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Sample records for glycolytically produced nadh

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

    Science.gov (United States)

    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.

  2. Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

    Science.gov (United States)

    Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

    2012-02-15

    In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Vemuri, Goutham; Eiteman, M.A; McEwen, J.E

    2007-01-01

    effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation...... Crabtree effect.’’ The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely...... respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree...

  4. Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo.

    Science.gov (United States)

    Yaseen, Mohammad A; Sutin, Jason; Wu, Weicheng; Fu, Buyin; Uhlirova, Hana; Devor, Anna; Boas, David A; Sakadžić, Sava

    2017-05-01

    Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

  5. 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.

  6. Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.

    2014-01-01

    Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyse substrate oxidation and as such it plays a key role in various biological processes such as aging, cell...... death and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labelled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining...... a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/ [lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD...

  7. Gravity Responsive NADH Oxidase of the Plasma Membrane

    Science.gov (United States)

    Morre, D. James (Inventor)

    2002-01-01

    A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

  8. 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

  9. Sources and implications of NADH/NAD+ redox imbalance in diabetes and its complications

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    Wu J

    2016-05-01

    Full Text Available Jinzi Wu,1Zhen Jin,1Hong Zheng,1,2Liang-Jun Yan1 1Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX, USA; 2Department of Basic Theory of Traditional Chinese Medicine, College of Basic Medicine, Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China Abstract: NAD+ is a fundamental molecule in metabolism and redox signaling. In diabetes and its complications, the balance between NADH and NAD+ can be severely perturbed. On one hand, NADH is overproduced due to influx of hyperglycemia to the glycolytic and Krebs cycle pathways and activation of the polyol pathway. On the other hand, NAD+ can be diminished or depleted by overactivation of poly ADP ribose polymerase that uses NAD+ as its substrate. Moreover, sirtuins, another class of enzymes that also use NAD+ as their substrate for catalyzing protein deacetylation reactions, can also affect cellular content of NAD+. Impairment of NAD+ regeneration enzymes such as lactate dehydrogenase in erythrocytes and complex I in mitochondria can also contribute to NADH accumulation and NAD+ deficiency. The consequence of NADH/NAD+ redox imbalance is initially reductive stress that eventually leads to oxidative stress and oxidative damage to macromolecules, including DNA, lipids, and proteins. Accordingly, redox imbalance-triggered oxidative damage has been thought to be a major factor contributing to the development of diabetes and its complications. Future studies on restoring NADH/NAD+ redox balance could provide further insights into design of novel antidiabetic strategies. Keywords: mitochondria, complex I, reactive oxygen species, polyol pathway, poly ADP ribosylation, sirtuins, oxidative stress, oxidative damage

  10. Pulse photolysis of NADH in the presence of cysteine

    International Nuclear Information System (INIS)

    Scheel, H.E.

    1976-01-01

    In the UV irradiation of NADH under anaerobic conditions, cysteine, which often acts as a radioprotective substance, has a sensitizing effect. With the aid of pulse photolysis, it was studied which reaction mechanisms in the presence or absence of cysteine are responsible for the damage to NADH in aqueous solution. In the absence of cysteine, the characteristic NADH absorption at 340 nm is reduced immediately after UV quanta have been absorbed by the adenine fraction of the molecules; in the presence of cysteine, a secondary reaction causes additional damage. The spectra of the intermediate products of NADH and cysteine have been recorded for different cysteine concentrations, and the reaction constants have been determined. These values suggest that the sensitizing effect is due to a reaction of NADH with radical anions produced by photolysis. (orig.) [de

  11. The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: role of NADH and consequences for insulin secretion.

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    Heart, Emma; Palo, Meridith; Womack, Trayce; Smith, Peter J S; Gray, Joshua P

    2012-01-15

    Pancreatic β-cells release insulin in response to elevation of glucose from basal (4-7mM) to stimulatory (8-16mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H(2)O(2)), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H(2)O(2) inhibit insulin secretion. Menadione, which produces H(2)O(2) via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H(2)O(2) production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1-10μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H(2)O(2) formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H(2)O(2) and menadione on insulin secretion. Published by Elsevier Inc.

  12. Frequent and recent retrotransposition of orthologous genes plays a role in the evolution of sperm glycolytic enzymes

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    de Villena Fernando

    2010-05-01

    Full Text Available Abstract Background The central metabolic pathway of glycolysis converts glucose to pyruvate, with the net production of 2 ATP and 2 NADH per glucose molecule. Each of the ten reactions in this pathway is typically catalyzed by multiple isozymes encoded by a multigene family. Several isozymes in this pathway are expressed only during spermatogenesis, and gene targeting studies indicate that they are essential for sperm function and male fertility in mouse. At least three of the novel glycolytic isozymes are encoded by retrogenes (Pgk2, Aldoart1, and Aldoart2. Their restricted expression profile suggests that retrotransposition may play a significant role in the evolution of sperm glycolytic enzymes. Results We conducted a comprehensive genomic analysis of glycolytic enzymes in the human and mouse genomes and identified several intronless copies for all enzymes in the pathway, except Pfk. Within each gene family, a single orthologous gene was typically retrotransposed frequently and independently in both species. Several retroposed sequences maintained open reading frames (ORFs and/or provided evidence of alternatively spliced exons. We analyzed expression of sequences with ORFs and Gpi1 transcript in mouse spermatogenic cells. Conclusions Our analysis detected frequent, recent, and lineage-specific retrotransposition of orthologous glycolytic enzymes in the human and mouse genomes. Retrotransposition events are associated with LINE/LTR and genomic integration is random. We found evidence for the alternative splicing of parent genes. Many retroposed sequences have maintained ORFs, suggesting a functional role for these genes.

  13. 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.

  14. Identification of NADH kinase activity in filamentous fungi and structural modelling of the novel enzyme from Fusarium oxysporum

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Papadakis, Emmanouil; Topakas, E.

    2008-01-01

    ATP-NADH kinase phosphorylates NADH to produce NADPH at the expense of ATP. The present study describes Fusarium oxysporum NADH kinase (ATP:NADH 2'-phosphotransferase, EC 2.7.1.86), a novel fungal enzyme capable of synthesizing NADPH using NADH as the preferred diphosphonicotinamide...

  15. 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.

  16. The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: Role of NADH and consequences for insulin secretion

    Energy Technology Data Exchange (ETDEWEB)

    Heart, Emma [Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543 (United States); Palo, Meridith; Womack, Trayce [Department of Science, United States Coast Guard Academy, New London, CT, 06320 (United States); Smith, Peter J.S. [Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543 (United States); Institute for Life Sciences, University of Southampton (United Kingdom); Gray, Joshua P., E-mail: Joshua.p.gray@uscga.edu [Cellular Dynamics Program, Marine Biological Laboratory, Woods Hole, MA, 02543 (United States); Department of Science, United States Coast Guard Academy, New London, CT, 06320 (United States)

    2012-01-15

    Pancreatic β-cells release insulin in response to elevation of glucose from basal (4–7 mM) to stimulatory (8–16 mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H{sub 2}O{sub 2}), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H{sub 2}O{sub 2} inhibit insulin secretion. Menadione, which produces H{sub 2}O{sub 2} via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H{sub 2}O{sub 2} production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1–10 μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H{sub 2}O{sub 2} formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H{sub 2}O{sub 2} and menadione on insulin secretion. -- Highlights: ► Menadione stimulation or inhibition of insulin secretion is dependent upon applied glucose levels. ► Menadione-dependent H{sub 2}O{sub 2} production is proportional to applied glucose levels. ► Quinone-mediated redox cycling

  17. The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: Role of NADH and consequences for insulin secretion

    International Nuclear Information System (INIS)

    Heart, Emma; Palo, Meridith; Womack, Trayce; Smith, Peter J.S.; Gray, Joshua P.

    2012-01-01

    Pancreatic β-cells release insulin in response to elevation of glucose from basal (4–7 mM) to stimulatory (8–16 mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H 2 O 2 ), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H 2 O 2 inhibit insulin secretion. Menadione, which produces H 2 O 2 via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H 2 O 2 production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1–10 μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H 2 O 2 formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H 2 O 2 and menadione on insulin secretion. -- Highlights: ► Menadione stimulation or inhibition of insulin secretion is dependent upon applied glucose levels. ► Menadione-dependent H 2 O 2 production is proportional to applied glucose levels. ► Quinone-mediated redox cycling is dependent on glycolysis

  18. Imaging of a glucose analog, calcium and NADH in neurons and astrocytes: dynamic responses to depolarization and sensitivity to pioglitazone

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    Pancani, Tristano; Anderson, Katie L.; Porter, Nada M.; Thibault, Olivier

    2011-01-01

    Neuronal Ca2+ dyshomeostasis associated with cognitive impairment and mediated by changes in several Ca2+ sources has been seen in animal models of both aging and diabetes. In the periphery, dysregulation of intracellular Ca2+ signals may contribute to the development of insulin resistance. In the brain, while it is well-established that type 2 diabetes mellitus is a risk factor for the development of dementia in the elderly, it is not clear whether Ca2+ dysregulation might also affect insulin sensitivity and glucose utilization. Here we present a combination of imaging techniques testing the disappearance of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as an indication of glycolytic activity in neurons and astrocytes. Our work shows that glucose utilization at rest is greater in neurons compared to astrocytes, and ceases upon activation in neurons with little change in astrocytes. Pretreatment of hippocampal cultures with pioglitazone, a drug used in the treatment of type 2 diabetes, significantly reduced glycolytic activity in neurons and enhanced it in astrocytes. This series of experiments, including FURA-2 and NADH imaging, provides results that are consistent with the idea that Ca2+ levels may rapidly alter glycolytic activity, and that downstream events beyond Ca2+ dysregulation with aging, may alter cellular metabolism in the brain. PMID:21978418

  19. Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie

    2009-01-01

    the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric...

  20. Influence of oxygen on NADH recycling and oxidative stress resistance systems in Lactobacillus panis PM1.

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

    2013-01-31

    Lactobacillus panis strain PM1 is an obligatory heterofermentative and aerotolerant microorganism that also produces 1,3-propanediol from glycerol. This study investigated the metabolic responses of L. panis PM1 to oxidative stress under aerobic conditions. Growth under aerobic culture triggered an early entrance of L. panis PM1 into the stationary phase along with marked changes in end-product profiles. A ten-fold higher concentration of hydrogen peroxide was accumulated during aerobic culture compared to microaerobic culture. This H2O2 level was sufficient for the complete inhibition of L. panis PM1 cell growth, along with a significant reduction in end-products typically found during anaerobic growth. In silico analysis revealed that L. panis possessed two genes for NADH oxidase and NADH peroxidase, but their expression levels were not significantly affected by the presence of oxygen. Specific activities for these two enzymes were observed in crude extracts from L. panis PM1. Enzyme assays demonstrated that the majority of the H2O2 in the culture media was the product of NADH: H2O2 oxidase which was constitutively-active under both aerobic and microaerobic conditions; whereas, NADH peroxidase was positively-activated by the presence of oxygen and had a long induction time in contrast to NADH oxidase. These observations indicated that a coupled NADH oxidase - NADH peroxidase system was the main oxidative stress resistance mechanism in L. panis PM1, and was regulated by oxygen availability. Under aerobic conditions, NADH is mainly reoxidized by the NADH oxidase - peroxidase system rather than through the production of ethanol (or 1,3-propanediol or succinic acid production if glycerol or citric acid is available). This system helped L. panis PM1 directly use oxygen in its energy metabolism by producing extra ATP in contrast to homofermentative lactobacilli.

  1. Identification of mitochondrial electron transport chain-mediated NADH radical formation by EPR spin-trapping techniques.

    Science.gov (United States)

    Matsuzaki, Satoshi; Kotake, Yashige; Humphries, Kenneth M

    2011-12-20

    The mitochondrial electron transport chain (ETC) is a major source of free radical production. However, due to the highly reactive nature of radical species and their short lifetimes, accurate detection and identification of these molecules in biological systems is challenging. The aim of this investigation was to determine the free radical species produced from the mitochondrial ETC by utilizing EPR spin-trapping techniques and the recently commercialized spin-trap, 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). We demonstrate that this spin-trap has the preferential quality of having minimal mitochondrial toxicity at concentrations required for radical detection. In rat heart mitochondria and submitochondrial particles supplied with NADH, the major species detected under physiological pH was a carbon-centered radical adduct, indicated by markedly large hyperfine coupling constant with hydrogen (a(H) > 2.0 mT). In the presence of the ETC inhibitors, the carbon-centered radical formation was increased and exhibited NADH concentration dependency. The same carbon-centered radical could also be produced with the NAD biosynthesis precursor, nicotinamide mononucleotide, in the presence of a catalytic amount of NADH. The results support the conclusion that the observed species is a complex I derived NADH radical. The formation of the NADH radical could be blocked by hydroxyl radical scavengers but not SOD. In vitro experiments confirmed that an NADH-radical is readily formed by hydroxyl radical but not superoxide anion, further implicating hydroxyl radical as an upstream mediator of NADH radical production. These findings demonstrate the identification of a novel mitochondrial radical species with potential physiological significance and highlight the diverse mechanisms and sites of production within the ETC.

  2. Crystallization of the NADH-oxidizing domain of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

    International Nuclear Information System (INIS)

    Tao, Minli; Türk, Karin; Diez, Joachim; Grütter, Markus G.; Fritz, Günter; Steuber, Julia

    2006-01-01

    The FAD domain of the NqrF subunit from the Na + -translocating NADH dehydrogenase from V. cholerae has been purified and crystallized. A complete data set was recorded at 3.1 Å. The Na + -translocating NADH:quinone oxidoreductase (Na + -NQR) from pathogenic and marine bacteria is a respiratory complex that couples the exergonic oxidation of NADH by quinone to the transport of Na + across the membrane. The NqrF subunit oxidizes NADH and transfers the electrons to other redox cofactors in the enzyme. The FAD-containing domain of NqrF has been expressed, purified and crystallized. The purified NqrF FAD domain exhibited high rates of NADH oxidation and contained stoichiometric amounts of the FAD cofactor. Initial crystallization of the flavin domain was achieved by the sitting-drop technique using a Cartesian MicroSys4000 robot. Optimization of the crystallization conditions yielded yellow hexagonal crystals with dimensions of 30 × 30 × 70 µm. The protein mainly crystallizes in long hexagonal needles with a diameter of up to 30 µm. Crystals diffract to 2.8 Å and belong to space group P622, with unit-cell parameters a = b = 145.3, c = 90.2 Å, α = β = 90, γ = 120°

  3. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    Science.gov (United States)

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

  4. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    International Nuclear Information System (INIS)

    Stoll, V.S.; Blanchard, J.S.

    1988-01-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [ 14 C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols

  5. NAD+/NADH and skeletal muscle mitochondrial adaptations to exercise

    Science.gov (United States)

    White, Amanda T.

    2012-01-01

    The pyridine nucleotides, NAD+ and NADH, are coenzymes that provide oxidoreductive power for the generation of ATP by mitochondria. In skeletal muscle, exercise perturbs the levels of NAD+, NADH, and consequently, the NAD+/NADH ratio, and initial research in this area focused on the contribution of redox control to ATP production. More recently, numerous signaling pathways that are sensitive to perturbations in NAD+(H) have come to the fore, as has an appreciation for the potential importance of compartmentation of NAD+(H) metabolism and its subsequent effects on various signaling pathways. These pathways, which include the sirtuin (SIRT) proteins SIRT1 and SIRT3, the poly(ADP-ribose) polymerase (PARP) proteins PARP1 and PARP2, and COOH-terminal binding protein (CtBP), are of particular interest because they potentially link changes in cellular redox state to both immediate, metabolic-related changes and transcriptional adaptations to exercise. In this review, we discuss what is known, and not known, about the contribution of NAD+(H) metabolism and these aforementioned proteins to mitochondrial adaptations to acute and chronic endurance exercise. PMID:22436696

  6. One-electron transfer reactions of the couple NAD./NADH

    International Nuclear Information System (INIS)

    Grodkowski, J.; Neta, P.; Carlson, B.W.; Miller, L.

    1983-01-01

    One-electron transfer reactions involving nicotinamide-adenine dinucleotide in its oxidized and reducd forms (NAD./NADH) were studied by pulse radiolysis in aqueous solutions. One-electron oxidation of NADH by various phenoxyl radicals and phenothiazine cation radicals was found to take place with rate constants in the range of 10 5 to 10 8 M -1 s -1 , depending on the redox potential of the oxidizing species. In all cases, NAD. is formed quantitatively with no indication for the existence of the protonated form (NADH + .). The spectrum of NAD., as well as the rates of oxidation of NADH by phenoxyl and by (chlorpromazine) + . were independent of pH between pH 4.5 and 13.5. Reaction of deuterated NADH indicated only a small kinetic isotope effect. All these findings point to an electron transfer mechanism. On the other hand, attempts to observe the reverse electron transfer, i.e., one-electron reduction of NAD. to NADH by radicals such as semiquinones, showed that k was less than 10 4 to 10 5 M -1 s -1 , so that it was unobservable. Consequently, it was not possible to achieve equilibrium conditions which would have permitted the direct measurement of the redox potential for NAD./NADH. One-electron reduction of NAD. appears to be an unlikely process. 1 table

  7. Redox-flexible NADH oxidase biosensor: A platform for various dehydrogenase bioassays and biosensors

    International Nuclear Information System (INIS)

    Serban, Simona; El Murr, Nabil

    2006-01-01

    A generic amperometric bioassay based on the enzymatic oxidation catalysed by the stable NADH oxidase (NAox) from Thermus thermophilus has been developed for NADH measurements. The NAox uses O 2 as its natural electron acceptor and produces H 2 O 2 in a two-electron process. Electrochemical and spectrophotometric experiments showed that the NAox used in this work, presents a very good activity towards its substrate and, in contrary to previously mentioned NADH oxidases, does not require the addition of any exogenous flavin cofactor neither to promote nor to maintain its activity. In addition, the NAox used also works with artificial electron acceptors like ferrocene derivatives. O 2 was successfully replaced by redox mediators such as hydroxymethyl ferrocene (FcCH 2 OH) for the regeneration of the active enzyme. Combining the NAox with the mediator and the horseradish peroxidase we developed an original, high sensitive 'redox-flexible' NADH amperometric bioassay working in a large window of applied potentials in both oxidation and reduction modes. The biosensor has a continuous and complementary linearity range permitting to measure NADH concentrations starting from 5 x 10 -6 M in reduction until 2 x 10 3 M in oxidation. This redox-flexibility allows choosing the applied potential in order to avoid electrochemical interferences. The association of the 'redox-flexible' concept with NADH dependent enzymes opens a novel strategy for dehydrogenases based bioassays and biosensors. The great number of dehydrogenases available makes the concept applicable for numerous substrates to analyse. Moreover it allows the development of a wide range of biosensors on the basis of a generic platform. This gives several advantages over the previous manufacturing techniques and offers a general and flexible scheme for the fabrication of biosensors presenting high sensitivities, wide calibration ranges and less affected by electrochemical interferences

  8. Improving methyl ketone production in Escherichia coli by heterologous expression of NADH-dependent FabG

    DEFF Research Database (Denmark)

    Goh, Ee Been; Chen, Yan; Petzold, Christopher J.

    2018-01-01

    balance, as fatty acid-derived pathways face the systematic metabolic challenge of net NADPH consumption (in large part, resulting from the key fatty acid biosynthetic enzyme FabG [β-ketoacyl-ACP reductase]) and net NADH production. In this study, we attempted to mitigate cofactor imbalance...... by heterologously expressing NADH-dependent, rather than NADPH-dependent, versions of FabG identified in previous studies. Of the four NADH-dependent versions of FabG tested in our previously best-reported methyl ketone-producing strain (EGS1895), the version from Acholeplasma laidlawii (Al_FabG) showed...... for the base strain (EGS1895) under fermentation conditions optimized in this study. Shotgun proteomic data for strains EGS2920 and EGS1895 during fed-batch fermentation were consistent with the goal of alleviating NADPH limitation through expression of Al_FabG. For example, relative to strain EGS1895, strain...

  9. Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

    Science.gov (United States)

    Löw, Hans; Crane, Frederick L; Morré, D James

    2012-11-01

    The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Genetically encoded probes for NAD+/NADH monitoring.

    Science.gov (United States)

    Bilan, Dmitry S; Belousov, Vsevolod V

    2016-11-01

    NAD + and NADH participate in many metabolic reactions. The NAD + /NADH ratio is an important parameter reflecting the general metabolic and redox state of different types of cells. For a long time, in situ and in vivo NAD + /NADH monitoring has been hampered by the lack of suitable tools. The recent development of genetically encoded indicators based on fluorescent proteins linked to specific nucleotide-binding domains has already helped to address this monitoring problem. In this review, we will focus on four available indicators: Peredox, Frex family probes, RexYFP and SoNar. Each indicator has advantages and limitations. We will also discuss the most important points that should be considered when selecting a suitable indicator for certain experimental conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  12. 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...

  13. A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

    Science.gov (United States)

    Wang, Jizhong; Yang, Chengli; Chen, Xing; Bao, Bingxin; Zhang, Xuan; Li, Dali; Du, Xingfan; Shi, Ruofu; Yang, Junfang; Zhu, Ronghui

    2016-08-01

    To find an efficient and cheap system for NAD(+) regeneration A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

  14. 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.

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

    Science.gov (United States)

    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.

  16. A radiometric method for the determination of NADH in subpicomole amounts

    International Nuclear Information System (INIS)

    Weber, G.; Rosenthal, W.; Oberdisse, E.

    1988-01-01

    A radiometric method has been devised for the determination of small quantities of NADH formed in preceding dehydrogenase reactions. In a coupled enzymatic reaction, phosphoglycerate kinase (PGK) catalyzes the transfer of [/sup 32/P]orthophosphate from [gamma-/sup 32/P]ATP to 3-phosphoglycerate; the intermediate, 1,3-[1-/sup 32/P]diphosphoglycerate, is dephosphorylated by glyceraldehyde-3-phosphate dehydrogenase (GAP-DH). [/sup 32/P]Orthophosphate is released proportionally to NADH and can be measured after adsorption of [gamma-/sup 32/P]ATP to activated charcoal. With this method, 0.2 pmol of NADH are detectable in the presence of a 10/sup 4/-fold excess of NAD over NADH

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

    Directory of Open Access Journals (Sweden)

    Robert W B Brown

    Full Text Available Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH and phosphoglycerate kinase (PGK: we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed.

  20. Lactate: link between glycolytic and oxidative metabolism.

    Science.gov (United States)

    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.

