. In X-linked recessive diseases, male patients with frameshift mutations typically have a severe or even lethal phenotype. We report a case of a 17-month-old boy with Menkes disease (NIM #309400), an X-linked recessive copper metabolism disorder caused by mutations in the ATP7A copper transporter gene. He exhibited an unexpectedly late onset and experienced milder symptoms. His genomic DNA showed a de novo two-nucleotide deletion in exon 4 of ATP7A, predicting a translational frameshift and premature stop codon, and a classic severe phenotype. Characterization of his ATP7A mRNA showed no abnormal splicing. We speculate that translation reinitiation could occur downstream to the premature termination codon and produce a partially functional ATP7A protein. Study of the child's fibroblasts found no evidence of translation reinitiation; however, the possibility remains that this phenomenon occurred in neural tissues and influenced the clinical phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of ATP in the regulation of cholesterol biogenesis

International Nuclear Information System (INIS)

Subba Rao, G.; Ramasarma, T.

1974-01-01

Intraperitoneal administration of glucose (4oomg/rat) stimulated the biogenesis of sterols in starved rats while citrate or pyruvate (20mg/rat) did not have any effect. ATP (10mg/ rat) administered intraperitoneally stimulated the incorporation of acetate-1- 14 C into sterols but not of mevalonate-2- 14 C into sterols in starved rats. The results indicate that ATP may play a role in regulating cholesterol biogenesis and it is not acting merely as an energy source. (author)

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

OpenAIRE

Khalid N. M. Abdelazeem; Yogesh Singh; Florian Lang; Madhuri S. Salker

2017-01-01

Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of ...

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

Science.gov (United States)

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.

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

International Nuclear Information System (INIS)

Ahluwalia, Unnati; Deep, Shashank

2012-01-01

Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures

Science.gov (United States)

Koizumi, Schuichi; Fujishita, Kayoko; Tsuda, Makoto; Shigemoto-Mogami, Yukari; Inoue, Kazuhide

2003-09-01

Originally ascribed passive roles in the CNS, astrocytes are now known to have an active role in the regulation of synaptic transmission. Neuronal activity can evoke Ca2+ transients in astrocytes, and Ca2+ transients in astrocytes can evoke changes in neuronal activity. The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between astrocytes and neurons. We demonstrate here that ATP, a primary mediator of intercellular Ca2+ signaling among astrocytes, also mediates intercellular signaling between astrocytes and neurons in hippocampal cultures. Mechanical stimulation of astrocytes evoked Ca2+ waves mediated by the release of ATP and the activation of P2 receptors. Mechanically evoked Ca2+ waves led to decreased excitatory glutamatergic synaptic transmission in an ATP-dependent manner. Exogenous application of ATP does not affect postsynaptic glutamatergic responses but decreased presynaptic exocytotic events. Finally, we show that astrocytes exhibit spontaneous Ca2+ waves mediated by extracellular ATP and that inhibition of these Ca2+ responses enhanced excitatory glutamatergic transmission. We therefore conclude that ATP released from astrocytes exerts tonic and activity-dependent down-regulation of synaptic transmission via presynaptic mechanisms.

An experimental study of the regulation of glycolytic oscillations in yeast

DEFF Research Database (Denmark)

Schrøder, Tine Daa; Özalp, Veli Cengiz; Lunding, Anita

2013-01-01

We have studied oscillating glycolysis in the strain BY4743 and isogenic strains with deletions of genes encoding enzymes in glycolysis, mitochondrial electron transport and ATP synthesis. We found that deletion of the gene encoding the hexokinase 1 isoform does not affect the oscillations while...... to processes within glycolysis also processes outside this pathway contribute to the control of the oscillatory behaviour....

Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells.

Directory of Open Access Journals (Sweden)

Ayako Nakano

Full Text Available Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2, KG-1 (ABCB1 and HepG2 cells (ABCB1 and ABCG2. Interestingly, although side population (SP cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

Expression of genes encoding F-1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis

DEFF Research Database (Denmark)

Købmann, Brian Jensen; Solem, Christian; Pedersen, M.B.

2002-01-01

of the genes encoding F-1-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F-1-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase...... threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions...

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

Science.gov (United States)

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

2017-04-26

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

A futile cycle, formed between two ATP-dependant γ-glutamyl cycle ...

Indian Academy of Sciences (India)

Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a ...