  1. 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.

  2. Dual utilization of NADPH and NADH cofactors enhances xylitol production in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Jo, Jung-Hyun; Oh, Sun-Young; Lee, Hyeun-Soo; Park, Yong-Cheol; Seo, Jin-Ho

    2015-12-01

    Xylitol, a natural sweetener, can be produced by hydrogenation of xylose in hemicelluloses. In microbial processes, utilization of only NADPH cofactor limited commercialization of xylitol biosynthesis. To overcome this drawback, Saccharomyces cerevisiae D452-2 was engineered to express two types of xylose reductase (XR) with either NADPH-dependence or NADH-preference. Engineered S. cerevisiae DWM expressing both the XRs exhibited higher xylitol productivity than the yeast strain expressing NADPH-dependent XR only (DWW) in both batch and glucose-limited fed-batch cultures. Furthermore, the coexpression of S. cerevisiae ZWF1 and ACS1 genes in the DWM strain increased intracellular concentrations of NADPH and NADH and improved maximum xylitol productivity by 17%, relative to that for the DWM strain. Finally, the optimized fed-batch fermentation of S. cerevisiae DWM-ZWF1-ACS1 resulted in 196.2 g/L xylitol concentration, 4.27 g/L h productivity and almost the theoretical yield. Expression of the two types of XR utilizing both NADPH and NADH is a promising strategy to meet the industrial demands for microbial xylitol production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Stabilized NADH as a Countermeasure for Jet Lag

    Science.gov (United States)

    Kay, Gary G.; Viirre, Erik; Clark, Jonathan

    2001-01-01

    Current remedies for jet lag (phototherapy, melatonin, stimulant, and sedative medications) are limited in efficacy and practicality. The efficacy of a stabilized, sublingual form of reduced nicotin amide adenine dinucleotide (NADH, ENADAlert, Menuco Corp.) as a countermeasure for jet lag was examined. Because NADH increases cellular production of ATP and facilitates dopamine synthesis, it may counteract the effects of jet lag on cognitive functioning and sleepiness. Thirty-five healthy, employed subjects participated in this double-blind, placebo-controlled study. Training and baseline testing were conducted on the West Coast before subjects flew overnight to the East Coast, where they would experience a 3-hour time difference. Upon arrival, individuals were randomly assigned to receive either 20 mg of sublingual stabilized ADH (n=18) or identical placebo tablets (n=17). All participants completed computer-administered tests (including CogScreen7) to assess changes in cognitive functioning, mood, and sleepiness in the morning and afternoon. Jet lag resulted in increased sleepiness for over half the participants and deterioration of cognitive functioning for approximately one third. The morning following the flight, subjects experienced lapses of attention in addition to disruptions in working memory, divided attention, and visual perceptual speed. Individuals who received NADH performed significantly better on 5 of 8 cognitive and psychomotor test measures (P less than or equal to 0.5) and showed a trend for better performance on the other three measures (P less than or equal to .l0). Subjects also reported less sleepiness compared with those who received placebo. No adverse effects were observed with NADH treatment. Stabilized NADH significantly reduced jet lag-induced disruptions of cognitive functioning, was easily administered, and was found to have no adverse side effects.

  4. Regulation of hydrogen production by Enterobacter aerogenes by external NADH and NAD{sup +}

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Chong; Ma, Kun; Xing, Xin-Hui [Department of Chemical Engineering, Tsinghua University, Beijing 100084 (China)

    2009-02-15

    Experiments involving the addition of external nicotinamide adenine dinucleotide, reduced form (NADH) or nicotinamide adenine dinucleotide (NAD{sup +}) have been designed to examine how the hydrogen in Enterobacter aerogenes is liberated by NADH or NAD{sup +}. The addition of external NADH or NAD{sup +} was found to regulate hydrogen production by E. aerogenes in resting cells, batch cultures, and chemostat cultures. Particularly in chemostat cultivation, with the external addition of NADH, hydrogen production via the NADH pathway was decreased, while that via the formate pathway was increased; in the end, the overall hydrogen p was decreased. The addition of NAD{sup +}, on the other hand, gave the opposite results. The membrane-bound hydrogenase was found to play a central role in regulating hydrogen production. The occurrence of NADH oxidation (NAD{sup +} reduction) on the cell membrane resulted in an electron flow across the membrane; this changed the oxidation state and metabolic pattern of the cells, which eventually affected the hydrogen evolution. (author)

  5. Overexpression of a novel endogenous NADH kinase in Aspergillus nidulans enhances growth

    DEFF Research Database (Denmark)

    Panagiotou, Gianni; Grotkjær, Thomas; Hofmann, Gerald

    2009-01-01

    .7.1.86) has been identified. The enzyme has a predicted molecular weight of 49 kDa. We characterised the role of this NADH kinase by genomic integration of the putative gene AN8837.2 under a strong constitutive promoter. The physiological effects of overexpressed NADH kinase in combination with different...... yield on glucose and the maximum specific growth rate increased from 0.47 g/g and 0.22 h(-1) (wild type) to 0.54 g/g and 0.26 h(-1) (NADH kinase overexpressed), respectively. The results suggest that overexpression of NADH kinase improves the growth efficiency of the cell by increasing the access...

  6. Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions.

    Science.gov (United States)

    Heux, Stéphanie; Sablayrolles, Jean-Marie; Cachon, Rémy; Dequin, Sylvie

    2006-09-01

    We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.

  7. Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

    Science.gov (United States)

    Marty-Teysset, C.; de la Torre, F.; Garel, J.-R.

    2000-01-01

    The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H2O2 oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen. PMID:10618234

  8. 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.

  9. Metabolic control by sirtuins and other enzymes that sense NAD+, NADH, or their ratio.

    Science.gov (United States)

    Anderson, Kristin A; Madsen, Andreas S; Olsen, Christian A; Hirschey, Matthew D

    2017-12-01

    NAD + is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD + , NADH, and the NAD + /NADH ratio have long been known to control the activity of several oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD + -dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD + , NADH, or NAD + /NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD + , but neither NADH nor the ratio. Finally, we identify future studies that might be informative to further interrogate physiological and pathophysiological changes in NAD + and NADH, as well as enzymes like sirtuins that sense and respond to redox changes in the cell. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Metabolic control by sirtuins and other enzymes that sense NAD(+), NADH, or their ratio

    DEFF Research Database (Denmark)

    Anderson, Kristin A; Madsen, Andreas S; Olsen, Christian A

    2017-01-01

    NAD(+) is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD(+), NADH, and the NAD(+)/NADH ratio have long been known to control the activity of several...... oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD(+)-dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD......(+), NADH, or NAD(+)/NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD(+), but neither NADH nor the ratio. Finally, we...

  11. Cofactor engineering to regulate NAD+/NADH ratio with its application to phytosterols biotransformation.

    Science.gov (United States)

    Su, Liqiu; Shen, Yanbing; Zhang, Wenkai; Gao, Tian; Shang, Zhihua; Wang, Min

    2017-10-30

    Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D

  12. Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

    Science.gov (United States)

    Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

    2015-02-01

    Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. Copyright © 2015 Elsevier GmbH. All rights reserved.

  13. Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

    Science.gov (United States)

    Danylovych, H V

    2016-01-01

    We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

  14. The glycolytic pathway of Trimastix pyriformis is an evolutionary mosaic

    Directory of Open Access Journals (Sweden)

    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

  15. Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence.

    Science.gov (United States)

    Li, Dong; Zheng, Wei; Qu, Jianan Y

    2008-10-15

    A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.

  16. The unique features of glycolytic pathways in Archaea.

    Science.gov (United States)

    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.

  17. NADH oxidation on screen-printed electrode modified with a new phenothiazine diazonium salt

    Energy Technology Data Exchange (ETDEWEB)

    Doumeche, Bastien; Blum, Loic J. [GEMBAS, Genie Enzymatique, Membranes Biomimetiques et Assemblages Supramoleculaires, ICBMS UMR 5246, Universite Lyon 1, 43 bd du 11 Novembre 1918, 69622 Villeurbanne (France)

    2010-10-15

    NADH oxidation catalysts are extremely important in the field of electrochemical biosensors and enzymatic biofuel cells. Based on the growing diazonium chemistry, we synthesized the diazonium salt of the well-known NADH mediator toluidine blue O. The electrochemical reduction of the diazonium moiety by cyclic voltammetry onto a screen-printed electrode leads to an electrocatalyst suitable for the oxidation of NADH. The amperometric response for its oxidation shows a maximal current of 1.2 {mu}A ([NADH] = 100 {mu}M). Based on electrochemical measurements, the surface coverage is found to be 3.78 x 10{sup -11} mol cm{sup -2} and the heterogeneous standard rate constant k{sub h} is 1.21 {+-} 0.16 s{sup -1}. The sensitive layer for the oxidation of NADH is improved by electrografting the diazonium salt with a potentiostatic method. Both the surface coverage and the heterogeneous standard rate constant k{sub h} are improved and found to be 6.08 {+-} 0.63 x 10{sup -11} mol cm{sup -2} and {proportional_to} 5.02 s{sup -} {sup 1}, respectively. The amperometric response is also improved by an 8 fold factor, reaching 9.87 {mu}A ([NADH] = 120 {mu}M). These remarkably high values for screen-printed electrodes are comparable to glassy carbon electrodes making this method suitable for low-cost bioelectronical devices. (author)

  18. Involvement of NADH Oxidase in Competition and Endocarditis Virulence in Streptococcus sanguinis.

    Science.gov (United States)

    Ge, Xiuchun; Yu, Yang; Zhang, Min; Chen, Lei; Chen, Weihua; Elrami, Fadi; Kong, Fanxiang; Kitten, Todd; Xu, Ping

    2016-05-01

    Here, we report for the first time that the Streptococcus sanguinis nox gene encoding NADH oxidase is involved in both competition with Streptococcus mutans and virulence for infective endocarditis. An S. sanguinis nox mutant was found to fail to inhibit the growth of Streptococcus mutans under microaerobic conditions. In the presence of oxygen, the recombinant Nox protein of S. sanguinis could reduce oxygen to water and oxidize NADH to NAD(+) The oxidation of NADH to NAD(+) was diminished in the nox mutant. The nox mutant exhibited decreased levels of extracellular H2O2; however, the intracellular level of H2O2 in the mutant was increased. Furthermore, the virulence of the nox mutant was attenuated in a rabbit endocarditis model. The nox mutant also was shown to be more sensitive to blood killing, oxidative and acid stresses, and reduced growth in serum. Thus, NADH oxidase contributes to multiple phenotypes related to competitiveness in the oral cavity and systemic virulence. Copyright © 2016 Ge et al.

  19. Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Vemuri, G. N.; Bao, X. M.

    2009-01-01

    of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase...

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    ,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

  1. Amperometric sensing of NADH and ethanol using a hybrid film electrode modified with electrochemically fabricated zirconia nanotubes and poly (acid fuchsin)

    International Nuclear Information System (INIS)

    Liu, X.; Li, B.; Zhan, G.; Liu, C.; Li, C.; Ma, M.

    2012-01-01

    We report on a glassy carbon electrode (GCE) modified with a film of chitosin containing acid fuchsin (AF) adsorbed onto zirconia nanotubes. The mixture was polymerized by cyclic voltammetric scannings in the potential range from - 0. 8 V to +1. 3 V in buffer solution to produce a hybrid film electrode (nano-ZrO 2 /PAF/GCE). The morphology of the hybrid film electrode surface was characterized by scanning electron microscopy. Its electrochemical properties were studied via electrochemical impedance spectroscopy. The electrochemical response of nicotinamide adenine dinucleotide (NADH) was investigated by differential pulse voltammetry and amperometry. The results indicated that the nano-ZrO 2 /PAF/GCE possesses well synergistic catalytic activity towards NADH. Compared to an unmodified GCE, the oxidation overpotential is negatively shifted by 224 mV, and the oxidation current is significantly increased. Under optimal conditions, the amperometric response is linearly proportional to the concentration of NADH in the 1. 0 - 100. 0 μM concentration range. Ethanol also can be determined by amperometry if alcohol dehydrogenase and NADH are added to the sample. Two linear relationships between current and alcohol concentration were obtained. They cover the range from 0. 03 to 1. 0 mM, and from 1. 0 to 12. 0 mM. (author)

  2. NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

    Science.gov (United States)

    Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

    2018-01-20

    The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

  3. Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses.

    Science.gov (United States)

    Bhat, Shabir A; Iqbal, Iram K; Kumar, Ashwani

    2016-01-01

    The NADH:NAD + ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD + ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD + ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD + levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD + ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD + ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further

  4. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

    Science.gov (United States)

    Titov, Denis V; Cracan, Valentin; Goodman, Russell P; Peng, Jun; Grabarek, Zenon; Mootha, Vamsi K

    2016-04-08

    A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state. Copyright © 2016, American Association for the Advancement of Science.

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

    Science.gov (United States)

    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.

  6. Characterization of vanadate-dependent NADH oxidation activity and isolation of yeast DNA which complements a class 1 vanadate resistance mutation

    International Nuclear Information System (INIS)

    Minasi, L.E.

    1989-01-01

    A vanadate-dependent NADH oxidation activity has been characterized in plasma membranes from the yeast S cerevisiae. NADH oxidation activity was maximally stimulated at pH 5.0 in phosphate buffer. NADH oxidation was not dependent on the concentration of plasma membranes. The vanadate-dependent NADH oxidation activity was abolished under anaerobic conditions and the concomitant uptake of oxygen occurred during NADH oxidation. The activity was inhibited by superoxide dismutase and stimulated by the presence of paraquat. These results indicate that the vanadate stimulation of NADH oxidation in yeast plasma membranes occurs as a result of the vanadate-dependent oxidation of NADH by superoxide, generated by a plasma membrane NADH oxidase. 51 V-NMR results indicated that a phosphate-vanadate anhydride was the stimulatory species in pH 5.0 and pH 7.0 phosphate buffer. Yeast DNA has been isolated which complements a class 1 vanadate resistance mutation

  7. Over-expression of NADH-dependent oxidoreductase (fucO) for increasing furfural or 5-hydroxymethylfurfural tolerance

    Science.gov (United States)

    Miller, Elliot N.; Zhang, Xueli; Yomano, Lorraine P.; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2015-10-13

    The subject invention pertains to the discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural. This allows for a new approach to improve furfural tolerance in bacterial and/or yeast cells used to produce desired products. Thus, novel biocatalysts (bacterial, fungal or yeast cells) exhibiting increased tolerance to furfural and 5-hydroxymethylfurfural (5-HMF) are provided as are methods of making and using such biocatalysts for the production of a desired product.

  8. Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

    Science.gov (United States)

    Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

    2011-01-01

    NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD+ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD+ biosynthesis in cells harboring the ntt4 gene. The minimal NAD+ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed. PMID:21742902

  9. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    use

    2011-12-07

    Dec 7, 2011 ... potent inhibitors of NADH oxidases, silver nitrate and potassium cyanide did not show any significant ... anaerobes, a class of organisms that have not been ... DNA and amino acid sequence analyses were performed using.

  10. Crystallization and preliminary crystallographic analysis of a flavoprotein NADH oxidase from Lactobacillus brevis

    International Nuclear Information System (INIS)

    Kuzu, Mutlu; Niefind, Karsten; Hummel, Werner; Schomburg, Dietmar

    2005-01-01

    The water-forming flavoenzyme NADH oxidase was crystallized successfully for the first time. The crystals diffract X-rays to at least 4.0 Å resolution. NADH oxidase (NOX) from Lactobacillus brevis is a homotetrameric flavoenzyme composed of 450 amino acids per subunit. The molecular weight of each monomer is 48.8 kDa. The enzyme catalyzes the oxidation of two equivalents of NADH and reduces one equivalent of oxygen to yield two equivalents of water, without releasing hydrogen peroxide after the reduction of the first equivalent of NADH. Crystals of this protein were grown in the presence of 34% polyethylene glycol monomethyl ether 2000, 0.1 M sodium acetate and 0.2 M ammonium sulfate at pH 5.4. They belong to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 Å, α = γ = 90, β = 103.8°. The current diffraction limit is 4.0 Å. The self-rotation function of the native data set is consistent with a NOX tetramer in the asymmetric unit

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

    Science.gov (United States)

    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.

  12. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  13. Alterations in cerebral metabolism observed in living rodents using fluorescence lifetime microscopy of intrinsic NADH (Conference Presentation)

    Science.gov (United States)

    Yaseen, Mohammad A.; Sakadžić, Sava; Sutin, Jason; Wu, Weicheng; Fu, Buyin; Boas, David A.

    2017-02-01

    Monitoring cerebral energy metabolism at a cellular level is essential to improve our understanding of healthy brain function and its pathological alterations. In this study, we resolve specific alterations in cerebral metabolism utilizing minimally-invasive 2-Photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence, collected in vivo from anesthetized rats and mice. Time-resolved lifetime measurements enables distinction of different components contributing to NADH autofluorescence. These components reportedly represent different enzyme-bound formulations of NADH. Our observations from this study confirm the hypothesis that NADH FLIM can identify specific alterations in cerebral metabolism. Using time-correlated single photon counting (TCSPC) equipment and a custom-built multimodal imaging system, 2-photon fluorescence lifetime imaging (FLIM) was performed in cerebral tissue with high spatial and temporal resolution. Multi-exponential fits for NADH fluorescence lifetimes indicate 4 distinct components, or 'species.' We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in anaerobic glycolysis and aerobic oxidative metabolism. Classification models developed with experimental data correctly predict the metabolic impairments associated with bicuculline-induced focal seizures in separate experiments. Compared to traditional intensity-based NADH measurements, lifetime imaging of NADH is less susceptible to the adverse effects of overlying blood vessels. Evaluating NADH measurements will ultimately lead to a deeper understanding of cerebral energetics and its pathology-related alterations. Such knowledge will likely aid development of therapeutic strategies for neurodegenerative diseases such as Alzheimer's Disease, Parkinson's disease, and stroke.

  14. Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

    OpenAIRE

    Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

    2011-01-01

    NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. co...

  15. Cold stress decreases the capacity for respiratory NADH oxidation in potato leaves

    DEFF Research Database (Denmark)

    Svensson, Å.S.; Johansson, F.I.; Møller, I.M.

    2002-01-01

    is 10% of the original level. This decrease is accompanied by specific decreases of immunodetected NDA protein and internal rotenone-insensitive NADH oxidation in mitochondria isolated from cold-treated plants. The alternative oxidase is not cold-induced neither at the protein nor at the activity level......Cold stress effects on the expression of genes for respiratory chain enzymes were investigated in potato (Solarium tuberosum L., cv. Desiree) leaves. The nda1 and ndb1 genes, homologues to genes encoding the non-proton-pumping respiratory chain NADH dehydrogenases of Escherichia coli and yeast......, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). Using a real-time PCR system, we demonstrate a specific and gradual decrease of the NDA1 transcript after exposing the plants to 5 C. After 6 days of cold treatment the NDA1 transcript abundance...

  16. Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Sporty, J; Kabir, M M; Turteltaub, K; Ognibene, T; Lin, S; Bench, G

    2008-01-10

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

  17. The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Westerhoff, H.V.; Snoep, J.L.

    2002-01-01

    Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch...... cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis...... (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells....

  18. Study of glycolytic intermediates in hereditary elliptocytosis with thalassemia

    Directory of Open Access Journals (Sweden)

    Pavri Roshan

    1977-01-01

    Full Text Available Glycolytic intermediates like ATP, DPG and GSH have been studied in a family with. hereditary elliptocytosis and thalassemia. Results indicate a fall in ATP with a concomitant rise in DPG in the Patient. Findings are discussed in relation to other data.

  19. Cloning and mRNA Expression of NADH Dehydrogenase during Ochlerotatus taeniorhynchus Development and Pesticide Response

    Science.gov (United States)

    NADH dehydrogenase, the largest of the respiratory complexes, is the first enzyme of the mitochondrial electron transport chain. We have cloned and sequenced cDNA of NADH dehydrogenase gene from Ochlerotatus (Ochlerotatus) taeniorhynchus (Wiedemann) adult (GeneBank Accession number: FJ458415). The ...

  20. Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)*

    Science.gov (United States)

    Elguindy, Mahmoud M.; Nakamaru-Ogiso, Eiko

    2015-01-01

    Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. PMID:26063804

  1. Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).

    Science.gov (United States)

    Elguindy, Mahmoud M; Nakamaru-Ogiso, Eiko

    2015-08-21

    Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions

    DEFF Research Database (Denmark)

    Kasimova, M.R.; Grigiene, J.; Krab, K.

    2006-01-01

    with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins...... metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism....

  4. 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

  5. Gene cloning and characterization of NADH oxidase from ...

    African Journals Online (AJOL)

    The genome search of Thermococcus kodakarensis revealed three open reading frames, Tk0304, Tk1299 and Tk1392 annotated as nicotinamide adenine dinucleotide (NADH) oxidases. This study deals with cloning, and characterization of Tk0304. The gene, composed of 1320 nucleotides, encodes a protein of 439 ...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  8. Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

    Science.gov (United States)

    Tejwani, Vijay; Schmitt, Franz-Josef; Wilkening, Svea; Zebger, Ingo; Horch, Marius; Lenz, Oliver; Friedrich, Thomas

    2017-01-01

    Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H 2 -driven production of biodegradable polymers and hydrocarbons. H 2 oxidation by R. eutropha takes place in the presence of O 2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H 2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H 2 oxidation with the reduction of NAD + to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD + pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD + ] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD + ] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD + ] ratios represents a novel and sensitive tool to determine the redox state of cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions.

    Science.gov (United States)

    Kasischke, Karl A; Lambert, Elton M; Panepento, Ben; Sun, Anita; Gelbard, Harris A; Burgess, Robert W; Foster, Thomas H; Nedergaard, Maiken

    2011-01-01

    Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO(2) range with a p(50) of 3.4 ± 0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution.

  10. Rotational barriers of 1,3-substitute pyridines and benzenes as models for the NAD+/NADH coenzyme

    NARCIS (Netherlands)

    Vanhommerig, S.A.M.; Meier, R.J.; Sluyterman, L.A.A.E.; Meijer, E.M.

    1994-01-01

    The NAD+/NADH coenzyme is involved in many enzyme-catalysed oxidation-reduction reactions. In order to obtain better insight in the catalytic mechanism of NAD+/NADH dependent dehydrogenases, conformational studies of 1,3-substituted pyridines and benzenes were carried out, using ab initio,

  11. Nikkaji Dictionary: デアミノNADH [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term デアミノNADH 名詞 一般 * * * * デアミノNADH ... Nikkaji J707.348H 200906099845094600 C CA06 UNKNOWN_2 デアミノ N AD H

  12. Investigation of the Ionization Mechanism of NAD+/NADH-Modified Gold Electrodes in ToF-SIMS Analysis.

    Science.gov (United States)

    Hua, Xin; Zhao, Li-Jun; Long, Yi-Tao

    2018-06-04

    Analysis of nicotinamide adenine dinucleotide (NAD + /NADH)-modified electrodes is important for in vitro monitoring of key biological processes. In this work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to analyze NAD + /NADH-modified gold electrodes. Interestingly, no obvious characteristic peaks of nicotinamide fragment could be observed in the mass spectra of NAD + /NADH in their neutral sodium pyrophosphate form. However, after acidification, the characteristic peaks for both NAD + and NADH were detected. This was due to the suppression effect of inner pyrophosphoric salts in both neutral molecules. Besides, it was proved that the suppression by inner salt was intramolecular. No obvious suppression was found between neighboring molecules. These results demonstrated the suppression effect of inner salts in ToF-SIMS analysis, providing useful evidence for the study of ToF-SIMS ionization mechanism of organic molecule-modified electrodes. Graphical Abstract ᅟ.

  13. The glycolytic flux in Escherichia coli is controlled by the demand for ATP

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Westerhoff, H.V.; Snoep, J.L.

    2002-01-01

    additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (> 75%) resides...... not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i...

  14. 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...

  15. 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....

  16. Preparation and electrochemical application of rutin biosensor for differential pulse voltammetric determination of NADH in the presence of acetaminophen

    Directory of Open Access Journals (Sweden)

    HAMID R. ZARE

    2010-10-01

    Full Text Available The electrocatalytic behavior of reduced nicotinamide adenine di-nucleotide (NADH was studied at the surface of a rutin biosensor, using various electrochemical methods. According to the results, the rutin biosensor had a strongly electrocatalytic effect on the oxidation of NADH with the overpotential being decreased by about 450 mV as compared to the process at a bare glassy carbon electrode, GCE. This value is significantly greater than the value of 220 mV that was reported for rutin embedded in a lipid-cast film. The kinetic parameters of the electron transfer coefficient, a, and the heterogeneous charge transfer rate constant, kh, for the electrocatalytic oxidation of NADH at the rutin biosensor were estimated. Furthermore, the linear dynamic range; sensitivity and limit of detection for NADH were evaluated using the differential pulse voltammetry method. The advantages of this biosensor for the determination of NADH are excellent catalytic activity and reproducibility, good detection limit and high exchange current density. The rutin biosensor could separate the oxidation peak potentials of NADH and acetaminophen present in the same solution while at a bare GCE, the peak potentials were indistinguishable.

  17. Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values.

    Science.gov (United States)

    Wilkening, Svea; Schmitt, Franz-Josef; Horch, Marius; Zebger, Ingo; Lenz, Oliver; Friedrich, Thomas

    2017-09-01

    The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D  ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD + concentration, while the apparent K D for NADH is only slightly affected. We found that NAD + has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD + dissociation constant of K I  ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD + hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD + depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD + concentrations below 100 µM.

  18. Characterization of water-forming NADH oxidases for co-factor regeneration

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; J. Charnock, Simon

    an environmentaland economic perspective [1]. Alcohol dehydrogenases (ADH) offer one such alternative. However, the reaction requires the oxidized nicotinamide co-factor (NAD+) that must be recycled due to its high cost contribution. One regeneration method that offers certain advantages is the oxidation of NADH......Traditional chemical methods for alcohol oxidation are often associated with issues such as high consumption of expensive oxidizing agents, generation of metal waste and the use of environmentally undesirable organic solvents. Developing green, selective catalysts is therefore important from...... using water forming NADH oxidases (NOX-2). The implementation of the ADH/NOX system for alcohol oxidation, however, requires consideration of several different issues. Enzyme activity and stability at relevant pH and temperature conditions, but also the tolerance to the substrates and products present...

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions

    OpenAIRE

    Kasischke, Karl A; Lambert, Elton M; Panepento, Ben; Sun, Anita; Gelbard, Harris A; Burgess, Robert W; Foster, Thomas H; Nedergaard, Maiken

    2010-01-01

    Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO2 range with a p ...

  1. 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

  2. The increase of NADH fluorescence lifetime is associated with the metabolic change during osteogenic differentiation of human mesenchymal stem cells (hMSCs)

    Science.gov (United States)

    Guo, Han Wen; Yu, Jia Sin; Hsu, Shu Han; Wei, Yau Huei; Lee, Oscar K.; Wang, Hsing Wen

    2011-03-01

    Fluorescence lifetime of NADH had been used as an optical marker for monitoring cellular metabolism. In our pervious studies, we have demonstrated that NADH lifetime of hMSCs increase gradually with time of osteogenic differentiation. In this study, we measured NADH lifetime of hMSCs from a different donor as well as the corresponding metabolic indices such as ATP level, oxygen consumption and lactate release. We also measure the quantity of Complex I, III, IV and V. The results show that during differentiation more oxygen consumed, higher ATP level expressed and less lactate released, and the increase of NADH lifetime was associated with ATP level. Higher expression of the total Complex protein was observed at 3 and 4 weeks after differentiation than controls. However, Complex I expression did not show significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime was associated with the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria after differentiation.

  3. Direct electrocatalytic reduction of coenzyme NAD{sup +} to enzymatically-active 1,4-NADH employing an iridium/ruthenium-oxide electrode

    Energy Technology Data Exchange (ETDEWEB)

    Ullah, Nehar, E-mail: nehar.ullah@mail.mcgill.ca; Ali, Irshad; Omanovic, Sasha

    2015-01-15

    A thermally prepared iridium/ruthenium-oxide coating (Ir{sub 0.8}Ru{sub 0.2}-oxide) formed on a titanium substrate was investigated as a possible electrode for direct electrochemical regeneration of enzymatically-active 1,4-NADH from its oxidized form NAD{sup +}, at various electrode potentials, in a batch electrochemical reactor. The coating surface was characterized by ‘cracked mud’ morphology, yielding a high surface roughness. The NADH regeneration results showed that the percentage of enzymatically-active 1,4-NADH present in the product mixture (i.e. recovery) is strongly dependent on the electrode potential, reaching a maximum (88%) at −1.70 V vs. MSE. The relatively high recovery was explained on the basis of availability of adsorbed ‘active’ hydrogen (H{sub ads}) on the Ir/Ru-oxide surface, i.e. on the basis of electrochemical hydrogenation. - Highlights: • Ir{sub 0.8}Ru{sub 0.2}-oxide coating was formed thermally on a Ti substrate. • Electrochemical regeneration of enzymatically-active 1,4-NADH was investigated. • The 1,4-NADH recovery percentage is strongly dependent on the electrode potential. • A highest recovery, 88%, was obtained at −1.70 V vs. MSE. • The NADH regeneration process involved electrochemical hydrogenation.

  4. The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

    OpenAIRE

    Jackson, R H; Cole, J A; Cornish-Bowden, A

    1981-01-01

    The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a m...

  5. 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

  6. 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.

  7. Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    Full Text Available Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

  8. 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.

  9. Intracellular NAD(H) levels control motility and invasion of glioma cells.

    NARCIS (Netherlands)

    Horssen, R. van; Willemse, M.P.; Haeger, A.; Attanasio, F.; Guneri, T.; Schwab, A.; Stock, C.M.; Buccione, R.; Fransen, J.A.M.; Wieringa, B.

    2013-01-01

    Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting

  10. 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).

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

    Science.gov (United States)

    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.

  12. Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH

    DEFF Research Database (Denmark)

    Madsen, Andreas Stahl; Andersen, Christian; Daoud, Mohammad Mahdi

    2016-01-01

    Protein lysine posttranslational modification by an increasing number of different acyl groups is becoming appreciated as a regulatory mechanism in cellular biology. Sirtuins are class III histone deacylases that use NAD+ as a co-substrate during amide bond hydrolysis. Several studies have...... described the sirtuins as sensors of the NAD+/NADH ratio, but it has not been formally tested for all the mammalian sirtuins in vitro. To address this problem, we first synthesized a wide variety of peptide-based probes, which were used to identify the range of hydrolytic activities of human sirtuins...... to be prone to hydrolytic cleavage by SIRT1-3 and SIRT6, supporting recent findings. We then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit these NAD+-dependent deacylase activities of the sirtuins. In the commonly used 7-amino-4-methylcoumarin-coupled fluorescence-based assay...

  13. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  14. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  15. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    International Nuclear Information System (INIS)

    Kohio, Hinissan P.; Adamson, Amy L.

    2013-01-01

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

  16. Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri.

    Science.gov (United States)

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K

    2008-02-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.

  17. 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.

  18. Lambda Red-mediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

    Science.gov (United States)

    Pohl, Thomas; Uhlmann, Mareike; Kaufenstein, Miriam; Friedrich, Thorsten

    2007-09-18

    The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

  19. Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants

    DEFF Research Database (Denmark)

    Hollinshead, Whitney D.; Rodriguez, Sarah; Martin, Hector Garcia

    2016-01-01

    Background: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps...... directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux...... in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium). Conclusions: We engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E...

  20. Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Xi, Wenjun [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    A capillary electrophoresis (CE) method is developed to determine both NAD+ and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD+ and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD+ and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD+ levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD+ and NADH levels with and without exposure to oxidative stress induced by H2O2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD+ and NADH levels, while astrocytes respond by increasing cellular NADH/NAD+ ratio.

  1. Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

    Science.gov (United States)

    Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

    2017-07-25

    Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

  2. 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...

  3. Engineering a synthetic anaerobic respiration for reduction of xylose to xylitol using NADH output of glucose catabolism by Escherichia coli AI21.

    Science.gov (United States)

    Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde

    2016-04-16

    Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over

  4. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    Science.gov (United States)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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...

  7. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor

    Science.gov (United States)

    Morre, D. James

    2002-01-01

    The cell-surface NADH oxidase (NOX) protein of plant and animal cells will utilize both NADH and NADPH as reduced electron donors for activity. The two activities are distinguished by a differential inhibition by the redox inhibitor diphenyleneiodonium chloride (DPI). Using both plasma membranes and cells, activity with NADPH as donor was markedly inhibited by DPI at submicromolar concentrations, whereas with NADH as donor, DPI was much less effective or had no effect on the activity. The possibility of the inhibition being the result of two different enzymes was eliminated by the use of a recombinant NOX protein. The findings support the concept that NOX proteins serve as terminal oxidases for plasma membrane electron transport involving cytosolic reduced pyridine nucleotides as the natural electron donors and with molecular oxygen as the electron acceptor.

  8. Activated barrier crossing dynamics in the non-radiative decay of NADH and NADPH

    Energy Technology Data Exchange (ETDEWEB)

    Blacker, Thomas S., E-mail: t.blacker@ucl.ac.uk [Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), University College London, London WC1E 6BT (United Kingdom); Department of Physics and Astronomy, University College London, London WC1E 6BT (United Kingdom); Research Department of Cell and Developmental Biology, University College London, London WC1E 6BT (United Kingdom); Marsh, Richard J., E-mail: richard.marsh@ucl.ac.uk [Department of Physics and Astronomy, University College London, London WC1E 6BT (United Kingdom); Duchen, Michael R., E-mail: m.duchen@ucl.ac.uk [Research Department of Cell and Developmental Biology, University College London, London WC1E 6BT (United Kingdom); Bain, Angus J., E-mail: a.bain@ucl.ac.uk [Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), University College London, London WC1E 6BT (United Kingdom); Department of Physics and Astronomy, University College London, London WC1E 6BT (United Kingdom)

    2013-08-30

    Highlights: ► NADH and NADPH have a high rate of non-radiative excited state decay. ► Conformational relaxation is shown to be a significant non-radiative pathway. ► The Kramers equation describes the barrier crossing dynamics of the relaxation. ► Conformational restriction upon enzyme binding will alter NAD(P)H lifetimes. - Abstract: In live tissue, alterations in metabolism induce changes in the fluorescence decay of the biological coenzyme NAD(P)H, the mechanism of which is not well understood. In this work, the fluorescence and anisotropy decay dynamics of NADH and NADPH were investigated as a function of viscosity in a range of water–glycerol solutions. The viscosity dependence of the non-radiative decay is well described by Kramers and Kramers–Hubbard models of activated barrier crossing over a wide viscosity range. Our combined lifetime and anisotropy analysis indicates common mechanisms of non-radiative relaxation in the two emitting states (conformations) of both molecules. The low frequencies associated with barrier crossing suggest that non-radiative decay is mediated by small scale motion (e.g. puckering) of the nicotinamide ring. Variations in the fluorescence lifetimes of NADH and NADPH when bound to different enzymes may therefore be attributed to differing levels of conformational restriction upon binding.

  9. The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

    Science.gov (United States)

    Jackson, R H; Cole, J A; Cornish-Bowden, A

    1981-01-01

    The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions. PMID:6279095

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri▿ †

    Science.gov (United States)

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K.

    2008-01-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0′ = −410 mV) with NADH (E0′ = −320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0′ = −10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper. PMID:17993531

  13. Carbon nanofiber vs. carbon microparticles as modifiers of glassy carbon and gold electrodes applied in electrochemical sensing of NADH.

    Science.gov (United States)

    Pérez, Briza; Del Valle, Manel; Alegret, Salvador; Merkoçi, Arben

    2007-12-15

    Carbon materials (CMs), such as carbon nanotubes (CNTs), carbon nanofibers (CNFs), and carbon microparticles (CMPs) are used as doping materials for electrochemical sensors. The efficiency of these materials (either before or after acidic treatments) while being used as electrocatalysts in electrochemical sensors is discussed for beta-nicotinamide adenine dinucleotide (NADH) detection using cyclic voltammetry (CV). The sensitivity of the electrodes (glassy carbon (GC) and gold (Au)) modified with both treated and untreated materials have been deeply studied. The response efficiencies of the GC and Au electrodes modified with CNF and CMP, using dimethylformamide (DMF) as dispersing agent are significantly different due to the peculiar physical and chemical characteristics of each doping material. Several differences between the electrocatalytic activities of CMs modified electrodes upon NADH oxidation have been observed. The CNF film promotes better the electron transfer of NADH minimizing the oxidation potential at +0.352 V. Moreover higher currents for the NADH oxidation peak have been observed for these electrodes. The shown differences in the electrochemical reactivities of CNF and CMP modified electrodes should be with interest for future applications in biosensors.

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

    Science.gov (United States)

    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.

  15. A fiber-optic sorbitol biosensor based on NADH fluorescence detection toward rapid diagnosis of diabetic complications.

    Science.gov (United States)

    Gessei, Tomoko; Arakawa, Takahiro; Kudo, Hiroyuki; Mitsubayashi, Kohji

    2015-09-21

    Accumulation of sorbitol in the tissue is known to cause microvascular diabetic complications. In this paper, a fiber-optic biosensor for sorbitol which is used as a biomarker of diabetic complications was developed and tested. The biosensor used a sorbitol dehydrogenase from microorganisms of the genus Flavimonas with high substrate specificity and detected the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) by the enzymatic reaction. An ultraviolet light emitting diode (UV-LED) was used as the excitation light source of NADH. The fluorescence of NADH was detected using a spectrometer or a photomultiplier tube (PMT). The UV-LED and the photodetector were coupled using a Y-shaped optical fiber. In the experiment, an optical fiber probe with a sorbitol dehydrogenase immobilized membrane was placed in a cuvette filled with a phosphate buffer containing the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). The changes in NADH fluorescence intensity were measured after adding a standard sorbitol solution. According to the experimental assessment, the calibration range of the sorbitol biosensor systems using a spectrometer and a PMT was 5.0-1000 μmol L(-1) and 1.0-1000 μmol L(-1), respectively. The sorbitol biosensor system using the sorbitol dehydrogenase from microorganisms of the genus Flavimonas has high selectivity and sensitivity compared with that from sheep liver. The sorbitol biosensor allows for point-of-care testing applications or daily health care tests for diabetes patients.

  16. 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.

  17. Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

    Science.gov (United States)

    Weinstein, Edward A.; Yano, Takahiro; Li, Lin-Sheng; Avarbock, David; Avarbock, Andrew; Helm, Douglas; McColm, Andrew A.; Duncan, Ken; Lonsdale, John T.; Rubin, Harvey

    2005-01-01

    Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics. PMID:15767566

  18. Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

    International Nuclear Information System (INIS)

    Yagi, T.

    1987-01-01

    The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and BacilLus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [ 14 C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [ 14 C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity

  19. A novel strategy involved in [corrected] anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network.

    Directory of Open Access Journals (Sweden)

    Ranji Singh

    Full Text Available The reduced nicotinamide adenine dinucleotide phosphate (NADPH is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH, a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC, malic enzyme (ME, malate dehydrogenase (MDH, malate synthase (MS, and isocitrate lyase (ICL that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK and the upregulation of pyruvate kinase (PK ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.

  20. Renewable Molecular Flasks with NADH Models: Combination of Light-Driven Proton Reduction and Biomimetic Hydrogenation of Benzoxazinones.

    Science.gov (United States)

    Zhao, Liang; Wei, Jianwei; Lu, Junhua; He, Cheng; Duan, Chunying

    2017-07-17

    Using small molecules with defined pockets to catalyze chemical transformations resulted in attractive catalytic syntheses that echo the remarkable properties of enzymes. By modulating the active site of a nicotinamide adenine dinucleotide (NADH) model in a redox-active molecular flask, we combined biomimetic hydrogenation with in situ regeneration of the active site in a one-pot transformation using light as a clean energy source. This molecular flask facilitates the encapsulation of benzoxazinones for biomimetic hydrogenation of the substrates within the inner space of the flask using the active sites of the NADH models. The redox-active metal centers provide an active hydrogen source by light-driven proton reduction outside the pocket, allowing the in situ regeneration of the NADH models under irradiation. This new synthetic platform, which offers control over the location of the redox events, provides a regenerating system that exhibits high selectivity and efficiency and is extendable to benzoxazinone and quinoxalinone systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. 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.

  2. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  3. Overexpression of a water-forming NADH oxidase improves the metabolism and stress tolerance of Saccharmyces cerevisiae in aerobic fermenation

    Directory of Open Access Journals (Sweden)

    Xinchi Shi

    2016-09-01

    Full Text Available Redox homeostasis is fundamental to the maintenance of metabolism. Redox imbalance can cause oxidative stress, which affects metabolism and growth. Water-forming NADH oxidase regulates the redox balance by oxidizing cytosolic NADH to NAD+, which relieves cytosolic NADH accumulation through rapid glucose consumption in Saccharomyces cerevisiae, thus decreasing the production of the byproduct glycerol in industrial ethanol production. Here, we studied the effects of overexpression of a water-forming NADH oxidase from Lactococcus lactis on the stress response of S. cerevisiae in aerobic batch fermentation, and we constructed an interaction network of transcriptional regulation and metabolic networks to study the effects of and mechanisms underlying NADH oxidase regulation. The oxidase-overexpressing strain (NOX showed increased glucose consumption, growth, and ethanol production, while glycerol production was remarkably lower. Glucose was exhausted by NOX at 26 h, while 18.92 ± 0.94 g/L residual glucose was left in the fermentation broth of the control strain (CON at this time point. At 29.5 h, the ethanol concentration for NOX peaked at 35.25 ± 1.76 g/L, which was 14.37 % higher than that for CON (30.82 ± 1.54 g/L. Gene expression involved in the synthesis of thiamine, which is associated with stress responses in various organisms, was increased in NOX. The transcription factor HAP4 was significantly upregulated in NOX at the late-exponential phase, indicating a diauxic shift in response to starvation. The apoptosis-inducing factor Nuc1 was downregulated while the transcription factor Sok2, which regulates the production of the small signaling molecule ammonia, was upregulated at the late-exponential phase, benefiting young cells on the rim. Reactive oxygen species production was decreased by 10% in NOX, supporting a decrease in apoptosis. The HOG pathway was not activated, although the osmotic stress was truly higher, indicating improved

  4. 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

  5. Multifunctional roles of enolase in Alzheimer's disease brain: beyond altered glucose metabolism.

    Science.gov (United States)

    Butterfield, D Allan; Lange, Miranda L Bader

    2009-11-01

    Enolase enzymes are abundantly expressed, cytosolic carbon-oxygen lyases known for their role in glucose metabolism. Recently, enolase has been shown to possess a variety of different regulatory functions, beyond glycolysis and gluconeogenesis, associated with hypoxia, ischemia, and Alzheimer's disease (AD). AD is an age-associated neurodegenerative disorder characterized pathologically by elevated oxidative stress and subsequent damage to proteins, lipids, and nucleic acids, appearance of neurofibrillary tangles and senile plaques, and loss of synapse and neuronal cells. It is unclear if development of a hypometabolic environment is a consequence of or contributes to AD pathology, as there is not only a significant decline in brain glucose levels in AD, but also there is an increase in proteomics identified oxidatively modified glycolytic enzymes that are rendered inactive, including enolase. Previously, our laboratory identified alpha-enolase as one the most frequently up-regulated and oxidatively modified proteins in amnestic mild cognitive impairment (MCI), early-onset AD, and AD. However, the glycolytic conversion of 2-phosphoglycerate to phosphoenolpyruvate catalyzed by enolase does not directly produce ATP or NADH; therefore it is surprising that, among all glycolytic enzymes, alpha-enolase was one of only two glycolytic enzymes consistently up-regulated from MCI to AD. These findings suggest enolase is involved with more than glucose metabolism in AD brain, but may possess other functions, normally necessary to preserve brain function. This review examines potential altered function(s) of brain enolase in MCI, early-onset AD, and AD, alterations that may contribute to the biochemical, pathological, clinical characteristics, and progression of this dementing disorder.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate▿

    OpenAIRE

    Wang, X.; Miller, E. N.; Yomano, L. P.; Zhang, X.; Shanmugam, K. T.; Ingram, L. O.

    2011-01-01

    Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low Km for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced eth...

  8. Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines.