High-altitude adaptation of Tibetan chicken from MT-COI and ATP-6 perspective.

Science.gov (United States)

Zhao, Xiaoling; Wu, Nan; Zhu, Qing; Gaur, Uma; Gu, Ting; Li, Diyan

2016-09-01

The problem of hypoxia adaptation in high altitudes is an unsolved brainteaser in the field of life sciences. As one of the best chicken breeds with adaptability to highland environment, the Tibetan chicken, is genetically different from lowland chicken breeds. In order to gain a better understanding of the mechanism of hypoxic adaptability in high altitude, in the present study, we focused on the MT-COI together with ATP-6 gene to explore the regulatory mechanisms for hypoxia adaptability in Tibet chicken. Here, we sequenced MT-COI of 29 Tibetan chickens and 30 Chinese domestic chickens and ATP-6 gene of 28 Tibetan chickens and 29 Chinese domestic chickens. In MT-COI gene, 9 single nucleotide polymorphisms (SNPs) were detected though none of these was a missense mutation, confirming the fact that MT-COI gene is a largely conservative sequence. In ATP-6 gene, 6 single nucleotide polymorphisms (SNPs) were detected and we found a missense mutation (m.9441G > A) in the ATP-6 gene of Tibetan chicken resulting in an amino acid substitution. Due to the critical role of ATP-6 gene in the proton translocation and energy metabolism, we speculated the possibility of this mutation playing an important role in easier energy conversion and metabolism in Tibetan chickens than Chinese domestic chickens so as to better adapt to the harsh environment of the high-altitude areas. The Median-joining profile also suggested that haplotype Ha2 has the ancestral position to the other haplotypes and has significant relationship with high-altitude adaptation in ATP-6 gene. Therefore, we considered that the polymorphism (m.9441G > A) in the ATP-6 gene may affect the specific functions of ATP-6 enzyme relating to high-altitude adaptation of Tibetan chicken and MT-COI gene is a largely conservative sequence.

Atp1a3-deficient heterozygous mice show lower rank in the hierarchy and altered social behavior.

Science.gov (United States)

Sugimoto, H; Ikeda, K; Kawakami, K

2017-10-23

Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3 +/- ) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3 +/- and compared the rank and hierarchy structure between Atp1a3 +/- and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3 +/- than wild-type mice, and Atp1a3 +/- approached Atp1a3 +/- mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3 +/- group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3 +/- and wild type. (4) Atp1a3 +/- showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3 +/- showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

A two-hour antibiotic susceptibility test by ATP-bioluminescence.

Science.gov (United States)

March Rosselló, Gabriel Alberto; García-Loygorri Jordán de Urries, María Cristina; Gutiérrez Rodríguez, María Purificación; Simarro Grande, María; Orduña Domingo, Antonio; Bratos Pérez, Miguel Ángel

2016-01-01

The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Biochemical endpoints of glucocorticoid hormone action

Energy Technology Data Exchange (ETDEWEB)

Young, D.A.; Nicholson, M.L.; Guyette, W.A.; Giddings, S.J.; Mendelsohn, S.L.; Nordeen, S.K.; Lyons, R.T.

1978-01-01

Both the rapidly evolving metabolic effects of glucocorticoids and the more slowly developing lethal actions appear to be initiated via the synthesis of new mRNAs and proteins. The chronic suppression of cell growth may be the consequence of suppression of overall rates of protein synthesis (and probably RNA and DNA synthesis as well) that in turn may represent the cellular response to the small changes in ratios of adenine nucleotides that result from the suppression of oxidative ATP production. The inhibition of glucose transport may also play a role here to prevent a compensatory increase in glycolytic ATP production. Some other hormone actions, the decrease in the ability of cells to concentrate AIB and the increase in nuclear fragility are unrelated to, and evolve separately from, the hormonal inhibitions on energy production. Cell killing is not the result of suppression of protein synthesis, nor of hormone-induced increases in calcium uptake. While the mechanisms are unknown, the increase in nuclear fragility appears to be the earliest measure of their operation. In tumor cells resistance to lethal actions of glucocorticoids may emerge via the selection of cells with hardier membranes, that are better able to withstand the intracellular destructive events set in motion by high levels of glucocorticoids.

Monitoring of glycolytic activity secondary to ischaemia in knee replacement surgery.