    Science.gov (United States)

    Schurig-Briccio, Lici A; Yano, Takahiro; Rubin, Harvey; Gennis, Robert B

    2014-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD(+)) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC50) values as low as 8μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Intracellular Redox State Revealed by In Vivo 31P MRS Measurement of NAD+ and NADH Contents in Brains

    Science.gov (United States)

    Lu, Ming; Zhu, Xiao-Hong; Zhang, Yi; Chen, Wei

    2015-01-01

    Purpose Nicotinamide adenine dinucleotide (NAD), in oxidized (NAD+) or reduced (NADH) form, plays key roles in cellular metabolism. Intracellular NAD+/NADH ratio represents the cellular redox state; however, it is difficult to measure in vivo. We report here a novel in vivo 31P MRS method for noninvasive measurement of intracellular NAD concentrations and NAD+/NADH ratio in the brain. Methods It uses a theoretical model to describe the NAD spectral patterns at a given field for quantification. Standard NAD solutions and independent cat brain measurements at 9.4 T and 16.4 T were used to evaluate this method. We also measured T1 values of brain NAD. Results Model simulation and studies of solutions and brains indicate that the proposed method can quantify submillimolar NAD concentrations with reasonable accuracy if adequate 31P MRS signal-to-noise ratio and linewidth were obtained. The NAD concentrations and NAD+/NADH ratio of cat brains measured at 16.4 T and 9.4 T were consistent despite the significantly different T1 values and NAD spectra patterns at two fields. Conclusion This newly established 31P MRS method makes it possible for the first time to noninvasively study the intracellular redox state and its roles in brain functions and diseases, and it can potentially be applied to other organs. PMID:23843330

  10. Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction.

    Science.gov (United States)

    Bekers, K M; Heijnen, J J; van Gulik, W M

    2015-08-01

    With the current quantitative metabolomics techniques, only whole-cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH-catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose-limited chemostats under steady-state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose-limited steady-state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Acrolein inhibits NADH-linked mitochondrial enzyme activity: implications for Alzheimer's disease.

    Science.gov (United States)

    Pocernich, Chava B; Butterfield, D Allan

    2003-01-01

    In Alzheimer's disease (AD) brain increased lipid peroxidation and decreased energy utilization are found. Mitochondria membranes contain a significant amount of arachidonic and linoleic acids, precursors of lipid peroxidation products, 4-hydroxynonenal (HNE) and 2-propen-1-al (acrolein), that are extremely reactive. Both alkenals are increased in AD brain. In this study, we examined the effects of nanomolar levels of acrolein on the activities of pyruvate dehydrogenase (PDH) and Alpha-ketoglutarate dehydrogenase (KGDH), both reduced nicotinamide adenine dinucleotide (NADH)-linked mitochondrial enzymes. Acrolein decreased PDH and KGDH activities significantly in a dose-dependent manner. Using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS), acrolein was found to bind lipoic acid, a component in both the PDH and KGDH complexes, most likely explaining the loss of enzyme activity. Acrolein also interacted with oxidized nicotinamide adenine dinucleotide (NAD(+)) in such a way as to decrease the production of NADH. Acrolein, which is increased in AD brain, may be partially responsible for the dysfunction of mitochondria and loss of energy found in AD brain by inhibition of PDH and KGDH activities, potentially contributing to the neurodegeneration in this disorder.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

    Science.gov (United States)

    Li, Min; Riddle, Suzette; Zhang, Hui; D'Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J; Hansen, Kirk C; Moldvan, Radu; McKeon, B Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Caánovas, Angela; Medrano, Juan F; Thomas, Milton G; Iloska, Dijana; Plecitá-Hlavatá, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A; El Kasmi, Karim C; Zhang, QingHong; Stenmark, Kurt R

    2016-10-11

    Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). RNA sequencing, quantitative polymerase chain reaction, 13 C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U- 13 C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD + ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD + ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly

  14. Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Co-Repressor C-terminal Binding Protein-1

    Science.gov (United States)

    Li, Min; Riddle, Suzette; Zhang, Hui; D’Alessandro, Angelo; Flockton, Amanda; Serkova, Natalie J.; Hansen, Kirk C.; Moldvan, Radu; McKeon, B. Alexandre; Frid, Maria; Kumar, Sushil; Li, Hong; Liu, Hongbing; Cánovas, Angela; Medrano, Juan F.; Thomas, Milton G.; Iloska, Dijana; Plecita-Hlavata, Lydie; Ježek, Petr; Pullamsetti, Soni; Fini, Mehdi A.; El Kasmi, Karim C.; Zhang, Qinghong; Stenmark, Kurt R.

    2016-01-01

    Background Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells including fibroblasts in pulmonary hypertension (PH). Herein, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and pro-inflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional co-repressor C-terminal binding protein 1 (CtBP1). Methods RNA-Sequencing, qPCR, 13C-NMR, fluorescence-lifetime imaging, mass spectrometry-based metabolomics and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure NADH/NAD+ ratio in bovine and human adventitial fibroblasts, and mouse lung tissues. Immunohistochemistry was utilized to assess CtBP1 expression in the whole lung tissues. CtBP1 siRNA and the pharmacologic inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were utilized to abrogate CtBP1 activity in cells and hypoxic mice. Results We found adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with IPAH (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with IPAH and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB, or genetically blocking CtBP1 using siRNA, upregulated the cyclin-dependent genes (p15 and p21) and pro-apoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. ChIP analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  17. Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability.

    Science.gov (United States)

    Shaw, Daniel J; Robb, Kirsty; Vetter, Beatrice V; Tong, Madeline; Molle, Virginie; Hunt, Neil T; Hoskisson, Paul A

    2017-07-05

    Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in V max for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.

  18. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Hopperton, Kathryn E., E-mail: kathryn.hopperton@mail.utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Duncan, Robin E., E-mail: robin.duncan@uwaterloo.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Bazinet, Richard P., E-mail: richard.bazinet@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Archer, Michael C., E-mail: m.archer@utoronto.ca [Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada); Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 (Canada)

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from {sup 14}C-labeled acetate to those supplied exogenously as {sup 14}C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare

  19. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity

    International Nuclear Information System (INIS)

    Hopperton, Kathryn E.; Duncan, Robin E.; Bazinet, Richard P.; Archer, Michael C.

    2014-01-01

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from 14 C-labeled acetate to those supplied exogenously as 14 C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2–3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. - Highlights: • Fatty acid synthase (FASN) is over-expressed in cancer but its function is unknown. • We compare utilization of

  20. Mechanism of 3,4-dihydroxybenzaldehyde electropolymerization at carbon paste electrodes : catalytic detection of NADH

    Directory of Open Access Journals (Sweden)

    Delbem Maria Flávia

    2002-01-01

    Full Text Available Cyclic voltammetry was used to study 3,4-dihydroxybenzaldehyde (3,4-DHB electropolymerization processes on carbon paste electrodes. The characteristics of the electropolymerized films were highly dependent on pH, anodic switching potential, scan rate, 3,4-DHB concentrations and number of cycles. Film stability was determined in citrate/phosphate buffer solutions at the same pH used during the electropolymerization process. The best conditions to prepare carbon paste modified electrodes were pH 7.8; 0.0 <= Eapl <= 0.25 V; 10 mV s-1; 0.25 mmol L-1 3,4-DHB and 10 scans. These carbon paste modified electrodes were used for NADH catalytic detection at 0.23 V in the range 0.015 <= [NADH] <= 0.21 mmol L-1. Experimental data were used to propose a mechanism for the 3,4--DHB electropolymerization processes, which involves initial phenoxyl radical formation.

  1. Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

    Science.gov (United States)

    Watanabe, Seiya; Abu Saleh, Ahmed; Pack, Seung Pil; Annaluru, Narayana; Kodaki, Tsutomu; Makino, Keisuke

    2007-09-01

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD(+)-dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52- and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k(cat)/K(m) with NADH)/(k(cat)/K(m) with NADPH)] compared with the wild-type (WT), which was due to decrease of k(cat) with NADPH in the R276H mutant and increase of K(m) with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP(+) and NADH/NAD(+) ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

  2. Unassigned MURF1 of kinetoplastids codes for NADH dehydrogenase subunit 2

    Directory of Open Access Journals (Sweden)

    Burger Gertraud

    2008-10-01

    Full Text Available Abstract Background In a previous study, we conducted a large-scale similarity-free function prediction of mitochondrion-encoded hypothetical proteins, by which the hypothetical gene murf1 (maxicircle unidentified reading frame 1 was assigned as nad2, encoding subunit 2 of NADH dehydrogenase (Complex I of the respiratory chain. This hypothetical gene occurs in the mitochondrial genome of kinetoplastids, a group of unicellular eukaryotes including the causative agents of African sleeping sickness and leishmaniasis. In the present study, we test this assignment by using bioinformatics methods that are highly sensitive in identifying remote homologs and confront the prediction with available biological knowledge. Results Comparison of MURF1 profile Hidden Markov Model (HMM against function-known profile HMMs in Pfam, Panther and TIGR shows that MURF1 is a Complex I protein, but without specifying the exact subunit. Therefore, we constructed profile HMMs for each individual subunit, using all available sequences clustered at various identity thresholds. HMM-HMM comparison of these individual NADH subunits against MURF1 clearly identifies this hypothetical protein as NAD2. Further, we collected the relevant experimental information about kinetoplastids, which provides additional evidence in support of this prediction. Conclusion Our in silico analyses provide convincing evidence for MURF1 being a highly divergent member of NAD2.

  3. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    OpenAIRE

    Kukiełka, E; Cederbaum, A I

    1995-01-01

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid...

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

    Science.gov (United States)

    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.

  5. Photochemical Properties and Reactivity of a Ru Compound Containing an NAD/NADH-Functionalized 1,10-Phenanthroline Ligand.

    Science.gov (United States)

    Kobayashi, Katsuaki; Ohtsu, Hideki; Nozaki, Koichi; Kitagawa, Susumu; Tanaka, Koji

    2016-03-07

    An NAD/NADH-functionalized ligand, benzo[b]pyrido[3,2-f][1,7]-phenanthroline (bpp), was newly synthesized. A Ru compound containing the bpp ligand, [Ru(bpp)(bpy)2](2+), underwent 2e(-) and 2H(+) reduction, generating the NADH form of the compound, [Ru(bppHH)(bpy)2](2+), in response to visible light irradiation in CH3CN/TEA/H2O (8/1/1). The UV-vis and fluorescent spectra of both [Ru(bpp)(bpy)2](2+) and [Ru(bppHH)(bpy)2](2+) resembled the spectra of [Ru(bpy)3](2+). Both complexes exhibited strong emission, with quantum yields of 0.086 and 0.031, respectively; values that are much higher than those obtained from the NAD/NADH-functionalized complexes [Ru(pbn)(bpy)2](2+) and [Ru(pbnHH)(bpy)2](2+) (pbn = (2-(2-pyridyl)benzo[b]-1.5-naphthyridine, pbnHH = hydrogenated form of pbn). The reduction potential of the bpp ligand in [Ru(bpp)(bpy)2](2+) (-1.28 V vs SCE) is much more negative than that of the pbn ligand in [Ru(pbn)(bpy)2](2+) (-0.74 V), although the oxidation potentials of bppHH and pbnHH are essentially equal (0.95 V). These results indicate that the electrochemical oxidation of the dihydropyridine moiety in the NADH-type ligand was independent of the π system, including the Ru polypyridyl framework. [Ru(bppHH)(bpy)2](2+) allowed the photoreduction of oxygen, generating H2O2 in 92% yield based on [Ru(bppHH)(bpy)2](2+). H2O2 production took place via singlet oxygen generated by the energy transfer from excited [Ru(bppHH)(bpy)2](2+) to triplet oxygen.

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

    Science.gov (United States)

    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

  7. Bioinspired Design of Alcohol Dehydrogenase@nano TiO2 Microreactors for Sustainable Cycling of NAD+/NADH Coenzyme

    Directory of Open Access Journals (Sweden)

    Sen Lin

    2018-02-01

    Full Text Available The bioinspired design and construction of enzyme@capsule microreactors with specific cell-like functionality has generated tremendous interest in recent years. Inspired by their fascinating complexity, scientists have endeavored to understand the essential aspects of a natural cell and create biomimicking microreactors so as to immobilize enzymes within the hierarchical structure of a microcapsule. In this study, simultaneous encapsulation of alcohol dehydrogenase (ADH was achieved during the preparation of microcapsules by the Pickering emulsion method using amphiphilic modified TiO2 nanoparticles (NPs as building blocks for assembling the photocatalytic microcapsule membrane. The ADH@TiO2 NP microreactors exhibited dual catalytic functions, i.e., spatially confined enzymatic catalysis and the membrane-associated photocatalytic oxidation under visible light. The sustainable cycling of nicotinamide adenine dinucleotide (NAD coenzyme between NADH and NAD+ was realized by enzymatic regeneration of NADH from NAD+ reduction, and was provided in a form that enabled further photocatalytic oxidation to NAD+ under visible light. This bioinspired ADH@TiO2 NP microreactor allowed the linking of a semiconductor mineral-based inorganic photosystem to enzymatic reactions. This is a first step toward the realization of sustainable biological cycling of NAD+/NADH coenzyme in synthetic functional microsystems operating under visible light irradiation.

  8. Effect of different NADH oxidase levels on glucose metabolism by Lactococus lactis : kinetics of intracellular metabolite pools determined by in vivo nuclear magnetic resonance

    NARCIS (Netherlands)

    Neves, A.R.; Ramos, A.; Costa, H.; Swam, van I.I.; Hugenholtz, J.; Kleerebezem, M.; Vos, de W.M.; Santos, H.

    2002-01-01

    Three isogenic strains of Lactococcus lactis with different levels of H2O-forming NADH oxidase activity were used to study the effect of oxygen on glucose metabolism: the parent strain L. lactis MG1363, a NOX- strain harboring a deletion of the gene coding for H2O-forming NADH oxidase, and a NOX

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Fatty acid synthase plays a role in cancer metabolism beyond providing fatty acids for phospholipid synthesis or sustaining elevations in glycolytic activity.

    Science.gov (United States)

    Hopperton, Kathryn E; Duncan, Robin E; Bazinet, Richard P; Archer, Michael C

    2014-01-15

    Fatty acid synthase is over-expressed in many cancers and its activity is required for cancer cell survival, but the role of endogenously synthesized fatty acids in cancer is unknown. It has been suggested that endogenous fatty acid synthesis is either needed to support the growth of rapidly dividing cells, or to maintain elevated glycolysis (the Warburg effect) that is characteristic of cancer cells. Here, we investigate both hypotheses. First, we compared utilization of fatty acids synthesized endogenously from (14)C-labeled acetate to those supplied exogenously as (14)C-labeled palmitate in the culture medium in human breast cancer (MCF-7 and MDA-MB-231) and untransformed breast epithelial cells (MCF-10A). We found that cancer cells do not produce fatty acids that are different from those derived from exogenous palmitate, that these fatty acids are esterified to the same lipid and phospholipid classes in the same proportions, and that their distribution within neutral lipids is not different from untransformed cells. These results suggest that endogenously synthesized fatty acids do not fulfill a specific function in cancer cells. Furthermore, we observed that cancer cells excrete endogenously synthesized fatty acids, suggesting that they are produced in excess of requirements. We next investigated whether lipogenic activity is involved in the maintenance of high glycolytic activity by culturing both cancer and non-transformed cells under anoxic conditions. Although anoxia increased glycolysis 2-3 fold, we observed no concomitant increase in lipogenesis. Our results indicate that breast cancer cells do not have a specific qualitative or quantitative requirement for endogenously synthesized fatty acids and that increased de novo lipogenesis is not required to sustain elevations in glycolytic activity induced by anoxia in these cells. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Ca2+ dependence of gluconeogenesis stimulation by glucagon at different cytosolic NAD+-NADH redox potentials

    Directory of Open Access Journals (Sweden)

    Marques-da-Silva A.C.

    1997-01-01

    Full Text Available The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD+-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD+-NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a gluconeogenesis from lactate plus pyruvate was not affected by Ca2+-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools; gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca2+-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100

  12. 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

  13. Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria.

    Science.gov (United States)

    Kawasaki, Shinji; Ishikura, Jun; Chiba, Daisuke; Nishino, Tomoko; Niimura, Youichi

    2004-04-01

    Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O(2). When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O(2)-free N(2) carrier-gas) to microoxic (sparged with 3% O(2)/97% N(2) mixed carrier-gas) growth conditions in the mid exponential phase (OD(660)=1.0). When the strain grew under 3% O(2)/97% N(2), the medium remains anoxic. Thirty minutes after beginning aeration with 3% O(2), the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H(2)O, and the estimated K(m) for oxygen was 61.9 microM. These data demonstrate that an O(2)-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.

  14. 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...

  15. Highly stable and reusable immobilized formate dehydrogenases: Promising biocatalysts for in situ regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Barış Binay

    2016-02-01

    Full Text Available This study aimed to prepare robust immobilized formate dehydrogenase (FDH preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150, Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU, and Immobead 150 support functionalized with aldehyde groups (FDHIALD. The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t1/2 of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.

  16. Occurrence of the malate-aspartate shuttle in various tumor types.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1976-04-01

    The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

  17. Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

    Directory of Open Access Journals (Sweden)

    Zhong Ying

    2008-01-01

    Full Text Available Abstract Background The p49/STRAP (or SRFBP1 protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. Results In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. Conclusion These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

  18. Development of an Electrochemical Sensor for NADH Determination Based on a Caffeic Acid Redox Mediator Supported on Carbon Black

    Directory of Open Access Journals (Sweden)

    Chiara Zanardi

    2015-04-01

    Full Text Available Screen-printed electrode (SPE modified with carbon black nanoparticles (CB has been tested as a new platform for the stable deposition of caffeic acid (CFA on the electrode surface. The electrochemical performance from varying the amount of CFA/CB composite has been tested with respect to NADH determination. The electrocatalytic activity of CFA/CB has also been compared with that of SPEs modified by a single component of the coating, i.e., either CFA or CB. Finally, glycerol dehydrogenase, a typical NADH-dependent enzyme, was deposited on the CFA/CB coating in order to test the applicability of the sensor in glycerol determination.

  19. Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype

    Directory of Open Access Journals (Sweden)

    Pluk Helma

    2009-07-01

    Full Text Available Abstract Background The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. Results Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. Conclusion The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

  20. NADH-fluorescence scattering correction for absolute concentration determination in a liquid tissue phantom using a novel multispectral magnetic-resonance-imaging-compatible needle probe

    Science.gov (United States)

    Braun, Frank; Schalk, Robert; Heintz, Annabell; Feike, Patrick; Firmowski, Sebastian; Beuermann, Thomas; Methner, Frank-Jürgen; Kränzlin, Bettina; Gretz, Norbert; Rädle, Matthias

    2017-07-01

    In this report, a quantitative nicotinamide adenine dinucleotide hydrate (NADH) fluorescence measurement algorithm in a liquid tissue phantom using a fiber-optic needle probe is presented. To determine the absolute concentrations of NADH in this phantom, the fluorescence emission spectra at 465 nm were corrected using diffuse reflectance spectroscopy between 600 nm and 940 nm. The patented autoclavable Nitinol needle probe enables the acquisition of multispectral backscattering measurements of ultraviolet, visible, near-infrared and fluorescence spectra. As a phantom, a suspension of calcium carbonate (Calcilit) and water with physiological NADH concentrations between 0 mmol l-1 and 2.0 mmol l-1 were used to mimic human tissue. The light scattering characteristics were adjusted to match the backscattering attributes of human skin by modifying the concentration of Calcilit. To correct the scattering effects caused by the matrices of the samples, an algorithm based on the backscattered remission spectrum was employed to compensate the influence of multiscattering on the optical pathway through the dispersed phase. The monitored backscattered visible light was used to correct the fluorescence spectra and thereby to determine the true NADH concentrations at unknown Calcilit concentrations. Despite the simplicity of the presented algorithm, the root-mean-square error of prediction (RMSEP) was 0.093 mmol l-1.

  1. Silver nanoparticles embedded in amine-functionalized silicate sol–gel network assembly for sensing cysteine, adenosine and NADH

    International Nuclear Information System (INIS)

    Maduraiveeran, Govindhan; Ramaraj, Ramasamy

    2011-01-01

    Silver nanoparticles embedded in amine-functionalized silicate sol–gel network were synthesized and used for sensing biomolecules such as cysteine, adenosine, and β-nicotinamide adenine dinucleotide (NADH). The sensing of these biomolecules by the assembly of silver nanoparticles was triggered by the optical response of the surface plasmon resonance (SPR) of the silver nanoparticles. The optical sensor exhibited the lowest detection limit (LOD) of 5, 20, and 5 μM for cysteine, adenosine, and NADH, respectively. The sensing of biomolecules in the micromolar range by using the amine-functionalized silicate sol–gel embedded silver nanoparticles was studied in the presence of interference molecules like uridine, glycine, guanine, and guanosine. Thus, the present approach might open up a new avenue for the development of silver nanoparticles-based optical sensor devices for biomolecules.

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

    Science.gov (United States)

    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.

  3. 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

  4. An EPR spin-probe and spin-trap study of the free radicals produced by plant plasma membranes

    Directory of Open Access Journals (Sweden)

    GORAN BACIC

    2005-02-01

    Full Text Available Plant plasma membranes are known to produce superoxide radicals, while the production of hydroxyl radical is thought to occur only in the cell wall. In this work it was demonstrated using combined spin-trap and spin-probe EPR spectroscopic techniques, that plant plasma membranes do produce superoxide and hydroxyl radicals but by kinetically different mechanisms. The results show that superoxide and hydroxyl radicals can be detected by DMPO spin-trap and that the mechanisms and location of their production can be differentiated using the reduction of spin-probes Tempone and 7-DS. It was shown that the mechanism of production of oxygen reactive species is NADH dependent and diphenylene iodonium inhibited. The kinetics of the reduction of Tempone, combined with scavengers or the absence of NADH indicates that hydroxyl radicals are produced by a mechanism independent of that of superoxide production. It was shown that a combination of the spin-probe and spin-trap technique can be used in free radical studies of biological systems, with a number of advantages inherent to them.

  5. Comparative Proteome Analysis between High Lipid-Producing Strain Mucor circinelloides WJ11 and Low Lipid-Producing Strain CBS 277.49.

    Science.gov (United States)

    Tang, Xin; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Song, Yuanda; Chen, Wei

    2017-06-21

    Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

    Science.gov (United States)

    Branny, P; de la Torre, F; Garel, J R

    1998-04-01

    The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.

  8. Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis.

    Directory of Open Access Journals (Sweden)

    Tingting Guo

    Full Text Available Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+ ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2O-forming NADH oxidase activity led to 76.95% lower H(2O(2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2O(2 accumulation and prolong cell survival.

  9. Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

    DEFF Research Database (Denmark)

    Knudsen, Jenny Dahl; Hägglöf, Cecilia; Weber, Nora

    2016-01-01

    yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated. RESULTS: A series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol...

  10. Detection of NADH via electrocatalytic oxidation at single-walled carbon nanotubes modified with Variamine blue

    International Nuclear Information System (INIS)

    Radoi, A.; Compagnone, D.; Valcarcel, M.A.; Placidi, P.; Materazzi, S.; Moscone, D.; Palleschi, G.

    2008-01-01

    Screen-printed electrodes (SPEs) modified with Variamine blue (VB), covalently attached to the oxidized single-walled carbon nanotubes (SWCNTs-COOH), were developed and used as chemical sensors for the detection of the reduced nicotinamide adenine dinucleotide (NADH). The Variamine blue redox mediator was covalently linked to the SWCNTs-COOH by the N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) chemistry. Infrared Fourier transform (FT-IR) spectroscopy revealed the presence of the amide bands situated at 1623 cm -1 (I band), 1577 cm -1 (II band) and 1437 cm -1 (III band) demonstrating the covalent linkage of Variamine blue to SWCNTs-COOH. The heterogeneous electron transfer rate, k obs. , was 13,850 M -1 s -1 , and the k s and α were 0.8 s -1 and 0.56, respectively. The pH dependence was also investigated. SPEs modified with Variamine blue by using the DCC/NHS conjugation method, showed a variation of -36 mV per pH unit. A successful application was the development of a lactate biosensor obtained by the immobilization of the L-lactate dehydrogenase on the NADH sensor

  11. Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

    OpenAIRE

    Weinstein, Edward A.; Yano, Takahiro; Li, Lin-Sheng; Avarbock, David; Avarbock, Andrew; Helm, Douglas; McColm, Andrew A.; Duncan, Ken; Lonsdale, John T.; Rubin, Harvey

    2005-01-01

    Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs...

  12. A strategy to promote the electroactive platform adopting poly(o-anisidine)-silver nanocomposites probed for the voltammetric detection of NADH and dopamine.

    Science.gov (United States)

    Sangamithirai, D; Munusamy, S; Narayanan, V; Stephen, A

    2017-11-01

    A study on the voltammetric detection of NADH (β-nicotinamide adenine dinucleotide), Dopamine (DA) and their simultaneous determination is presented in this work. The electrochemical sensor was fabricated with the hybrid nanocomposites of poly(o-anisidine) and silver nanoparticles prepared by simple and cost-effective insitu chemical oxidative polymerization technique. The nanocomposites were synthesized with different (w/w) ratios of o-anisidine and silver by increasing the amount of o-anisidine in each, by keeping silver at a fixed quantity. The XRD patterns revealed the semi-crystalline nature of poly(o-anisidine) and the face centered cubic structure of silver. The presence of silver in its metallic state and the formation of nanocomposite were established by XPS analysis. Raman studies suggested the presence of site-selective interaction between poly(o-anisidine) and silver. HRTEM studies revealed the formation of polymer matrix type nanocomposite with the embedment of silver nanoparticles. The sensing performance of the materials were studied via cyclic voltammetry, differential pulse voltammetry and chronoamperometry techniques. Fabricated sensor with 3:1 (w/w) ratio of poly(o-anisidine) and silver exhibited good catalytic activity towards the detection of NADH and DA in terms of potential and current response, when compared to others. Several important electrochemical parameters regulating the performance of the sensor have been evaluated. Under the optimum condition, differential pulse voltammetry method exhibited the linear response in the range of 0.03 to 900μM and 5 to 270μM with a low detection limit of 0.006μM and 0.052μM for NADH and DA, respectively. The modified electrodes exhibited good sensitivity, stability, reproducibility and selectivity with well-separated oxidation peaks for NADH and DA in the simultaneous determination of their binary mixture. The analytical performance of the nanocomposite as an electrochemical sensor was also

  13. Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely re...

  14. 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 ...

  15. 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.

  16. 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)

  17. Catalytic properties of nickel ferrites for oxidation of glucose, β-nicotiamide adenine dinucleotide (NADH) and methanol

    Energy Technology Data Exchange (ETDEWEB)

    Galindo, R. [Departamento de Química, Universidad de Guanajuato, Cerro de la Venada s/n, Pueblito de Rocha, C.P. 36040 Guanajuato, Gto (Mexico); Departamento de Química Física Aplicada, Universidad Autónoma de Madrid, Cantoblanco s/n, C.P. 28049 Madrid (Spain); Gutiérrez, S. [Departamento de Química, Universidad de Guanajuato, Cerro de la Venada s/n, Pueblito de Rocha, C.P. 36040 Guanajuato, Gto (Mexico); Menéndez, N. [Departamento de Química Física Aplicada, Universidad Autónoma de Madrid, Cantoblanco s/n, C.P. 28049 Madrid (Spain); Herrasti, P., E-mail: pilar.herrasti@uam.es [Departamento de Química Física Aplicada, Universidad Autónoma de Madrid, Cantoblanco s/n, C.P. 28049 Madrid (Spain)

    2014-02-15

    Highlights: ► NiFe{sub 2}O{sub 4} nanoparticles obtained by electrochemical method are effective catalyst. ► A partially inverse spinel was obtained with 57% Fe{sup 3+} in tetrahedral position. ► A non-enzymatic electrode using NiFe{sub 2}O{sub 4} nanoparticles has been manufactured. -- Abstract: Nickel ferrite nanoparticles (NiFe{sub 2}O{sub 4}) were synthesized by electrochemical method and used as catalyst for direct oxidation of glucose, NADH and methanol. Characterization of these nanoparticles was carried out by X-ray diffraction, Mössbauer spectroscopy, and colloidal properties such as hydrodynamic radius and Zeta potential. To evaluate the catalytic properties of these nanoparticles against the oxidation process, paste graphite electrodes mixing nickel ferrites and different conductive materials (graphite, carbon nanotubes) and binders agents (mineral oil, 1-octylpyridinium hexafluorophosphate (nOPPF6)) were used. The results prove good catalytic properties of these materials, with an oxidation potential around 0.75, 0.5 and 0.8 V for glucose, NADH, and methanol, respectively.

  18. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

    International Nuclear Information System (INIS)

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-01-01

    A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Proteomic evidences for rex regulation of metabolism in toxin-producing Bacillus cereus ATCC 14579.

    Directory of Open Access Journals (Sweden)

    Sabrina Laouami

    Full Text Available The facultative anaerobe, Bacillus cereus, causes diarrheal diseases in humans. Its ability to deal with oxygen availability is recognized to be critical for pathogenesis. The B. cereus genome comprises a gene encoding a protein with high similarities to the redox regulator, Rex, which is a central regulator of anaerobic metabolism in Bacillus subtilis and other Gram-positive bacteria. Here, we showed that B. cereus rex is monocistronic and down-regulated in the absence of oxygen. The protein encoded by rex is an authentic Rex transcriptional factor since its DNA binding activity depends on the NADH/NAD+ ratio. Rex deletion compromised the ability of B. cereus to cope with external oxidative stress under anaerobiosis while increasing B. cereus resistance against such stress under aerobiosis. The deletion of rex affects anaerobic fermentative and aerobic respiratory metabolism of B. cereus by decreasing and increasing, respectively, the carbon flux through the NADH-recycling lactate pathway. We compared both the cellular proteome and exoproteome of the wild-type and Δrex cells using a high throughput shotgun label-free quantitation approach and identified proteins that are under control of Rex-mediated regulation. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000886. The data suggest that Rex regulates both the cross-talk between metabolic pathways that produce NADH and NADPH and toxinogenesis, especially in oxic conditions.

  1. New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

    Directory of Open Access Journals (Sweden)

    Rocha-Martín Javier

    2011-11-01

    Full Text Available Abstract Background The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds. Results The NADH-oxidase (NOX presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C. The hyperactivated form presented a high specific activity (37.5 U/mg at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme. The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols. Conclusion We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.

  2. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    Science.gov (United States)

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle.

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

    Science.gov (United States)

    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.

  4. 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

  5. Contribution of the NADH-oxidase (Nox) to the aerobic life of Lactobacillus sanfranciscensis DSM20451T.

    Science.gov (United States)

    Jänsch, André; Freiding, Simone; Behr, Jürgen; Vogel, Rudi F

    2011-02-01

    Lactobacillus sanfranciscensis is the key bacterium in traditional sourdough fermentation. The molecular background of its oxygen tolerance was investigated by comparison of wild type and NADH-oxidase (Nox) knock out mutants. The nox gene of L. sanfranciscensis DSM20451(T) coding for a NADH-oxidase (Nox) was inactivated by single crossover integration to yield strain L. sanfranciscensis DSM20451Δnox. By inactivation of the native NADH-oxidase gene, it was ensured that besides fructose, O(2) can react as an electron acceptor. In aerated cultures the mutant strain was only able to grow in MRS media supplemented with fructose as electron acceptor, whereas the wild type strain showed a fructose independent growth response. The use of oxygen as an external electron acceptor enables L. sanfranciscensis to shift from acetyl-phosphate into the acetate branch and gain an additionally ATP, while the reduced cofactors were regenerated by Nox-activity. In aerated cultures the wild type strain formed a fermentation ratio of lactate to acetate of 1.09 in MRS supplemented with fructose after 24 h of fermentation, while the mutant strain formed a fermentation ratio of 3.05. Additionally, L. sanfranciscensis showed manganese-dependent growth response in aerated cultures, the final OD and growth velocity was increased in media supplemented with manganese. The expression of two predicted Mn(2+)/Fe(2+) transporters MntH1 and MntH2 in L. sanfranciscensis DSM20451(T) was verified by amplification of a 318 bp fragment of MntH1 and a 239 bp fragment of MntH2 from cDNA library obtained from aerobically, exponentially growing cells of L. sanfranciscensis DSM20451(T) in MRS. Moreover, the mutant strain DSM20451Δnox was more sensitive to the superoxide generating agent paraquat and showed inhibition of growth on diamide-treated MRS-plates without fructose supplementation. Copyright © 2010 Elsevier Ltd. All rights reserved.

  6. A potential mechanism of energy-metabolism oscillation in an aerobic chemostat culture of the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Xu, Zhaojun; Tsurugi, Kunio

    2006-04-01

    The energy-metabolism oscillation in aerobic chemostat cultures of yeast is a periodic change of the respiro-fermentative and respiratory phase. In the respiro-fermentative phase, the NADH level was kept high and respiration was suppressed, and glucose was anabolized into trehalose and glycogen at a rate comparable to that of catabolism. On the transition to the respiratory phase, cAMP levels increased triggering the breakdown of storage carbohydrates and the increased influx of glucose into the glycolytic pathway activated production of glycerol and ethanol consuming NADH. The resulting increase in the NAD(+)/NADH ratio stimulated respiration in combination with a decrease in the level of ATP, which was consumed mainly in the formation of biomass accompanying budding, and the accumulated ethanol and glycerol were gradually degraded by respiration via NAD(+)-dependent oxidation to acetate and the respiratory phase ceased after the recovery of NADH and ATP levels. However, the mRNA levels of both synthetic and degradative enzymes of storage carbohydrates were increased around the early respiro-fermentative phase, when storage carbohydrates are being synthesized, suggesting that the synthetic enzymes were expressed directly as active forms while the degradative enzymes were activated late by cAMP. In summary, the energy-metabolism oscillation is basically regulated by a feedback loop of oxido-reductive reactions of energy metabolism mediated by metabolites like NADH and ATP, and is modulated by metabolism of storage carbohydrates in combination of post-translational and transcriptional regulation of the related enzymes. A potential mechanism of energy-metabolism oscillation is proposed.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. A Class I UV-Blocking (senofilcon A) Soft Contact Lens Prevents UVA-induced Yellow Fluorescence and NADH loss in the Rabbit Lens Nucleus in vivo

    Science.gov (United States)

    Giblin, Frank J.; Lin, Li-Ren; Simpanya, Mukoma F.; Leverenz, Victor R.; Fick, Catherine E.

    2012-01-01

    It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm2 on the cornea) for 1 hour using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 hour. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear

  9. 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...

  10. 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...

  11. 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...

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    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.

  14. JST Thesaurus Headwords and Synonyms: NADH,NADPHオキシドレダクターゼ [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term NADH,NADPHオキシドレダクターゼ 名詞 一般 * * * ...* NAD(P)Hオキシドレダクターゼ NAD(P)Hオキシドレダクターゼ エヌエイディー(ピー)エイチオキシドレダクターゼ Thesaurus2015 200906027745899279 C LS38 UNKNOWN_2 NADH , NADPH オキシドレダクターゼ

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

    Directory of Open Access Journals (Sweden)

    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

  16. One-step construction of an electrode modified with electrodeposited Au/SiO2 nanoparticles, and its application to the determination of NADH and ethanol

    International Nuclear Information System (INIS)

    Liu, X.; Li, B.; Wang, X.; Li, C.

    2010-01-01

    A new electrode was developed by one-step potentiostatic electrodeposition (at -2. 0 V for 20 s) of Au/SiO 2 nanoparticles on a glassy carbon electrode. The resulting electrode (nano-Au/SiO 2 /GCE) was characterized by scanning electronic microscopy, X-ray photoelectron spectroscopy and electrochemical techniques. The electrochemical behavior of dihydronicotinamide adenine dinucleotide (NADH) at the nano-Au/SiO 2 /GCE were thoroughly investigated. Compared to the unmodified electrode, the overpotential decreased by about 300 mV, and the current response significantly increased. These changes indicated that the modified electrode showed excellent catalytic activity in the oxidation of NADH. A linear relationship was obtained in the NADH concentration range from 1. 0 x 10 -6 to 1. 0 x 10 -4 mol L -1 . In addition, amperometric sensing of ethanol at the nano-Au/SiO 2 /GCE in combination with alcohol dehydrogenase and nicotinamide adenine dinucleotide was successfully demonstrated. A wide linear response was also found for ethanol in the range from 5. 0 x 10 -5 to 1. 0 x 10 -3 mol L -1 and 1. 0 x 10 -3 to 1. 0 x 10 -2 mol L -1 , respectively. The method was successfully applied to determine ethanol in beer and biological samples. (author)

  17. Preparation of N-Graphdiyne Nanosheets at Liquid/Liquid Interface for Photocatalytic NADH Regeneration.

    Science.gov (United States)

    Pan, Qingyan; Liu, Hui; Zhao, Yingjie; Chen, Siqi; Xue, Bo; Kan, Xiaonan; Huang, Xiaowen; Liu, Jian; Li, Zhibo

    2018-04-16

    Two-dimensional (2D) N-graphdiyne (N-GDY) nanosheets containing different number of N were synthesized by polymerization of triazine, pyrazine, and pyridine-based monomers at liquid/liquid interface. The configurations and nanostructures of N-GDY were well-characterized. The wettability changed to more hydrophilic as the N contents increased. The collected N-GDY was further employed as metal-free photocatalyst for NADH regeneration. The catalytic performance was related with the N content in the graphdiyne. The N3-GDY demonstrated the best activity. This strategy provided a new promising platform of designing unique 2D N-GDY with tunable performance in biorelated catalysis.

  18. Tributyltin-induced apoptosis requires glycolytic adenosine trisphosphate production.

    Science.gov (United States)

    Stridh, H; Fava, E; Single, B; Nicotera, P; Orrenius, S; Leist, M

    1999-10-01

    The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.

  19. Crystallization and preliminary X-ray analysis of the complex of NADH and 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Sachiyo [Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522 (Japan); Nakamura, Shota; Ohkubo, Tadayasu [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ueda, Shigeru [Diagnostics Department, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni, Shizuoka 410-2321 (Japan); Uchiyama, Susumu [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kobayashi, Yuji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Oda, Masayuki, E-mail: oda@kpu.ac.jp [Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522 (Japan)

    2006-06-01

    The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 has been crystallized and X-ray diffraction data have been collected to 1.8 Å resolution. The NAD(P){sup +}-dependent enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 Å. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 Å, and contained two molecules, reflecting dimer formation of 3α-HSD, in the asymmetric unit.

  20. The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited: I. Proposed consequences for electron transfer in the enzyme

    NARCIS (Netherlands)

    Albracht, S.P.J.

    2010-01-01

    Bovine NADH:ubiquinone oxidoreductase (Complex I) is the first complex in the mitochondrial respiratory chain. It has long been assumed that it contained only one FMN group. However, as demonstrated in 2003, the intact enzyme contains two FMN groups. The second FMN was proposed to be located in a

  1. Oxidação eletrocatalítica de NADH empregando um novo sensor à base de um complexo macrocíclico n4 sobre grafeno e DNA

    OpenAIRE

    Ribeiro, Izabela Aparecida Luiz

    2015-01-01

    Área de concentração: Química Analítica. β-Nicotinamida adenina dinucleotídeo (NADH) e sua forma oxidada correspondente (NAD+) representam uma coenzima de grande importância em centenas de reações naturais catalisadas por enzimas desidrogenase, sendo vitais no metabolismo respiratório mitocondrial por estimular a produção de energia e atuam como principais transportadoras de cargas centrais em células vivas. Por estes motivos, a determinação eletroquímica de NADH tem atraído considerável a...

  2. Pathogenesis of Chronic Hyperglycemia: From Reductive Stress to Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Liang-Jun Yan

    2014-01-01

    Full Text Available Chronic overnutrition creates chronic hyperglycemia that can gradually induce insulin resistance and insulin secretion impairment. These disorders, if not intervened, will eventually be followed by appearance of frank diabetes. The mechanisms of this chronic pathogenic process are complex but have been suggested to involve production of reactive oxygen species (ROS and oxidative stress. In this review, I highlight evidence that reductive stress imposed by overflux of NADH through the mitochondrial electron transport chain is the source of oxidative stress, which is based on establishments that more NADH recycling by mitochondrial complex I leads to more electron leakage and thus more ROS production. The elevated levels of both NADH and ROS can inhibit and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH, respectively, resulting in blockage of the glycolytic pathway and accumulation of glycerol 3-phospate and its prior metabolites along the pathway. This accumulation then initiates all those alternative glucose metabolic pathways such as the polyol pathway and the advanced glycation pathways that otherwise are minor and insignificant under euglycemic conditions. Importantly, all these alternative pathways lead to ROS production, thus aggravating cellular oxidative stress. Therefore, reductive stress followed by oxidative stress comprises a major mechanism of hyperglycemia-induced metabolic syndrome.

  3. Gold electrodes modified with 16H, 18H-dibenzo[c,l]-7,9-dithia-16,18-diazapentacene for electrocatalytic oxidation of NADH

    NARCIS (Netherlands)

    Rosca, V.; Muresan, L.; Popescu, I.C.; Cristea, C.; Silberg, I.A.

    2001-01-01

    16H,18H-Dibenzo[c,l]-7,9-dithia-16,18-diazapentacene (DDDP), a new phenothiazine derivative containing two linearly condensed phenothiazine rings, strongly adsorbs on polyoriented gold resulting in a modified electrode with electrocatalytic activity for ß-nicotinamide adenine dinucleotide (NADH)

  4. On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

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

    2007-01-01

    We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found...... studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolarization of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane...... potential oscillations and delocalized DiOC(2)(3), while glycolysis continued to oscillate unaffected....

  5. 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 ...

  6. The reaction of NADPH with bovine mitochondrial NADH:ubiquinone oxidoreductase revisited: II. Comparison of the proposed working hypothesis with literature data.

    NARCIS (Netherlands)

    Albracht, S.P.J.

    2010-01-01

    The first purification of bovine NADH:ubiquinone oxidoreductase (Complex I) was reported nearly half a century ago (Hatefi et al. J Biol Chem 237:1676-1680, 1962). The pathway of electron-transfer through the enzyme is still under debate. A major obstacle is the assignment of EPR signals to the

  7. A Class I UV-blocking (senofilcon A) soft contact lens prevents UVA-induced yellow fluorescence and NADH loss in the rabbit lens nucleus in vivo.

    Science.gov (United States)

    Giblin, Frank J; Lin, Li-Ren; Simpanya, Mukoma F; Leverenz, Victor R; Fick, Catherine E

    2012-09-01

    It is known that fluorescence, much of it caused by UVA light excitation, increases in the aging human lens, resulting in loss of sharp vision. This study used an in vivo animal model to investigate UVA-excited fluorescence in the rabbit lens, which contains a high level of the UVA chromophore NADH, existing both free and bound to λ-crystallin. Also, the ability of a Class I (senofilcon A) soft contact lens to protect against UVA-induced effects on the rabbit lens was tested. Rabbit eyes were irradiated with UVA light in vivo (100 mW/cm(2) on the cornea) for 1 h using monochromatic 365 nm light. Irradiation was conducted in the presence of either a senofilcon A contact lens, a minimally UV-absorbing lotrafilcon A contact lens, or no contact lens at all. Eyes irradiated without a contact lens showed blue 365 nm-excited fluorescence initially, but this changed to intense yellow fluorescence after 1 h. Isolated, previously irradiated lenses exhibited yellow fluorescence originating from the lens nucleus when viewed under 365 nm light, but showed normal blue fluorescence arising from the cortex. Previously irradiated lenses also exhibited a faint yellow color when observed under visible light. The senofilcon A contact lens protected completely against the UVA-induced effects on fluorescence and lens yellowing, whereas the lotrafilcon A lens showed no protection. The UVA-exposure also produced a 53% loss of total NADH (free plus bound) in the lens nucleus, with only a 13% drop in the anterior cortex. NADH loss in the nucleus was completely prevented with use of a senofilcon A contact lens, but no significant protection was observed with a lotrafilcon A lens. Overall, the senofilcon A lens provided an average of 67% protection against UVA-induced loss of four pyridine nucleotides in four different regions of the lens. HPLC analysis with fluorescence detection indicated a nearly six-fold increase in 365 nm-excited yellow fluorescence arising from lens nuclear

  8. 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.

  9. Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels.

    Science.gov (United States)

    Baker, J L; Derr, A M; Karuppaiah, K; MacGilvray, M E; Kajfasz, J K; Faustoferri, R C; Rivera-Ramos, I; Bitoun, J P; Lemos, J A; Wen, Z T; Quivey, R G

    2014-06-01

    NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. 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.)