Science.gov (United States)

León-Muñoz, V J; Lisón-Almagro, A J; Hernández-García, C H; López-López, M

2018-04-17

To non-invasively assess tissue lesion secondary to ischaemia applied during knee replacement surgery. Secondary objectives: to assess whether this lesion correlates with the duration of ischaemia and whether instrumental and gender variables influence it. Prospective cohort study. Pre and postoperative serum lactate levels have been determined as an indicator of glycolytic activity secondary to ischaemia in 88 patients. Serum lactate determination was performed by reactive strips of enzymatic-amperometric detection on capillary blood. Preoperative serum lactate levels (mean and SD): 2.467±1.036 mmol/L. Postoperative serum lactate levels: 3.938±2.018 mmol/L. Ischaemia time 102.98±18.25minutes. Postoperative serum lactate levels were significantly higher than preoperative lactate levels. There are no statistical differences according to the time that the ischaemia was prolonged, gender or type of instrumentation used. In our study, postoperative serum lactate values were significantly higher than preoperative lactate values, with no correlation to the duration of ischaemia during knee replacement surgery. Copyright © 2018 SECOT. Publicado por Elsevier España, S.L.U. All rights reserved.

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.

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 ...

Effect of aspirin and prostaglandins on the carbohydrate metabolism in albino rats.: glucose oxidation through different pathways and glycolytic enzymes

International Nuclear Information System (INIS)

Balasubramanian, A.; Ramakrishnan, S.

1980-01-01

The effect of chronic and acute doses of aspirin and prostaglandins F2α and E2 individually on the oxidation of glucose through Embden Meyerhof-TCA cycle and pentose phosphate pathways and some key glycolytic enzymes of liver were studied in male albino rats. Studies were extended to find the combined effect of PGF2α and E2 with an acute dose of aspirin. There was increased utilisation of both 1- 14 C glucose and 6- 14 C glucose on aspirin treatment. However, the metabolism through the EM-TCA pathway was more pronounced as shown by a reduced ratio of 14 CO 2 from 1- 14 C and 6- 14 C glucose. Two hepatic key glycolytic enzymes viz. hexokinase and pyruvate kinase were increased due to aspirin treatment. Withdrawal of aspirin corrected the above impaired carbohydrate metabolism in liver. Prostaglandin F2α also caused a reduction in the utilisation of 1- 14 C glucose, while PGE2 recorded an increase in the utilisation of both 1- 14 C and 6- 14 C glucose when compared to controls, indicating that different members of prostaglandins could affect metabolisms and differently. Administration of the PGs and aspirin together showed an increase in the utilisation of 6- 14 C glucose. (auth.)

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch

Directory of Open Access Journals (Sweden)

Steven Zhao

2016-10-01

Full Text Available Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY, cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch.

Science.gov (United States)

Zhao, Steven; Torres, AnnMarie; Henry, Ryan A; Trefely, Sophie; Wallace, Martina; Lee, Joyce V; Carrer, Alessandro; Sengupta, Arjun; Campbell, Sydney L; Kuo, Yin-Ming; Frey, Alexander J; Meurs, Noah; Viola, John M; Blair, Ian A; Weljie, Aalim M; Metallo, Christian M; Snyder, Nathaniel W; Andrews, Andrew J; Wellen, Kathryn E

2016-10-18

Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-coenzyme A (CoA) plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here, we show that, upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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.

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.

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

Intestinal bile salt absorption in Atp8b1 deficient mice

NARCIS (Netherlands)

Groen, Annemiek; Kunne, Cindy; Paulusma, Coen C.; Kramer, Werner; Agellon, Luis B.; Bull, Laura N.; Elferink, Ronald P. J. Oude

2007-01-01

BACKGROUND/AIMS: Mutations in the ATP8B1 gene can cause Progressive Familial Intrahepatic Cholestasis type 1. We have previously reported that Atp8b1(G308V/G308V) mice, a model for PFIC1, have slightly, but significantly, higher baseline serum bile salt (BS) concentrations compared to wt mice. Upon

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

Science.gov (United States)

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

Science.gov (United States)

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

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.

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

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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.