  11. Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Chao [School of Chemistry and Chemical Engineering, Guizhou University, Guiyang 550025 (China); Wu, Hai-Long, E-mail: hlwu@hnu.edu.cn [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China); Zhou, Chang; Xiang, Shou-Xia; Zhang, Xiao-Hua; Yu, Yong-Jie; Yu, Ru-Qin [State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (China)

    2016-03-03

    The metabolic coenzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are the primary electron donor and acceptor respectively, participate in almost all biological metabolic pathways. This study develops a novel method for the quantitative kinetic analysis of the degradation reaction of NADH and the formation reaction of FAD in human plasma containing an uncalibrated interferent, by using three-way calibration based on multi-way fluorescence technique. In the three-way analysis, by using the calibration set in a static manner, we directly predicted the concentrations of both analytes in the mixture at any time after the start of their reactions, even in the presence of an uncalibrated spectral interferent and a varying background interferent. The satisfactory quantitative results indicate that the proposed method allows one to directly monitor the concentration of each analyte in the mixture as the function of time in real-time and nondestructively, instead of determining the concentration after the analytical separation. Thereafter, we fitted the first-order rate law to their concentration data throughout their reactions. Additionally, a four-way calibration procedure is developed as an alternative for highly collinear systems. The results of the four-way analysis confirmed the results of the three-way analysis and revealed that both the degradation reaction of NADH and the formation reaction of FAD in human plasma fit the first-order rate law. The proposed methods could be expected to provide promising tools for simultaneous kinetic analysis of multiple reactions in complex systems in real-time and nondestructively. - Highlights: • A novel three-way calibration method for the quantitative kinetic analysis of NADH and FAD in human plasma is proposed. • The method can directly monitor the concentration of each analyte in the reaction in real-time and nondestructively. • The method has the second-order advantage. • A

  12. Quantitative fluorescence kinetic analysis of NADH and FAD in human plasma using three- and four-way calibration methods capable of providing the second-order advantage

    International Nuclear Information System (INIS)

    Kang, Chao; Wu, Hai-Long; Zhou, Chang; Xiang, Shou-Xia; Zhang, Xiao-Hua; Yu, Yong-Jie; Yu, Ru-Qin

    2016-01-01

    The metabolic coenzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are the primary electron donor and acceptor respectively, participate in almost all biological metabolic pathways. This study develops a novel method for the quantitative kinetic analysis of the degradation reaction of NADH and the formation reaction of FAD in human plasma containing an uncalibrated interferent, by using three-way calibration based on multi-way fluorescence technique. In the three-way analysis, by using the calibration set in a static manner, we directly predicted the concentrations of both analytes in the mixture at any time after the start of their reactions, even in the presence of an uncalibrated spectral interferent and a varying background interferent. The satisfactory quantitative results indicate that the proposed method allows one to directly monitor the concentration of each analyte in the mixture as the function of time in real-time and nondestructively, instead of determining the concentration after the analytical separation. Thereafter, we fitted the first-order rate law to their concentration data throughout their reactions. Additionally, a four-way calibration procedure is developed as an alternative for highly collinear systems. The results of the four-way analysis confirmed the results of the three-way analysis and revealed that both the degradation reaction of NADH and the formation reaction of FAD in human plasma fit the first-order rate law. The proposed methods could be expected to provide promising tools for simultaneous kinetic analysis of multiple reactions in complex systems in real-time and nondestructively. - Highlights: • A novel three-way calibration method for the quantitative kinetic analysis of NADH and FAD in human plasma is proposed. • The method can directly monitor the concentration of each analyte in the reaction in real-time and nondestructively. • The method has the second-order advantage. • A

  13. 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

  14. A novel nine base deletion mutation in NADH-cytochrome b5 reductase gene in an Indian family with recessive congenital methemoglobinemia-type-II

    Directory of Open Access Journals (Sweden)

    Prashant Warang

    2015-12-01

    Full Text Available Recessive hereditary methemoglobinemia (RCM associated with severe neurological abnormalities is a very rare disorder caused by NADH- cytochrome b5 reductase (cb5r deficiency (Type II. We report a case of 11 month old male child who had severe mental retardation, microcephaly and gross global developmental delay with methemoglobin level of 61.1%. The diagnosis of NADH-CYB5R3 deficiency was made by the demonstration of significantly reduced NADH-CYB5R3 activity in the patient and intermediate enzyme activity in both the parents. Mutation analysis of the CYB5R gene revealed a novel nine nucleotide deletion in exon 6 leading to the elimination of 3 amino acid residues (Lys173, Ser174 and Val 175. To confirm that this mutation was not an artifact, we performed PCR-RFLP analysis using the restriction enzyme Drd I. As the normal sequence has a restriction recognition site for Drd I which was eliminated by the deletion, a single band of 603-bp was seen in the presence of the homozygous mutation. Molecular modeling analysis showed a significant effect of these 3 amino acids deletion on the protein structure and stability leading to a severe clinical presentation. A novel homozygous 9 nucleotide deletion (p.K173–p.V175del3 is shown to be segregated with the disease in this family. Knowing the profile of mutations would allow us to offer prenatal diagnosis in families with severe neurological disorders associated with RCM — Type II.

  15. In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH.

    Science.gov (United States)

    Yaseen, Mohammad A; Sakadžić, Sava; Wu, Weicheng; Becker, Wolfgang; Kasischke, Karl A; Boas, David A

    2013-02-01

    Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

  16. Effects of exogenous vitamins A, C, and E and NADH supplementation on proliferation, cytokines release, and cell redox status of lymphocytes from healthy aged subjects.

    Science.gov (United States)

    Bouamama, Samia; Merzouk, Hafida; Medjdoub, Amel; Merzouk-Saidi, Amel; Merzouk, Sid Ahmed

    2017-06-01

    Aging is an inevitable biological event that is associated with immune alterations. These alterations are related to increased cellular oxidative stress and micronutrient deficiency. Antioxidant supplementation could improve these age-related abnormalities. The aim of this study was to determine in vitro effects of vitamin A, vitamin C, vitamin E, and nicotinamide adenine dinucleotide (NADH) on T cell proliferation, cytokine release, and cell redox status in the elderly compared with young adults. Peripheral blood lymphocytes were isolated using a density gradient of Histopaque. They were cultured in vitro and stimulated with concanavalin A in the presence or absence of vitamins. Cell proliferation was determined by conducting MTT assays, and based on interleukin-2 and interleukin-4 secretions. Cell oxidant/antioxidant balance was assessed by assaying reduced glutathione (GSH), malondialdehyde, carbonyl protein levels, and catalase activity. The present study demonstrated that T-lymphocyte proliferation was decreased with aging and was associated with cytokine secretion alterations, GSH depletion, and intracellular oxidative stress. In the elderly, vitamin C, vitamin E, and NADH significantly improved lymphocyte proliferation and mitigated cellular oxidative stress, whereas vitamin A did not affect cell proliferation or cell redox status. In conclusion, vitamin C, vitamin E, and NADH supplementation improved T-lymphocytes response in the elderly, and could contribute to the prevention of age-related immune alterations. Consumption of food items containing these vitamins is recommended, and further investigation is necessary to evaluate the effect of vitamin supplementation in vivo.

  17. Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.

    Science.gov (United States)

    Finel, M; Skehel, J M; Albracht, S P; Fearnley, I M; Walker, J E

    1992-11-24

    NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.

  18. 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

  19. 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

  20. 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

  1. Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH.

    Science.gov (United States)

    Yuan, Yufeng; Huang, Kehan; Chang, Mengfang; Qin, Cuifang; Zhang, Sanjun; Pan, Haifeng; Chen, Yan; Xu, Jianhua

    2016-02-01

    Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD(+) (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD(+) concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD(+) and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I395/I550), fitting by a single-exponential decay function, can efficiently detect various NAD(+) levels from 100 to 4000 μM, as well as label NAD(+)/NADH (reduced form of NAD) ratios in the range of 1-50. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. GLYCOLYTIC AND ENERGETIC RESOURCES AS THE DETERMINANTS OF MEAT QUALITY OF DUROC FATTENERS

    Directory of Open Access Journals (Sweden)

    Andrzej Zybert

    2015-03-01

    Full Text Available The aim of this study was to determine the diagnostic value of glycolityc and energetical quantities on selected meat quality characteristics of Duroc fatteners. A total of 40 Duroc porkers were investigated. Among two analysed sets of determinants (R1 with glycogen and R1 with lactate measured in 45 min after slaughter, the best diagnostic value for meat quality characteristics exhibit a set involving R1 and glycogen that composed determination coefficient (RC2 was 0.66 for pH measured in 45 min up to 144 h post mortem. Also, with currently used meat diagnostic methods the most useful one that explains the glycolytic and energetic quantities in the highest degree is method that exploits 5 determinants, i.e. pH1, pH24, EC2, EC24 and L*24. Higher composed determination coefficient and canonical correlation (CR for this method was obtained for set involving R1 and lactate – 0.41 and 0.64** respectively.

  3. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate.

    Science.gov (United States)

    Wang, X; Miller, E N; Yomano, L P; Zhang, X; Shanmugam, K T; Ingram, L O

    2011-08-01

    Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low K(m) for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms.

  4. Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate▿

    Science.gov (United States)

    Wang, X.; Miller, E. N.; Yomano, L. P.; Zhang, X.; Shanmugam, K. T.; Ingram, L. O.

    2011-01-01

    Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low Km for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms. PMID:21685167

  5. A complicated biocomputing system based on multi-responsive P(NIPAM-co-APBA) copolymer film electrodes and electrocatalysis of NADH.

    Science.gov (United States)

    Liang, Jiying; Yu, Xue; Yang, Tiangang; Li, Menglu; Shen, Li; Jin, Yue; Liu, Hongyun

    2017-08-23

    In this paper, poly(N-isopropylacrylamide-co-3-aminophenylboronic acid) (P(NIPAM-co-APBA)) copolymer films were successfully electropolymerized on the Au electrode surface. The electroactive probe ferrocene carboxylic acid (FCA) in solution showed reversible thermal-, glucose- and pH-responsive on-off cyclic voltammetric (CV) behaviors at the film electrodes. The comparative experiments demonstrated that the thermo-responsive property of the film electrode was ascribed to the PNIPAM component of the films, whereas the glucose- and pH-sensitive behaviors came from the PAPBA constituent. The reduced form of nicotinamide adenine dinucleotide (NADH) could be electrocatalytically oxidized by FCA at the film electrodes, which would greatly amplify the multi-responsive CV signal difference between the on and off states. On the basis of these results, a binary 4-input/4-output logic circuit was fabricated with temperature, glucose, pH and NADH as inputs and the CV responses at 4 different levels as outputs. Moreover, a ternary CONSENSUS logic circuit was established on the same platform, which was the first report on the combination of ternary logic gate and bioelectrocatalysis without using enzymes. This work provided a novel idea for constructing complicated biocomputing systems by increasing the number of inputs/outputs with multi-sensitive interfaces and by designing new types of multi-valued logic gates on the basis of bioelectrocatalysis.

  6. A water-forming NADH oxidase from Lactobacillus pentosus and its potential application in the regeneration of synthetic biomimetic cofactors

    Directory of Open Access Journals (Sweden)

    Claudia eNowak

    2015-09-01

    Full Text Available The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox. Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13 % FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyse the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as a by-product.

  7. Regulation of expression of Na+ -translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae.

    Science.gov (United States)

    Fadeeva, Maria S; Yakovtseva, Evgenia A; Belevich, Galina A; Bertsova, Yulia V; Bogachev, Alexander V

    2007-10-01

    The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na(+)-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.

  8. A highly sensitive NADH sensor based on a mycelium-like nanocomposite using graphene oxide and multi-walled carbon nanotubes to co-immobilize poly(luminol) and poly(neutral red) hybrid films.

    Science.gov (United States)

    Chiang Lin, Kuo; Yu Lai, Szu; Ming Chen, Shen

    2014-08-21

    Hybridization of poly(luminol) (PLM) and poly(neutral red) (PNR) has been successfully performed and further enhanced by a conductive and steric hybrid nanotemplate using graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs). The morphology of the PLM-PNR-MWCNT-GO mycelium-like nanocomposite is studied by SEM and AFM and it is found to be electroactive, pH-dependent, and stable in the electrochemical system. It shows electrocatalytic activity towards NADH with a high current response and low overpotential. Using amperometry, it has been shown to have a high sensitivity of 288.9 μA mM(-1) cm(-2) to NADH (Eapp. = +0.1 V). Linearity is estimated in a concentration range of 1.33 × 10(-8) to 1.95 × 10(-4) M with a detection limit of 1.33 × 10(-8) M (S/N = 3). Particularly, it also shows another linear range of 2.08 × 10(-4) to 5.81 × 10(-4) M with a sensitivity of 151.3 μA mM(-1) cm(-2). The hybridization and activity of PLM and PNR can be effectively enhanced by MWCNTs and GO, resulting in an active hybrid nanocomposite for determination of NADH.

  9. Fast-twitch glycolytic skeletal muscle is predisposed to age-induced impairments in mitochondrial function

    DEFF Research Database (Denmark)

    Jacobs, Robert A; Díaz, Víctor; Soldini, Lavinia

    2013-01-01

    The etiology of mammalian senescence is suggested to involve the progressive impairment of mitochondrial function; however, direct observations of age-induced alterations in actual respiratory chain function are lacking. Accordingly, we assessed mitochondrial function via high-resolution respirom......The etiology of mammalian senescence is suggested to involve the progressive impairment of mitochondrial function; however, direct observations of age-induced alterations in actual respiratory chain function are lacking. Accordingly, we assessed mitochondrial function via high......-resolution respirometry and mitochondrial protein expression in soleus, quadricep, and lateral gastrocnemius skeletal muscles, which represent type 1 slow-twitch oxidative muscle (soleus) and type 2 fast-twitch glycolytic muscle (quadricep and gastrocnemius), respectively, in young (10-12 weeks) and mature (74-76 weeks......) mice. Electron transport through mitochondrial complexes I and III increases with age in quadricep and gastrocnemius, which is not observed in soleus. Mitochondrial coupling efficiency during respiration through complex I also deteriorates with age in gastrocnemius and shows a tendency (p = .085...

  10. Cell-Intrinsic Glycogen Metabolism Supports Early Glycolytic Reprogramming Required for Dendritic Cell Immune Responses.

    Science.gov (United States)

    Thwe, Phyu M; Pelgrom, Leonard; Cooper, Rachel; Beauchamp, Saritha; Reisz, Julie A; D'Alessandro, Angelo; Everts, Bart; Amiel, Eyal

    2017-09-05

    Dendritic cell (DC) activation by Toll-like receptor (TLR) agonists causes rapid glycolytic reprogramming that is required to meet the metabolic demands of their immune activation. Recent efforts in the field have identified an important role for extracellular glucose sourcing to support DC activation. However, the contributions of intracellular glucose stores to these processes have not been well characterized. We demonstrate that DCs possess intracellular glycogen stores and that cell-intrinsic glycogen metabolism supports the early effector functions of TLR-activated DCs. Inhibition of glycogenolysis significantly attenuates TLR-mediated DC maturation and impairs their ability to initiate lymphocyte activation. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon

    Directory of Open Access Journals (Sweden)

    Sylvia A. Reimann

    2011-01-01

    Full Text Available Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that the E. coli ompR malTcon double mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalTcon, the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism.

  12. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Mädje Katharina

    2012-01-01

    Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

  13. Redox specificity of 2-hydroxyacid-coupled NAD(+/NADH dehydrogenases: a study exploiting "reactive" arginine as a reporter of protein electrostatics.

    Directory of Open Access Journals (Sweden)

    Pooja Gupta

    Full Text Available With "reactive" arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD(+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs and cytoplasmic and mitochondrial malate dehydrogenases (MDHs are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in "reactivity" of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on "reactive" arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of "reactive" arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure or reduce NAD(+ (cationic nicotinamide structure. The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide or reduced (neutral nicotinamide coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme.

  14. Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

    International Nuclear Information System (INIS)

    Campos, Dijon Henrique Salomé de; Leopoldo, André Soares; Lima-Leopoldo, Ana Paula; Nascimento, André Ferreira do; Oliveira-Junior, Silvio Assis de; Silva, Danielle Cristina Tomaz da; Sugizaki, Mario Mateus; Padovani, Carlos Roberto; Cicogna, Antonio Carlos

    2014-01-01

    Obesity is defined by excessive accumulation of body fat relative to lean tissue. Studies during the last few years indicate that cardiac function in obese animals may be preserved, increased or diminished. Study the energy balance of the myocardium with the hypothesis that the increase in fatty acid oxidation and reduced glucose leads to cardiac dysfunction in obesity. 30-day-old male Wistar rats were fed standard and hypercaloric diet for 30 weeks. Cardiac function and morphology were assessed. In this paper was viewed the general characteristics and comorbities associated to obesity. The structure cardiac was determined by weights of the heart and left ventricle (LV). Myocardial function was evaluated by studying isolated papillary muscles from the LV, under the baseline condition and after inotropic and lusitropic maneuvers: myocardial stiffness; postrest contraction; increase in extracellular Ca2+ concentration; change in heart rate and inhibitor of glycolytic pathway. Compared with control group, the obese rats had increased body fat and co-morbities associated with obesity. Functional assessment after blocking iodoacetate shows no difference in the linear regression of DT, however, the RT showed a statistically significant difference in behavior between the control and the obese group, most notable being the slope in group C. The energy imbalance on obesity did not cause cardiac dysfunction. On the contrary, the prioritization of fatty acids utilization provides protection to cardiac muscle during the inhibition of glycolysis, suggesting that this pathway is fewer used by obese cardiac muscle

  15. Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Campos, Dijon Henrique Salomé de, E-mail: dijoncampos@gmail.com [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu da Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil); Leopoldo, André Soares; Lima-Leopoldo, Ana Paula [Departamento de Esportes - Centro de Educação Física e Desportos da Universidade Federal do Espírito Santo (UFES), Vitória, ES (Brazil); Nascimento, André Ferreira do [Instituto de Ciências da Saúde da Universidade Federal do Mato Grosso (UFMT), Sinop, MT (Brazil); Oliveira-Junior, Silvio Assis de [Escola de Fisioterapia da Universidade Federal do Mato Grosso do Sul (UFMS), Campo Grande, MS (Brazil); Silva, Danielle Cristina Tomaz da [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu da Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil); Sugizaki, Mario Mateus [Instituto de Ciências da Saúde da Universidade Federal do Mato Grosso (UFMT), Sinop, MT (Brazil); Padovani, Carlos Roberto [Departamento de Bioestatística, Instituto de Ciências Biológicas da Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil); Cicogna, Antonio Carlos, E-mail: dijoncampos@gmail.com [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu da Universidade Estadual Paulista (UNESP), Botucatu, SP (Brazil)

    2014-10-15

    Obesity is defined by excessive accumulation of body fat relative to lean tissue. Studies during the last few years indicate that cardiac function in obese animals may be preserved, increased or diminished. Study the energy balance of the myocardium with the hypothesis that the increase in fatty acid oxidation and reduced glucose leads to cardiac dysfunction in obesity. 30-day-old male Wistar rats were fed standard and hypercaloric diet for 30 weeks. Cardiac function and morphology were assessed. In this paper was viewed the general characteristics and comorbities associated to obesity. The structure cardiac was determined by weights of the heart and left ventricle (LV). Myocardial function was evaluated by studying isolated papillary muscles from the LV, under the baseline condition and after inotropic and lusitropic maneuvers: myocardial stiffness; postrest contraction; increase in extracellular Ca2+ concentration; change in heart rate and inhibitor of glycolytic pathway. Compared with control group, the obese rats had increased body fat and co-morbities associated with obesity. Functional assessment after blocking iodoacetate shows no difference in the linear regression of DT, however, the RT showed a statistically significant difference in behavior between the control and the obese group, most notable being the slope in group C. The energy imbalance on obesity did not cause cardiac dysfunction. On the contrary, the prioritization of fatty acids utilization provides protection to cardiac muscle during the inhibition of glycolysis, suggesting that this pathway is fewer used by obese cardiac muscle.

  16. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

    Science.gov (United States)

    Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

    2015-09-15

    Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

  17. Metabolic imaging of tumor for diagnosis and response for therapy

    Science.gov (United States)

    Zagaynova, Elena; Shirmanova, Marina; Lukina, Maria; Dudenkova, Varvara; Ignatova, Nadezgda; Elagin, Vadim; Shlivko, Irena; Scheslavsky, Vladislav; Orlinskay, Natalia

    2018-02-01

    Nonlinear optical microscopy combined with fluorescence lifetime imaging is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a noninvasive investigation of the biological tissue with subcellular resolution. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. In this study features of tumor metabolism in different systems of organization (from cell culture to patient lesion) was showed. The observed differences in the relative contributions of free NAD(P)H and FAD testify to an increased a glycolytic metabolism in cancer cells compare to fibroblasts. In 3D spheroids, the cells of the proliferating zone had greater a1 and lower tm values than the cells of the quiescent zone, which likely is a consequence of their higher glycolytic rate. During the growth of colorectal cancer in the experimental mouse model, the contribution of the free component of NAD(P)H was increased. Dysplastic nevus and melanoma is characterized by raised contribution of free NADH compare to healthy skin. Therefore, melanoma cells had very short value of τ1.

  18. Aspartic acid 397 in subunit B of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae forms part of a sodium-binding site, is involved in cation selectivity, and affects cation-binding site cooperativity.

    Science.gov (United States)

    Shea, Michael E; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

    2013-10-25

    The Na(+)-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC).

  19. Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD+ and monitoring NAD+/NADH based enzymatic reactions.

    Science.gov (United States)

    Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

    2017-03-01

    A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ 660 nm . Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD + strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD + were 0.201 L mol -1 and 3.6, respectively. A dynamic NAD + detection range of 50-500 μM with a limit of detection of 22.3 μM was discerned. The NAD + mediated quenching of AgNC was not interfered by NADH, NADP + , monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD + /NADH based enzyme catalyzed reactions in a turn-on/turn-off approach. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Redox Specificity of 2-Hydroxyacid-Coupled NAD+/NADH Dehydrogenases: A Study Exploiting “Reactive” Arginine as a Reporter of Protein Electrostatics

    Science.gov (United States)

    Durani, Susheel

    2013-01-01

    With “reactive” arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as NAD+-reducing or NADH-oxidizing enzymes. Specifically, M4 and H4 lactate dehydrogenases (LDHs) and cytoplasmic and mitochondrial malate dehydrogenases (MDHs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. The enzymes from diverse eukaryote and prokaryote sources thus are assessed in “reactivity” of functionally-critical arginine as a function of salt concentration and pH. Electrostatic calculations were performed on “reactive” arginines and found good correspondence with experiment. The reductive and oxidative LDHs and MDHs are assessed in their count over ionizable residues and in placement details of the residues in their structures as proteins. The variants found to be high or low in ΔpKa of “reactive” arginine are found to be also strong or weak cations that preferentially oxidize NADH (neutral nicotinamide structure) or reduce NAD+ (cationic nicotinamide structure). The ionized groups of protein structure may thus be important to redox specificity of the enzyme on basis of electrostatic preference for the oxidized (cationic nicotinamide) or reduced (neutral nicotinamide) coenzyme. Detailed comparisons of isozymes establish that the residues contributing in their redox specificity are scrambled in structure of the reductive enzyme. PMID:24391777

  1. NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms

    Science.gov (United States)

    Wang, S.; Pogue, R.; Morre, D. M.; Morre, D. J.

    2001-01-01

    NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

  2. Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5.

    Science.gov (United States)

    Kollipara, Sireesha; Tatireddy, Shivakishore; Pathirathne, Thusitha; Rathnayake, Lasantha K; Northrup, Scott H

    2016-08-25

    Brownian dynamics (BD) simulations provide here a theoretical atomic-level treatment of the reduction of human ferric cytochrome b5 (cyt b5) by NADH-cytochrome b5 reductaste (cyt b5r) and several of its mutants. BD is used to calculate the second-order rate constant of electron transfer (ET) between the proteins for direct correlation with experiments. Interestingly, the inclusion of electrostatic forces dramatically increases the reaction rate of the native proteins despite the overall negative charge of both proteins. The role played by electrostatic charge distribution in stabilizing the ET complexes and the role of mutations of several amino acid residues in stabilizing or destabilizing the complexes are analyzed. The complex with the shortest ET reaction distance (d = 6.58 Å) from rigid body BD is further subjected to 1 ns of molecular dynamics (MD) in a periodic box of TIP3P water to produce a more stable complex allowed by flexibility and with a shorter average reaction distance d = 6.02 Å. We predict a docking model in which the following ion-ion interactions are dominant (cyt b5r/cyt b5): Lys162-Heme O1D/Lys163-Asp64/Arg91-Heme O1A/Lys125-Asp70.

  3. Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

    Directory of Open Access Journals (Sweden)

    Dijon Henrique Salomé de Campos

    2014-10-01

    Full Text Available Background: Obesity is defined by excessive accumulation of body fat relative to lean tissue. Studies during the last few years indicate that cardiac function in obese animals may be preserved, increased or diminished. Objective: Study the energy balance of the myocardium with the hypothesis that the increase in fatty acid oxidation and reduced glucose leads to cardiac dysfunction in obesity. Methods: 30-day-old male Wistar rats were fed standard and hypercaloric diet for 30 weeks. Cardiac function and morphology were assessed. In this paper was viewed the general characteristics and comorbities associated to obesity. The structure cardiac was determined by weights of the heart and left ventricle (LV. Myocardial function was evaluated by studying isolated papillary muscles from the LV, under the baseline condition and after inotropic and lusitropic maneuvers: myocardial stiffness; postrest contraction; increase in extracellular Ca2+ concentration; change in heart rate and inhibitor of glycolytic pathway. Results: Compared with control group, the obese rats had increased body fat and co-morbities associated with obesity. Functional assessment after blocking iodoacetate shows no difference in the linear regression of DT, however, the RT showed a statistically significant difference in behavior between the control and the obese group, most notable being the slope in group C. Conclusion: The energy imbalance on obesity did not cause cardiac dysfunction. On the contrary, the prioritization of fatty acids utilization provides protection to cardiac muscle during the inhibition of glycolysis, suggesting that this pathway is fewer used by obese cardiac muscle.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    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

  6. Characterization of phosphofructokinase activity in Mycobacterium tuberculosis reveals that a functional glycolytic carbon flow is necessary to limit the accumulation of toxic metabolic intermediates under hypoxia.

    Directory of Open Access Journals (Sweden)

    Wai Yee Phong

    Full Text Available Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7-10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when

  7. The plasma membrane-associated NADH oxidase (ECTO-NOX) of mouse skin responds to blue light

    Science.gov (United States)

    Morre, D. James; Morre, Dorothy M.

    2003-01-01

    NADH oxidases of the external plasma membrane surface (ECTO-NOX proteins) are characterized by oscillations in activity with a regular period length of 24 min. Explants of mouse skin exhibit the oscillatory activity as estimated from the decrease in A(340) suggesting that individual ECTO-NOX molecules must somehow be induced to function synchronously. Transfer of explants of mouse skin from darkness to blue light (495 nm, 2 min, 50 micromol m(-1) s(-1)) resulted in initiation of a new activity maximum (entrainment) with a midpoint 36 min after light exposure followed by maxima every 24 min thereafter. Addition of melatonin resulted in a new maximum 24 min after melatonin addition. The findings suggest that the ECTO-NOX proteins play a central role in the entrainment of the biological clock both by light and by melatonin.

  8. RNA sequencing reveals a slow to fast muscle fiber type transition after olanzapine infusion in rats.

    Directory of Open Access Journals (Sweden)

    Christopher J Lynch

    Full Text Available Second generation antipsychotics (SGAs, like olanzapine, exhibit acute metabolic side effects leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. Understanding how SGAs affect the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats were infused intravenously with vehicle or olanzapine for 24h using a dose leading to a mild hyperglycemia. RNA-Seq was performed on gastrocnemius muscle, followed by alignment of the data with the Rat Genome Assembly 5.0. Olanzapine altered expression of 1347 out of 26407 genes. Genes encoding skeletal muscle fiber-type specific sarcomeric, ion channel, glycolytic, O2- and Ca2+-handling, TCA cycle, vascularization and lipid oxidation proteins and pathways, along with NADH shuttles and LDH isoforms were affected. Bioinformatics analyses indicate that olanzapine decreased the expression of slower and more oxidative fiber type genes (e.g., type 1, while up regulating those for the most glycolytic and least metabolically flexible, fast twitch fiber type, IIb. Protein turnover genes, necessary to bring about transition, were also up regulated. Potential upstream regulators were also identified. Olanzapine appears to be rapidly affecting the muscle transcriptome to bring about a change to a fast-glycolytic fiber type. Such fiber types are more susceptible than slow muscle to atrophy, and such transitions are observed in chronic metabolic diseases. Thus these effects could contribute to the altered body composition and metabolic disease olanzapine causes. A potential interventional strategy is implicated because aerobic exercise, in contrast to resistance exercise, can oppose such slow to fast fiber transitions.

  9. Plasma membrane NADH oxidase of maize roots responds to gravity and imposed centrifugal forces

    Science.gov (United States)

    Bacon, E.; Morre, D. J.

    2001-01-01

    NADH oxidase activities measured with excised roots of dark-grown maize (Zea mays) seedlings and with isolated plasma membrane vesicles from roots of dark-grown maize oscillated with a regular period length of 24 min and were inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic [correction of dichorophenoxyacetic] acid. The activities also responded to orientation with respect to gravity and to imposed centrifugal forces. Turning the roots upside down resulted in stimulation of the activity with a lag of about 10 min. Returning the sections to the normal upright position resulted in a return to initial rates. The activity was stimulated reversibly to a maximum of about 2-fold with isolated plasma membrane vesicles, when subjected to centrifugal forces of 25 to 250 x g for 1 to 4 min duration. These findings are the first report of a gravity-responsive enzymatic activity of plant roots inhibited by auxin and potentially related to the gravity-induced growth response. c2001 Editions scientifiques et medicales Elsevier SAS.

  10. Poly(neutral red) as a NAD{sup +} reduction catalyst and a NADH oxidation catalyst: Towards the development of a rechargeable biobattery

    Energy Technology Data Exchange (ETDEWEB)

    Arechederra, Marguerite N.; Addo, Paul K. [Department of Chemistry, Saint Louis University, 3501 Laclede Ave., St. Louis, MO 63103 (United States); Minteer, Shelley D., E-mail: minteers@slu.ed [Department of Chemistry, Saint Louis University, 3501 Laclede Ave., St. Louis, MO 63103 (United States)

    2011-01-01

    In this paper, we have established that poly(neutral red), PNR, functions as an electrocatalyst for the reduction and oxidation of NAD{sup +}/NADH in a rechargeable biobattery environment. The reversibility of this catalyst was possible only with the addition of Zn{sup 2+} for complexation to the redox polymer. The zinc ion complexation with the polymer facilitates electron and proton transfer to/from the substrate and the NAD{sup +}/NADH coenzyme without forming covalent bonds between the nicotinamide and the substrate surface. This research presents use of this reversible catalyst in a rechargeable biobattery. The rechargeable battery includes a Prussian blue cathode and a bioanode including NAD{sup +}-dependent alcohol dehydrogenase and zinc complexed PNR. This bioanode was coupled to the cathode with Nafion 212 acting as the ion exchange membrane separator between the two compartments. The biobattery has an open circuit potential of 0.545({+-}0.009) V when first assembled and 0.053({+-}0.005) V when fully discharged. However, when fully charged, the biobattery has an open circuit potential of 1.263({+-}0.051) V, a maximum power density of 16.3({+-}4.03) {mu}W cm{sup -3} and a maximum current density of 221({+-}13.2) {mu}A cm{sup -3}. The efficiency and stability of the biobattery were studied by cycling continuously at a discharging rate of 1 C and the results obtained showed reasonable stability over 50 cycles.

  11. Effects of mouthrinses with chlorhexidine and zinc ions combined with fluoride on the viability and glycolytic activity of dental plaque.

    Science.gov (United States)

    Giertsen, E; Scheie, A A

    1995-10-01

    Inhibition of plaque acidogenicity by a mouthrinse with chlorhexidine (CHX) or zinc ions has been ascribed to a prolonged bacteriostasis due to substantive properties of the agents. The present aim was to study the effects of mouthrinses with CHX and Zn ions combined with fluoride on the viability and glycolytic activity of dental plaque in order to assess the bacteriostatic versus possible bactericidal effects. Following 2 d of plaque accumulation, 4 groups of 10 students rinsed with either 12 mM NaF (F), 0.55 mM CHX diacetate+F (F-CHX), 10 mM Zn acetate+F (F-Zn), or with the three agents in combination (F-CHX-Zn). Plaque samples were collected before and 90 min after mouthrinsing. Thereafter, the in vivo plaque pH response to sucrose was monitored in each student using touch microelectrodes. F-CHX and F-CHX-Zn reduced the in vivo pH fall significantly as compared with F, whereas F-Zn exerted a non-significant inhibition. Pooled pre- and post-rinse plaque samples were used to measure the pH fall during fermentation of [14C]-glucose, and the glycolytic profiles were analyzed by HPLC. Bacterial viability was assessed by counting the colony-forming units (CFU). All mouthrinses except F reduced glucose consumption and acid formation and thus the pH fall. F-CHX reduced the CFU equal to the reduction of glucose consumption, indicating that inhibition of plaque acidogenicity was due to a bactericidal rather than a bacteriostatic effect. F and F-Zn did not reduce the CFU, thus F-Zn decreased glucose metabolism without affecting plaque viability. F-CHX-Zn reduced both the CFU and glucose metabolism of surviving plaque microorganisms.

  12. Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

    Science.gov (United States)

    Chueh, Pin-Ju; Kim, Chinpal; Cho, NaMi; Morre, Dorothy M.; Morre, D. James

    2002-01-01

    NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

  13. Studies on cell-free metabolism: ethanol production by a yeast glycolytic system reconstituted from purified enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Welch, P; Scopes, R K

    1985-07-01

    A reconstituted glycolytic system has been established from individually purified enzymes to simulate the conversion of glucose to ethanol plus CO/sub 2/ by yeast. Sustained and extensive conversion occurred provided that input of glucose matched the rate of ATP degradation appropriately. ATPase activity could be replaced by arsenate, which uncoupled ATP synthesis from glycolysis. The mode of uncoupling was investigated, and it was concluded that the artificial intermediate, 1-arseno-3-phosphoglycerate, has a half-life of no more than a few milliseconds. Arsenate at 4 mM concentration could simulate the equivalent of 10 ..mu..mol/ml min. of ATPase activity. The reconstituted enzyme system was capable of totally degrading one M (18% w/v) glucose in 8 hours giving 9% (w/v) ethanol. The levels of metabolites during metabolism were measured to detect rate-limiting steps. The successful operation of the reconstituted enzyme system demonstrates that it is possible to carry out complex chemical transformations with multiple enzyme systems in vitro. 36 references.

  14. Exploiting mitochondrial dysfunction for effective elimination of imatinib-resistant leukemic cells.

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    Jérome Kluza

    Full Text Available Challenges today concern chronic myeloid leukemia (CML patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.

  15. In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (−)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase

    International Nuclear Information System (INIS)

    Çelik, Haydar; Koşar, Müberra; Arinç, Emel

    2013-01-01

    Highlights: • We assessed inhibitory effects of 8 dietary flavonoids on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase. • The flavonol myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. • We investigated kinetics of myricetin-induced inhibition in detail. • We explored the structure–inhibitory activity relationship of compounds. • Modulation of cytochrome b5 reduction indicates a potential for myricetin to lead to some food–drug/xenobiotic interactions. - Abstract: The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC 50 value of 0.35 μM. Myricetin inhibited b5 reductase noncompetitively with a K i of 0.21 μM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis–Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC 50 = 9.8 μM). The remaining flavonoids were inactive within the concentration range tested (1–50 μM). Analysis of structure–activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics

  16. Global ablation of the mitochondrial calcium uniporter increases glycolysis in cortical neurons subjected to energetic stressors.

    Science.gov (United States)

    Nichols, Matthew; Elustondo, Pia A; Warford, Jordan; Thirumaran, Aruloli; Pavlov, Evgeny V; Robertson, George S

    2017-08-01

    The effects of global mitochondrial calcium (Ca 2+ ) uniporter (MCU) deficiency on hypoxic-ischemic (HI) brain injury, neuronal Ca 2+ handling, bioenergetics and hypoxic preconditioning (HPC) were examined. Forebrain mitochondria isolated from global MCU nulls displayed markedly reduced Ca 2+ uptake and Ca 2+ -induced opening of the membrane permeability transition pore. Despite evidence that these effects should be neuroprotective, global MCU nulls and wild-type (WT) mice suffered comparable HI brain damage. Energetic stress enhanced glycolysis and depressed Complex I activity in global MCU null, relative to WT, cortical neurons. HI reduced forebrain NADH levels more in global MCU nulls than WT mice suggesting that increased glycolytic consumption of NADH suppressed Complex I activity. Compared to WT neurons, pyruvate dehydrogenase (PDH) was hyper-phosphorylated in MCU nulls at several sites that lower the supply of substrates for the tricarboxylic acid cycle. Elevation of cytosolic Ca 2+ with glutamate or ionomycin decreased PDH phosphorylation in MCU null neurons suggesting the use of alternative mitochondrial Ca 2+ transport. Under basal conditions, global MCU nulls showed similar increases of Ca 2+ handling genes in the hippocampus as WT mice subjected to HPC. We propose that long-term adaptations, common to HPC, in global MCU nulls compromise resistance to HI brain injury and disrupt HPC.

  17. Overexpression of CYB5R3 and NQO1, two NAD+ -producing enzymes, mimics aspects of caloric restriction.

    Science.gov (United States)

    Diaz-Ruiz, Alberto; Lanasa, Michael; Garcia, Joseph; Mora, Hector; Fan, Frances; Martin-Montalvo, Alejandro; Di Francesco, Andrea; Calvo-Rubio, Miguel; Salvador-Pascual, Andrea; Aon, Miguel A; Fishbein, Kenneth W; Pearson, Kevin J; Villalba, Jose Manuel; Navas, Placido; Bernier, Michel; de Cabo, Rafael

    2018-04-28

    Calorie restriction (CR) is one of the most robust means to improve health and survival in model organisms. CR imposes a metabolic program that leads to increased stress resistance and delayed onset of chronic diseases, including cancer. In rodents, CR induces the upregulation of two NADH-dehydrogenases, namely NAD(P)H:quinone oxidoreductase 1 (Nqo1) and cytochrome b 5 reductase 3 (Cyb5r3), which provide electrons for energy metabolism. It has been proposed that this upregulation may be responsible for some of the beneficial effects of CR, and defects in their activity are linked to aging and several age-associated diseases. However, it is unclear whether changes in metabolic homeostasis solely through upregulation of these NADH-dehydrogenases have a positive impact on health and survival. We generated a mouse that overexpresses both metabolic enzymes leading to phenotypes that resemble aspects of CR including a modest increase in lifespan, greater physical performance, a decrease in chronic inflammation, and, importantly, protection against carcinogenesis, one of the main hallmarks of CR. Furthermore, these animals showed an enhancement of metabolic flexibility and a significant upregulation of the NAD + /sirtuin pathway. The results highlight the importance of these NAD + producers for the promotion of health and extended lifespan. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  18. Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

    Directory of Open Access Journals (Sweden)

    NED A SETAYESH

    2009-01-01

    Full Text Available The present work aims to study a new NADH-cytochrome b5 reductase (cb5r from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73% with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3. The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.

  19. Comparative Effects of Phosphoenolpyruvate, a Glycolytic Intermediate, as an Organ Preservation Agent with Glucose and N-Acetylcysteine against Organ Damage during Cold Storage of Mouse Liver and Kidney

    OpenAIRE

    Ishitsuka, Yoichi; Fukumoto, Yusuke; Kondo, Yuki; Irikura, Mitsuru; Kadowaki, Daisuke; Narita, Yuki; Hirata, Sumio; Moriuchi, Hiroshi; Maruyama, Toru; Hamasaki, Naotaka; Irie, Tetsumi

    2013-01-01

    We evaluated the usefulness of phosphoenolpyruvate (PEP), a glycolytic intermediate with antioxidative and energy supplementation potentials, as an organ preservation agent. Using ex vivo mouse liver and kidney of a static cold storage model, we compared the effects of PEP against organ damage and oxidative stress during cold preservation with those of glucose or N-acetylcysteine (NAC). Lactate dehydrogenase (LDH) leakage, histological changes, and oxidative stress parameters (measured as thi...

  20. Estimation of the rate of energy production of rat mast cells in vitro

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    Rat mast cells were treated with glycolytic and respiratory inhibitors. The rate of adenosine triphosphate depletion of cells incubated with both types of inhibitors and the rate of lactate produced in presence of antimycin A and glucose were used to estimate the rate of oxidative and glycolytic...

  1. Temperature induces significant changes in both glycolytic reserve and mitochondrial spare respiratory capacity in colorectal cancer cell lines

    International Nuclear Information System (INIS)

    Mitov, Mihail I.; Harris, Jennifer W.; Alstott, Michael C.; Zaytseva, Yekaterina Y.; Evers, B. Mark; Butterfield, D. Allan

    2017-01-01

    Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32 °C, 37 °C and 42 °C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32 °C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37 °C. Mitochondrial stress test for SW480 cells at 37 °C vs 42 °C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37 °C vs 42 °C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

  2. Temperature induces significant changes in both glycolytic reserve and mitochondrial spare respiratory capacity in colorectal cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Mitov, Mihail I., E-mail: m.mitov@uky.edu [Markey Cancer Center, University of Kentucky, Lexington, KY 40536 (United States); Harris, Jennifer W. [Department of Surgery, University of Kentucky, Lexington, KY 40506 (United States); Alstott, Michael C.; Zaytseva, Yekaterina Y. [Markey Cancer Center, University of Kentucky, Lexington, KY 40536 (United States); Evers, B. Mark [Markey Cancer Center, University of Kentucky, Lexington, KY 40536 (United States); Department of Surgery, University of Kentucky, Lexington, KY 40506 (United States); Butterfield, D. Allan [Markey Cancer Center, University of Kentucky, Lexington, KY 40536 (United States); Department of Chemistry, University of Kentucky, Lexington, KY 40506 (United States)

    2017-05-15

    Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32 °C, 37 °C and 42 °C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32 °C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37 °C. Mitochondrial stress test for SW480 cells at 37 °C vs 42 °C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37 °C vs 42 °C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

  3. 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.

  4. Lumba-Lumba Hidung Botol Laut Jawa Adalah Tursiops aduncus Berdasar Sekuen Gen NADH Dehidrogenase Subunit 6 (VERIFICATION BOTTLENOSE DOLPHINS FROM JAVA SEA IS TURSIOPS ADUNCUS BASED ON GENE SEQUENCES OF NADH DEHYDROGENASE SUBUNIT 6

    Directory of Open Access Journals (Sweden)

    Rini Widayanti

    2014-05-01

    Full Text Available Bottlenose dolphins (Tursiops sp. is one of the aquatic mammals widely spread in the marines ofIndonesia archipelago, especially the Java Sea. The taxonomy of the genus Tursiops is still  controversial.The purpose of this study was to examine the molecular basis of Tursiops sp of Java sea marine origin onthe basis of its NADH dehydrogenase gene subunit 6 (ND6 sequences. Samples of blood were collectedfrom five male bottle nose dolphins from captivity of PT. Wersut Seguni Indonesia. DNA was isolated,amplified by polymerase chain reaction (PCR, sequenced, and analyzed the data using the MEGA v. 5.1program. The results of PCR amplification was 868 base pairs (bp, DNA sequencing showed that 528nucleotides were ND6 gene, nucleotide at the position of 387 could be used to distinguish the bottle nosedolphins Java marine origin with T. aduncus.   Filogram using Neighbor joining method based on thenucleotide sequence of the gene ND6, showed that bottle nose dolphins Java marine origin belong to groupof T. aduncus.

  5. 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

  6. In vivo (31) P MRS assessment of intracellular NAD metabolites and NAD(+) /NADH redox state in human brain at 4 T.

    Science.gov (United States)

    Lu, Ming; Zhu, Xiao-Hong; Chen, Wei

    2016-07-01

    NAD(+) and NADH play key roles in cellular respiration. Intracellular redox state defined by the NAD(+) /NADH ratio (RX) reflects the cellular metabolic and physiopathological status. By taking advantage of high/ultrahigh magnetic field strengths, we have recently established a novel in vivo (31) P MRS-based NAD assay for noninvasive and quantitative measurements of intracellular NAD concentrations and redox state in animal and human brains at 16.4 T, 9.4 T and 7 T. To explore its potential for clinical application, in this study we investigated the feasibility of assessing the NAD metabolism and redox state in human brain at a lower field of 4 T by incorporating the (1) H-decoupling technique with the in vivo (31) P NAD assay. The use of (1) H decoupling significantly narrowed the linewidths of NAD and α-ATP resonances, resulting in higher sensitivity and better spectral resolution as compared with the (1) H-coupled (31) P spectrum. These improvements made it possible to reliably quantify cerebral NAD concentrations and RX, consistent with previously reported results obtained from similar age human subjects at 7 T. In summary, this work demonstrates the capability and utility of the (1) H-decoupled (31) P MRS-based NAD assay at lower field strength; thus, it opens new opportunities for studying intracellular NAD metabolism and redox state in human brain at clinical settings. This conclusion is supported by the simulation results, indicating that similar performance and reliability as observed at 4T can be achieved at 3 T with the same signal-to-noise ratio. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and in glycolytic flux

    DEFF Research Database (Denmark)

    Andersen, Heidi Winterberg; Solem, Christian; Hammer, Karin

    2001-01-01

    reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions, One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one...... kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further...... of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth fate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6- phosphate...