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

Energy Technology Data Exchange (ETDEWEB)

Zhdanov, Alexander V., E-mail: a.zhdanov@ucc.ie [School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork (Ireland); Waters, Alicia H.C. [School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork (Ireland); Golubeva, Anna V. [Alimentary Pharmabiotic Centre, University College Cork, Bioscience Institute, Western Road, Cork (Ireland); Papkovsky, Dmitri B. [School of Biochemistry and Cell Biology, University College Cork, Cavanagh Pharmacy Building, College Road, Cork (Ireland)

2015-01-01

Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

International Nuclear Information System (INIS)

Zhdanov, Alexander V.; Waters, Alicia H.C.; Golubeva, Anna V.; Papkovsky, Dmitri B.

2015-01-01

Changes in availability and utilisation of O 2 and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O 2 . Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O 2 and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O 2 and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O 2 and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells

Reduction in reactive oxygen species production by mitochondria from elderly subjects with normal and impaired glucose tolerance.

Science.gov (United States)

Ghosh, Sangeeta; Lertwattanarak, Raweewan; Lefort, Natalie; Molina-Carrion, Marjorie; Joya-Galeana, Joaquin; Bowen, Benjamin P; Garduno-Garcia, Jose de Jesus; Abdul-Ghani, Muhammad; Richardson, Arlan; DeFronzo, Ralph A; Mandarino, Lawrence; Van Remmen, Holly; Musi, Nicolas

2011-08-01

Aging increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes. It has been proposed that increased reactive oxygen species (ROS) generation by dysfunctional mitochondria could play a role in the pathogenesis of these metabolic abnormalities. We examined whether aging per se (in subjects with normal glucose tolerance [NGT]) impairs mitochondrial function and how this relates to ROS generation, whether older subjects with IGT have a further worsening of mitochondrial function (lower ATP production and elevated ROS generation), and whether exercise reverses age-related changes in mitochondrial function. Mitochondrial ATP and ROS production were measured in muscle from younger individuals with NGT, older individuals with NGT, and older individuals with IGT. Measurements were performed before and after 16 weeks of aerobic exercise. ATP synthesis was lower in older subjects with NGT and older subjects with IGT versus younger subjects. Notably, mitochondria from older subjects (with NGT and IGT) displayed reduced ROS production versus the younger group. ATP and ROS production were similar between older groups. Exercise increased ATP synthesis in the three groups. Mitochondrial ROS production also increased after training. Proteomic analysis revealed downregulation of several electron transport chain proteins with aging, and this was reversed by exercise. Old mitochondria from subjects with NGT and IGT display mitochondrial dysfunction as manifested by reduced ATP production but not with respect to increased ROS production. When adjusted to age, the development of IGT in elderly individuals does not involve changes in mitochondrial ATP and ROS production. Lastly, exercise reverses the mitochondrial phenotype (proteome and function) of old mitochondria.

Degradation of ATP and glycogen in cod ( Gadus morhua ) muscle during freezing

DEFF Research Database (Denmark)

Cappeln, Gertrud; Jessen, Flemming

2001-01-01

Changes in ATP, IMP, lactate and glycogen contents in the muscle of cod were followed during freezing at temperatures of -20C and -45C. ATP degradation was accompanied by a corresponding increase in IMP content. Simultaneous measurement of temperature showed that at both freezing rates......, the greatest decrease in ATP content was observed when the temperature reached -0.8C. Glycolysis occurred during freezing of cod as indicated by an increase in lactate content. The changes found in all measured metabolites were more pronounced when freezing was performed at a slow rate compared to a fast rate...

Pharmacological and molecular comparison of K(ATP) channels in rat basilar and middle cerebral arteries

DEFF Research Database (Denmark)

Ploug, Kenneth Beri; Edvinsson, Lars; Olesen, Jes

2006-01-01

, we studied the possible involvement of endothelial K(ATP) channels by pressurized arteriography after luminal administration of synthetic K(ATP) channel openers to rat basilar and middle cerebral arteries. Furthermore, we examined the mRNA and protein expression profile of K(ATP) channels to rat...... basilar and middle cerebral arteries using quantitative real-time PCR (Polymerase Chain Reaction) and Western blotting, respectively. In the perfusion system, we found no significant responses after luminal application of three K(ATP) channel openers to rat basilar and middle cerebral arteries...

A self-referencing biosensor for real-time monitoring of physiological ATP transport in plant systems.

Science.gov (United States)

Vanegas, Diana C; Clark, Greg; Cannon, Ashley E; Roux, Stanley; Chaturvedi, Prachee; McLamore, Eric S

2015-12-15

The objective of this study was to develop a self-referencing electrochemical biosensor for the direct measurement of ATP flux into the extracellular matrix by living cells/organisms. The working mechanism of the developed biosensor is based on the activity of glycerol kinase and glycerol-3-phosphate oxidase. A stratified bi-enzyme nanocomposite was created using a protein-templated silica sol gel encapsulation technique on top of graphene-modified platinum electrodes. The biosensor exhibited excellent electrochemical performance with a sensitivity of 2.4±1.8 nA/µM, a response time of 20±13 s and a lower detection limit of 1.3±0.7 nM. The self-referencing biosensor was used to measure exogenous ATP efflux by (i) germinating Ceratopteris spores and (ii) growing Zea mays L. roots. This manuscript demonstrates the first development of a non-invasive ATP micro-biosensor for the direct measurement of eATP transport in living tissues. Before this work, assays of eATP have not been able to record the temporally transient movement of ATP at physiological levels (nM and sub-nM). The method demonstrated here accurately measured [eATP] flux in the immediate vicinity of plant cells. Although these proof of concept experiments focus on plant tissues, the technique developed herein is applicable to any living tissue, where nanomolar concentrations of ATP play a critical role in signaling and development. This tool will be invaluable for conducting hypothesis-driven life science research aimed at understanding the role of ATP in the extracellular environment. Copyright © 2015 Elsevier B.V. All rights reserved.

Distinct age and differentiation-state dependent metabolic profiles of oligodendrocytes under optimal and stress conditions.

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Vijayaraghava T S Rao

Full Text Available Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that under optimal conditions in vitro, the respiratory activity of human adult brain-derived oligodendrocytes is lower and more predominantly glycolytic compared to oligodendrocytes differentiated in vitro from post natal rat brain oligodendrocyte progenitor cells. In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.

Inhibition of chemokine expression in rat inflamed paws by systemic use of the antihyperalgesic oxidized ATP

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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.

Comparative effect of lidocaine and bupivacaine on glucose uptake and lactate production in the perfused working rat heart

International Nuclear Information System (INIS)

Cronau, L.H. Jr.; Merin, R.G.; Aboulish, E.; Steenberg, M.L.; Maljorda, A.

1986-01-01

It has been suggested that at equivalent therapeutic concentrations, lidocaine and bupivacaine may have different cardiotoxic potency. In the isolated working rat heart preparation, the effect of a range of lidocaine and bupivacaine concentrations on glucose uptake and lactate production (LP) were observed. Insulin was added, 10 μ/L, to Ringer's solution containing 3 H-labeled glucose to measure the glycolytic flux (GF). The effect of the local anesthetics on LP at the indicated concentrations were similar. Lidocaine appears to depress the glycolytic flux from exogenous glucose to a lesser degree. Bupivacaine, 10 mg/L, depresses VO 2 to a greater degree than does lidocaine, 40 mg/L

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus

Energy Technology Data Exchange (ETDEWEB)

Nomata, Jiro [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601 (Japan); Terauchi, Kazuki [Department of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577 (Japan); Fujita, Yuichi, E-mail: fujita@agr.nagoya-u.ac.jp [Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601 (Japan)

2016-02-12

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN–BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e{sup –}) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratio of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase. - Highlights: • The stoichiometry of nitrogenase-like protochlorophyllide reductase was determined. • The minimal ATP/2e{sup –} ratio was 4, which coincides with that of nitrogenase. • The ATP/2e{sup –} ratio increases with increasing L-protein/NB-protein molar ratio. • DPOR has an intrinsic property, but retains features shared with nitrogenase.

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.

Ballast water compliance monitoring: A new application for ATP

Science.gov (United States)

Lo Curto, A.; Stehouwer, P.; Gianoli, C.; Schneider, G.; Raymond, M.; Bonamin, V.

2018-03-01

The coming into force of the USCG ballast water regulations and the IMO ballast water management convention resulted in the development of several technologies approved for the treatment of ballast water. To ensure compliance of these technologies, the development of rapid and robust analysis methods was necessary. In collaboration with the SGS Group (Switzerland) and LuminUltra (Canada), Aqua-tools (France) has developed an innovative Ballast Water Treatment Monitoring (BWTM) kit for rapid onboard testing. The affordable kit provides results in less than 1 h, is easy to use and durable ensuring that the ballast water treatment system on the ship is fully compliant with the discharge standards upon arrival in port. The core of this method is a combination of high-quality reagents (lysis solution and ATP 2G Luminase™ enzyme) not inhibited by salinity and a patented fast homogenizing method for ATP extraction developed for a higher ATP recovery from zooplankton and phytoplankton. Compared to traditional analysis methods, the BWTM Kit provides fast and accurate results for all three fractions of microorganisms (≥ 50 μm, ≥ 10 ÷ < 50 μm and bacteria). Preliminary tests carried out in cooperation with SGS showed that the proposed method was able to detect onboard the efficiency of the treatment systems used. Compliance limits were established for all size fractions and a correlation between the standard methods (microscopy, plate count, MPN) and ATP was evaluated. The BWTM kit can provide a fast indication of compliance or gross exceedance. The rare borderline cases, when encountered, of course require additional confirmation.

Diversity and regulation of ATP sulfurylase in photosynthetic organisms

Directory of Open Access Journals (Sweden)

Laura ePrioretti

2014-11-01

Full Text Available ATP sulfurylase (ATPS catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper challenge this view and suggest that ATPSes may have a crucial regulatory role in sulfate assimilation, at least in algae.In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s expression. Special attention is given to algae ATPSes. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPSes revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. For instance, algae ATPSes show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. It is interesting that, at least with respect to the number of cysteines, the ATPSes of eukaryotic algae are closer to the marine cyanobacteria of the genera Synechococcus and Prochlorococcus and are more distant from freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater.

Coupling of kinesin ATP turnover to translocation and microtubule regulation: one engine, many machines.

Science.gov (United States)

Friel, Claire T; Howard, Jonathon

2012-12-01

The cycle of ATP turnover is integral to the action of motor proteins. Here we discuss how variation in this cycle leads to variation of function observed amongst members of the kinesin superfamily of microtubule associated motor proteins. Variation in the ATP turnover cycle among superfamily members can tune the characteristic kinesin motor to one of the range of microtubule-based functions performed by kinesins. The speed at which ATP is hydrolysed affects the speed of translocation. The ratio of rate constants of ATP turnover in relation to association and dissociation from the microtubule influence the processivity of translocation. Variation in the rate-limiting step of the cycle can reverse the way in which the motor domain interacts with the microtubule producing non-motile kinesins. Because the ATP turnover cycle is not fully understood for the majority of kinesins, much work remains to show how the kinesin engine functions in such a wide variety of molecular machines.

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

Science.gov (United States)

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

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.

Optimum ratio of AET, ATP and serotonin applied in combinations determined with a reference to their toxicity

International Nuclear Information System (INIS)

Benova, D.K.; P''tev, I.Kh.

1985-01-01

In experiments on mice, a study was made of the quantitative dependence of toxicity of AET, ATP and serotonin applied in combinations. The toxicity decreased when ATP was combined with AET and increased when ATP of AET were combined with serotonin. The toxicity of a combination of all three substances was reduced by introducing high doses of ATP

Radioprotective effectiveness and toxicity of ATP, AET and serotonin applied individually or simultaneously to mice. Pt. 4

International Nuclear Information System (INIS)

Benova, D.K.; Putev, I.K.

1979-01-01

Interactions occuring between three drugs - AET, serotonin, and ATP - in simultaneous administration were studied quantitatively. Using isobologram techniques, paired drug combination were examined for synergism in protective action against radiation. For ATP+AET pairs, increase in ATP fraction enhanced protection. For ATP+serotonin pairs, peak effect was observed at 360 mg/kg b.w. of ATP and 12 mg/kg b.w. of serotonin. Higher ATP fractions lowered the effectiveness. The highest degree of synergism was found for AET+serotonin, with peak effect at 17 to 33 mg/kg of AET plus 11 to 7 mg/kg of serotonin. By applying a method specially elaborated to enable prediction of interactions between three drugs administered simultaneously and by making use of three-dimensional diagrams, the parts played by individual components of triple combinations in total effect were estimated and the component dose ratio providing maximum protection identified. The determining components in protection were found to be AET and serotonin, the latter being of greater importance. The rhole of ATP in total effect is small and enhancement may be noted only up to ATP doses of no more than 156 mg/kg. The maximum effectiveness dose ratio of serotonin:AET:ATP was identified as 1:2:7.5-9. (orig.) [de

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-12-18

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

NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages

Science.gov (United States)

Venter, Gerda; Oerlemans, Frank T. J. J.; Willemse, Marieke; Wijers, Mietske; Fransen, Jack A. M.; Wieringa, Bé

2014-01-01

Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+’s cytosolic role in the regulation of morphofunctional characteristics of macrophages. PMID:24824795

NAMPT-mediated salvage synthesis of NAD+ controls morphofunctional changes of macrophages.

Directory of Open Access Journals (Sweden)

Gerda Venter

Full Text Available Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H (i.e. NAD+ and NADH and NADP(H (i.e. NADP+ and NADPH play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT, found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+'s cytosolic role in the regulation of morphofunctional characteristics of macrophages.

Opposite effect of ATP on contraction force of tonic and phasic skeletal muscles in frogs.

Science.gov (United States)

Grishin, S N; Kamaliev, R R; Teplov, A Yu; Ziganshin, A U

2011-07-01

Experiments in vitro showed that ATP and adenosine equally suppressed contractions of frog m. sartorius, which belongs to the phasic type muscles. Adenosine receptors antagonist 8-SPT abolished the effect of adenosine, but did not change the effect of ATP. This fact proves the independence of signaling pathways of these purines. ATP produced an opposite effect on the tonic muscle m. cruralis and increased the force of its contraction. Adenosine produced an inhibitory effect on the force of m. cruralis contration. In this case, 8-SPT also eliminated the effect of adenosine, but did not change the effect of ATP. The potentiating effect of ATP was blocked by suramin, a nonselective antagonist of P2 receptors, which attests to their involvement into the effects of this purine. The opposite effects of purinergic regulation reflect fundamental differences in functional organization of phasic and tonic muscular systems. It was hypothesized that the increase in contraction force under the effect of ATP is a mechanism providing maitenance of the contracted state of tonic muscle without appreciable metabolic costs.

Glycolysis in contracting rat skeletal muscle is controlled by factors related to energy state

DEFF Research Database (Denmark)

Ørtenblad, Niels; Macdonald, Will A; Sahlin, Kent

2009-01-01

The control of glycolysis in contracting muscle is not fully understood. The aim of the present study was to examine whether activation of glycolysis is mediated by factors related to the energy state or by a direct effect of Ca2+ on the regulating enzymes. Extensor digitorum longus muscles from...... and 58% of those in Con respectively. Glycolytic rate in BTS was only 51% of that in Con but the relative contribution of ATP derived from PCr (phosphocreatine) and glycolysis and the relation between muscle contents of PCr and Lac (lactate) were not different. Prolonged cyanide incubation of quiescent...... contribution of energy delivered from PCr and glycolysis during both conditions suggests that the glycolytic rate is controlled by factors related to energy state....

Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP.

Science.gov (United States)

Carlier, M F; Criquet, P; Pantaloni, D; Korn, E D

1986-02-15

Cytochalasin D strongly inhibits the faster components in the reactions of actin filament depolymerization and elongation in the presence of 10 mM Tris-Cl-, pH 7.8, 0.2 mM dithiothreitol, 1 mM MgCl2, 0.1 mM CaCl2, and 0.2 mM ATP or ADP. Assuming an exclusive and total capping of the barbed end by the drug, the kinetic parameters derived at saturation by cytochalasin D refer to the pointed end and are 10-15-fold lower than at the barbed end. In ATP, the critical concentration increases with cytochalasin D up to 12-fold its value when both ends are free; as a result of the lowering of the free energy of nucleation by cytochalasin D, short oligomers of F-actin exist just above and below the critical concentration. Cytochalasin D interacts strongly with the barbed ends independently of the ADP-G-actin concentration (K = 0.5 nM-1). In contrast, the affinity of cytochalasin D decreases cooperatively with increasing ATP-G-actin concentration. These data are equally well accounted for by two different models: either cytochalasin D binds very poorly to ATP-capped filament ends whose proportion increases with actin concentration, or cytochalasin D binds equally well to ATP-ends and ADP-ends and also binds to actin dimers in ATP but not in ADP. A linear actin concentration dependence of the rate of growth was found at the pointed end, consistent with the virtual absence of an ATP cap at that end.

Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

Science.gov (United States)

Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Ray, Sougata Sinha; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

2015-03-01

Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

Double-lock ratchet mechanism revealing the role of  SER-344 in FoF1 ATP synthase

KAUST Repository

Beke-Somfai, T.

2011-03-07

In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi→ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γP-OADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.