  8. 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

  9. A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid.

    Science.gov (United States)

    Castro, Maite A; Beltrán, Felipe A; Brauchi, Sebastián; Concha, Ilona I

    2009-07-01

    In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K(+)]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD(+)/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.

  10. Engineering redox homeostasis to develop efficient alcohol-producing microbial cell factories.

    Science.gov (United States)

    Zhao, Chunhua; Zhao, Qiuwei; Li, Yin; Zhang, Yanping

    2017-06-24

    The biosynthetic pathways of most alcohols are linked to intracellular redox homeostasis, which is crucial for life. This crucial balance is primarily controlled by the generation of reducing equivalents, as well as the (reduction)-oxidation metabolic cycle and the thiol redox homeostasis system. As a main oxidation pathway of reducing equivalents, the biosynthesis of most alcohols includes redox reactions, which are dependent on cofactors such as NADH or NADPH. Thus, when engineering alcohol-producing strains, the availability of cofactors and redox homeostasis must be considered. In this review, recent advances on the engineering of cellular redox homeostasis systems to accelerate alcohol biosynthesis are summarized. Recent approaches include improving cofactor availability, manipulating the affinity of redox enzymes to specific cofactors, as well as globally controlling redox reactions, indicating the power of these approaches, and opening a path towards improving the production of a number of different industrially-relevant alcohols in the near future.

  11. Mono and bimetallic nanoparticles of gold, silver and palladium-catalyzed NADH oxidation-coupled reduction of Eosin-Y

    Science.gov (United States)

    Santhanalakshmi, J.; Venkatesan, P.

    2011-02-01

    Mono metallic (Au, Ag, Pd) and bimetallic (Au-Ag, Ag-Pd, Au-Pd) with 1:1 mol stoichiometry, nanoparticles are synthesized using one-pot, temperature controlled chemical method using cetyltrimethylammonium bromide (CTAB) as the capping agent. The particle sizes (Au = 5.6, Ag = 5.0, Pd = 6.0, Au-Ag = 9.2, Ag-Pd = 9.6, Au-Pd = 9.4 nm) are characterized by UV-Vis, HRTEM, and XRD measurements, respectively. CTAB bindings onto mono and bimetallic nanoparticles are analyzed by FTIR spectra. The catalytic activities of mono and bimetallic nanoparticles are tested on the reaction between NADH oxidation and Eosin-Y reduction. The effects of base, pH, ionic strength, nature of mono and bimetallic catalysts are studied and the reaction conditions are optimized. Bimetallic nanoparticles exhibited better catalysis than the mono metallic nanoparticles, which may be due to the electronic effects of the core to shell metal atoms.

  12. Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

    Directory of Open Access Journals (Sweden)

    Flora H.F. D'Angelis

    2014-09-01

    Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

  13. CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors.

    Science.gov (United States)

    Le Floch, Renaud; Chiche, Johanna; Marchiq, Ibtissam; Naiken, Tanesha; Naïken, Tanesha; Ilc, Karine; Ilk, Karine; Murray, Clare M; Critchlow, Susan E; Roux, Danièle; Simon, Marie-Pierre; Pouysségur, Jacques

    2011-10-04

    Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H(+)/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res(-)) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res(-) cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H(+) symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin(high) cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.

  14. Mono and bimetallic nanoparticles of gold, silver and palladium-catalyzed NADH oxidation-coupled reduction of Eosin-Y

    International Nuclear Information System (INIS)

    Santhanalakshmi, J.; Venkatesan, P.

    2011-01-01

    Mono metallic (Au, Ag, Pd) and bimetallic (Au–Ag, Ag–Pd, Au–Pd) with 1:1 mol stoichiometry, nanoparticles are synthesized using one-pot, temperature controlled chemical method using cetyltrimethylammonium bromide (CTAB) as the capping agent. The particle sizes (Au = 5.6, Ag = 5.0, Pd = 6.0, Au–Ag = 9.2, Ag–Pd = 9.6, Au–Pd = 9.4 nm) are characterized by UV–Vis, HRTEM, and XRD measurements, respectively. CTAB bindings onto mono and bimetallic nanoparticles are analyzed by FTIR spectra. The catalytic activities of mono and bimetallic nanoparticles are tested on the reaction between NADH oxidation and Eosin-Y reduction. The effects of base, pH, ionic strength, nature of mono and bimetallic catalysts are studied and the reaction conditions are optimized. Bimetallic nanoparticles exhibited better catalysis than the mono metallic nanoparticles, which may be due to the electronic effects of the core to shell metal atoms.Graphical Abstract

  15. The plasma membrane-associated NADH oxidase of spinach leaves responds to blue light

    Science.gov (United States)

    Morre, D. James; Penel, Claude; Greppin, Hubert; Morre, Dorothy M.

    2002-01-01

    The plasma membrane-associated NADH oxidase (NOX) of spinach leaf disks is characterized by oscillations in activity with a regular period length of ca. 24 min. Within a single population of plants exposed to light at the same time, NOX activities of all plants function synchronously. Exposure of plants transferred from darkness to blue light (495 nm, 2 min, 50 micromoles m-2 s-1) resulted in a complex response pattern but with a new maximum in the rate of NOX activity 36 (24+12) min after illumination and then with maxima in the rate of NOX activity every 24 min thereafter. Transient maxima in NOX activity were observed as well after 9.3 + /- 1.4 and 20.7 +/- 2.1 min. The blue light response differed from the response to red (650 nm, 10 min, 50 micromoles m-2 s-1) or white light where activity maxima were initiated 12 min after the light exposure followed by maxima every 24 min thereafter. Green or yellow light was ineffective. The light response was independent of the time in the 24-min NOX cycle when the light was given. The net effects of blue and red light were ultimately the same with a new maximum in the rate of NOX activity at 12+24=36 min (and every 24 min thereafter), but the mechanisms appear to be distinct.

  16. An azoreductase, aerobic NADH-dependent flavoprotein discovered from Bacillus sp.: functional expression and enzymatic characterization.

    Science.gov (United States)

    Ooi, Toshihiko; Shibata, Takeshi; Sato, Reiko; Ohno, Hiroaki; Kinoshita, Shinichi; Thuoc, Tran Linh; Taguchi, Seiichi

    2007-05-01

    The gene coding for an azoreductase, designated as an azrA, was cloned by polymerase chain reaction amplification from the genomic DNA of Bacillus sp. strain B29 isolated from soil. The azrA encoded a protein of 208 amino acids with calculated molecular mass of 22,766 Da. The enzyme was heterologously expressed in Escherichia coli with a strong band of 23 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Purified recombinant AzrA was a homodimer with a native molecular mass of 48 kDa containing two molecules of flavin mononucleotide (FMN; oxidized). This activity was oxygen insensitive and was nicotinamide adenine dinucleotide (reduced form; NADH) dependent. Recombinant AzrA exhibited a broad pH stability between 6 and 10 with a temperature optimum of 60-80 degrees C. The enzyme cleaved the model azo compound of methyl red [MR, 4'-(dimethylamino)-azobenzene-2-carboxylic acid] into 2-aminobenzoic acid and N, N'-dimethyl-p-phenylenediamine by ping-pong mechanism. The enzyme was not only able to decolorize MR but also able to decolorize sulfonated azo dyes such as Orange I and Acid Red 88.

  17. Size controlled synthesis of biocompatible gold nanoparticles and their activity in the oxidation of NADH

    International Nuclear Information System (INIS)

    Chandran, Parvathy R; Sandhyarani, N; Naseer, M; Udupa, N

    2012-01-01

    Size and shape controlled synthesis remains a major bottleneck in the research on nanoparticles even after the development of different methods for their preparation. By tuning the size and shape of a nanoparticle, the intrinsic properties of the nanoparticle can be controlled leading tremendous potential applications in different fields of science and technology. We describe a facile route for the one pot synthesis of gold nanoparticles in water using monosodium glutamate as the reducing and stabilizing agent in the absence of seed particles. The particle diameter can be easily controlled by varying the pH of the reaction medium. Nanoparticles were characterized using scanning electron microscopy, UV–vis absorption spectroscopy, cyclic voltammetry, and dynamic light scattering. Zeta potential measurements were made to compare the stability of the different nanoparticles. The results suggest that lower pH favours a nucleation rate giving rise to smaller particles and higher pH favours a growth rate leading to the formation of larger particles. The synthesized nanoparticles are found to be stable and biocompatible. The nanoparticles synthesized at high pH exhibited a good electrocatalytic activity towards oxidation of nicotinamide adenine dinucleotide (NADH).

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  19. Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation.

    Directory of Open Access Journals (Sweden)

    Christian Berrios

    2016-11-01

    Full Text Available Merkel cell polyomavirus (MCPyV is an etiological agent of Merkel cell carcinoma (MCC, a highly aggressive skin cancer. The MCPyV small tumor antigen (ST is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1. Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis.

  20. Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism.

    Science.gov (United States)

    Yu, Li; Sun, Yifan; Li, Jingjing; Wang, Yan; Zhu, Yuxing; Shi, Yong; Fan, Xiaojun; Zhou, Jianda; Bao, Ying; Xiao, Jie; Cao, Ke; Cao, Peiguo

    2017-08-15

    Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.

  1. Scale-down of continuous protein producing Saccharomyces cerevisiae cultivations using a two compartment system

    DEFF Research Database (Denmark)

    Wright, Naia Risager; Rønnest, Nanna Petersen; Thykær, Jette

    2016-01-01

    ml standard continuous cultivations. It was found that substrate gradients within a limited range result in increased productivity of the heterologous protein under regulation of the glycolytic TPI promoter and delay the decrease of protein and trehalose production during continuous cultivation...

  2. Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

    Science.gov (United States)

    Sacco, James C; Trepanier, Lauren A

    2010-01-01

    NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (Phydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

  3. Isolation of a high malic and low acetic acid-producing sake yeast Saccharomyces cerevisiae strain screened from respiratory inhibitor 2,4-dinitrophenol (DNP)-resistant strains.

    Science.gov (United States)

    Kosugi, Shingo; Kiyoshi, Keiji; Oba, Takahiro; Kusumoto, Kenichi; Kadokura, Toshimori; Nakazato, Atsumi; Nakayama, Shunichi

    2014-01-01

    We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+). Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Ketose induced respiratory inhibition in isolated hepatocytes.

    Science.gov (United States)

    Martínez, P; Carrascosa, J M; Núñez de Castro, I

    1987-06-01

    The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.

  5. Probing the redox metabolism in the strictly anaerobic, extremely thermophilic, hydrogen-producing Caldicellulosiruptor saccharolyticus using amperometry

    DEFF Research Database (Denmark)

    Kostesha, Natalie; Willquist, Karin; Emnéus, Jenny

    2011-01-01

    Changes in the redox metabolism in the anaerobic, extremely thermophilic, hydrogen-forming bacterium Caldicellulosiruptor saccharolyticus were probed for the first time in vivo using mediated amperometry with ferricyanide as a thermotolerant external mediator. Clear differences in the intracellul...... in the intracellular electron flow and to probe redox enzyme properties of a strictly anaerobic thermophile in vivo.......Changes in the redox metabolism in the anaerobic, extremely thermophilic, hydrogen-forming bacterium Caldicellulosiruptor saccharolyticus were probed for the first time in vivo using mediated amperometry with ferricyanide as a thermotolerant external mediator. Clear differences in the intracellular...... the NADH-dependent lactate dehydrogenase, upon which more NADH was directed to membrane-associated enzymes for ferricyanide reduction, leading to a higher electrochemical signal. The method is noninvasive and the results presented here demonstrate that this method can be used to accurately detect changes...

  6. Are bacteria the major producers of extracellular glycolytic enzymes in aquatic environments?

    Czech Academy of Sciences Publication Activity Database

    Vrba, Jaroslav; Callieri, C.; Bittl, T.; Šimek, Karel; Bertoni, R.; Filandr, P.; Hartman, Petr; Hejzlar, Josef; Macek, Miroslav; Nedoma, Jiří

    2004-01-01

    Roč. 89, č. 1 (2004), s. 102-117 ISSN 1434-2944 R&D Projects: GA AV ČR(CZ) IBS6017004; GA ČR(CZ) GA206/99/0028; GA ČR(CZ) GA206/00/0063 Institutional research plan: CEZ:AV0Z6017912 Keywords : ectoenzyme activity * diatoms * Daphnia longispina Subject RIV: DA - Hydrology ; Limnology Impact factor: 0.742, year: 2004

  7. Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mechanism of lipid accumulation at the proteomic level.

    Science.gov (United States)

    Tang, Xin; Zan, Xinyi; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda; Ratledge, Colin

    2016-02-11

    The oleaginous fungus, Mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. Nitrogen (N) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. Although a simple pathway from N depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon N depletion have not been investigated. In this study, we have systematically analyzed the changes at the levels of protein expression in M. circinelloides WJ11, a high lipid-producing strain (36 %, lipid/cell dry weight), during lipid accumulation. Proteomic analysis demonstrated that N depletion increased the expression of glutamine synthetase, involved in ammonia assimilation, for the supply of cellular nitrogen but decreased the metabolism of amino acids. Upon N deficiency, many proteins (e.g., fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase) involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle (e.g., isocitrate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydratase) were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, glucose-6-phosphate dehydrogenase, transaldolase and transketolase, which participate in the pentose phosphate pathway, were up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency. N limitation increased those expressions of the proteins involved in ammonia assimilation but decreased that

  8. Autoimmunity against a glycolytic enzyme as a possible cause for persistent symptoms in Lyme disease.

    Science.gov (United States)

    Maccallini, Paolo; Bonin, Serena; Trevisan, Giusto

    2018-01-01

    Some patients with a history of Borrelia burgdorferi infection develop a chronic symptomatology characterized by cognitive deficits, fatigue, and pain, despite antibiotic treatment. The pathogenic mechanism that underlines this condition, referred to as post-treatment Lyme disease syndrome (PTLDS), is currently unknown. A debate exists about whether PTLDS is due to persistent infection or to post-infectious damages in the immune system and the nervous system. We present the case of a patient with evidence of exposure to Borrelia burgdorferi sl and a long history of debilitating fatigue, cognitive abnormalities and autonomic nervous system issues. The patient had a positive Western blot for anti-basal ganglia antibodies, and the autoantigen has been identified as γ enolase, the neuron-specific isoenzyme of the glycolytic enzyme enolase. Assuming Borrelia own surface exposed enolase as the source of this autoantibody, through a mechanism of molecular mimicry, and given the absence of sera reactivity to α enolase, a bioinformatical analysis was carried out to identify a possible cross-reactive conformational B cell epitope, shared by Borrelia enolase and γ enolase, but not by α enolase. Taken that evidence, we hypothesize that this autoantibody interferes with glycolysis in neuronal cells, as the physiological basis for chronic symptoms in at least some cases of PTLDS. Studies investigating on the anti-γ enolase and anti-Borrelia enolase antibodies in PTLDS are needed to confirm our hypotheses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. 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

  10. 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.

  11. Upregulation of glycolytic enzymes, mitochondrial dysfunction and increased cytotoxicity in glial cells treated with Alzheimer's disease plasma.

    Directory of Open Access Journals (Sweden)

    Tharusha Jayasena

    Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each from healthy controls, individuals with amnestic mild cognitive impairment (aMCI and Alzheimer's disease (AD on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.

  12. The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

    Directory of Open Access Journals (Sweden)

    Sven Vilain

    2012-01-01

    Full Text Available Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC; however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I-associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling.

  13. Efficient electrochemical regeneration of nicotinamide cofactors using a cyclopentadienyl-rhodium complex on functionalized indium tin oxide electrodes

    International Nuclear Information System (INIS)

    Kim, Soojin; Lee, Ga Ye; Lee, Jungha; Rajkumar, Eswaran; Baeg, Jin-Ook; Kim, Jinheung

    2013-01-01

    Functionalized ITO electrodes are used to regenerate NADH using [Cp*Rh(bpy)(H 2 O)] 2+ (Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine) electrochemically in a buffer solution. Amino- and mercapto-functionalized electrodes featured higher activity and stability for electrocatalytic generation of NADH than a bare ITO electrode. Effect of metal nanoparticles was also studied on modified ITO electrodes and the addition of platinum nanoparticles even resulted in improved activity. The electrochemical regeneration was somewhat affected in the presence of dioxygen, but not significantly. In addition, a conversion of carbon dioxide was carried out utilizing the electrochemically generated NADH and formate dehydrogenase to produce formic acid

  14. Isolation and characterization of novel 1,3-propanediol-producing Lactobacillus panis PM1 from bioethanol thin stillage.

    Science.gov (United States)

    Khan, Nurul H; Kang, Tae Sun; Grahame, Douglas A S; Haakensen, Monique C; Ratanapariyanuch, Kornsulee; Reaney, Martin J; Korber, Darren R; Tanaka, Takuji

    2013-01-01

    Conversion of glycerol to 1,3-propanediol (1,3-PDO) is an attractive option to increase the economic efficiency of the biofuel industry. A bacterial strain that produced 1,3-PDO in the presence of glycerol was isolated from thin stillage, the fermentation residue of bioethanol production. This 1,3-PDO-producing organism was identified as Lactobacillus panis through biochemical characteristics and by 16S rRNA sequencing. Characterization of the L. panis strain hereafter designated as PM1 revealed it was an aerotolerant acidophilic anaerobe able to grow over a wide range of temperatures; tolerant to high concentrations of sodium chloride, ethanol, acetic acid, and lactic acid; and resistant to many common antibiotics. L. panis PM1 could utilize glucose, lactose, galactose, maltose, xylose, and arabinose, but could not grow on sucrose or fructose. Production of 1,3-PDO by L. panis PM1 occurred only when glucose was available as the carbon source in the absence of oxygen. These metabolic characteristics strongly suggested NADH recycling for glucose metabolism is achieved through 1,3-PDO production by this strain. These characteristics classified L. panis PM1 within the group III heterofermentative lactic acid bacteria, which includes the well-characterized 1,3-PDO-producing strain, Lactobacillus reuteri. Metabolite production profiles showed that L. panis PM1 produced considerable amounts of succinic acid (~11-12 mM) from normal MRS medium, which distinguishes this strain from L. reuteri strains.

  15. Understanding start-up problems in yeast glycolysis.

    Science.gov (United States)

    Overal, Gosse B; Teusink, Bas; Bruggeman, Frank J; Hulshof, Josephus; Planqué, Robert

    2018-05-01

    Yeast glycolysis has been the focus of research for decades, yet a number of dynamical aspects of yeast glycolysis remain poorly understood at present. If nutrients are scarce, yeast will provide its catabolic and energetic needs with other pathways, but the enzymes catalysing upper glycolytic fluxes are still expressed. We conjecture that this overexpression facilitates the rapid transition to glycolysis in case of a sudden increase in nutrient concentration. However, if starved yeast is presented with abundant glucose, it can enter into an imbalanced state where glycolytic intermediates keep accumulating, leading to arrested growth and cell death. The bistability between regularly functioning and imbalanced phenotypes has been shown to depend on redox balance. We shed new light on these phenomena with a mathematical analysis of an ordinary differential equation model, including NADH to account for the redox balance. In order to gain qualitative insight, most of the analysis is parameter-free, i.e., without assigning a numerical value to any of the parameters. The model has a subtle bifurcation at the switch between an inviable equilibrium state and stable flux through glycolysis. This switch occurs if the ratio between the flux through upper glycolysis and ATP consumption rate of the cell exceeds a fixed threshold. If the enzymes of upper glycolysis would be barely expressed, our model predicts that there will be no glycolytic flux, even if external glucose would be at growth-permissable levels. The existence of the imbalanced state can be found for certain parameter conditions independent of the mentioned bifurcation. The parameter-free analysis proved too complex to directly gain insight into the imbalanced states, but the starting point of a branch of imbalanced states can be shown to exist in detail. Moreover, the analysis offers the key ingredients necessary for successful numerical continuation, which highlight the existence of this bistability and the

  16. Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli.

    Science.gov (United States)

    Song, Hun-Suk; Jeon, Jong-Min; Kim, Hyun-Joong; Bhatia, Shashi Kant; Sathiyanarayanan, Ganesan; Kim, Junyoung; Won Hong, Ju; Gi Hong, Yoon; Young Choi, Kwon; Kim, Yun-Gon; Kim, Wooseong; Yang, Yung-Hun

    2017-12-01

    To reduce the furfural toxicity for biochemical production in E. coli, a new strategy was successfully applied by supplying NAD(P)H through the nicotine amide salvage pathway. To alleviate the toxicity, nicotinamide salvage pathway genes were overexpressed in recombinant, isobutanol-producing E. coli. Gene expression of pncB and nadE respectively showed increased tolerance to furfural among these pathways. The combined expression of pncB and nadE was the most effective in increasing the tolerance of the cells to toxic aldehydes. By comparing noxE- and fdh-harbouring strains, the form of NADH, rather than NAD + , was the major effector of furfural tolerance. Overall, this study is the application of the salvage pathway to isobutanol production in the presence of furfural, and this system seems to be applicable to alleviate furfural toxicity in the production of other biochemical. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Redox conditions and protein oxidation in plant mitochondria

    DEFF Research Database (Denmark)

    Møller, Ian Max; Kasimova, Marina R.; Krab, Klaas

    2005-01-01

    Redox conditions and protein oxidation in plant mitochondria NAD(P)H has a central position in respiratory metabolism. It is produced by a large number of enzymes, e.g. the Krebs cycle dehydrogenases, in the mitochondrial matrix and is oxidised by, amongst others, the respiratory chain. Most...... of this NAD(P)H appears to be bound to proteins, in fact free NAD(P)H – an important parameter in metabolic regulation - has never been observed in mitochondria. We have estimated free and bound NAD(P)H in isolated plant mitochondria under different metabolic conditions. The fluorescence spectra of free...... and bound NADH was determined and used to deconvolute fluorescence spectra of actively respiring mitochondria. Most of the mitochondrial NADH is bound in states 2 and 4. The amount of free NADH is lower but relatively constant even increasing a little in state 3 where it is about equal to bound NADH...

  18. Effects of the deletion of the Escherichia coli frataxin homologue CyaY on the respiratory NADH:ubiquinone oxidoreductase

    Directory of Open Access Journals (Sweden)

    Grauman Peter L

    2007-07-01

    Full Text Available Abstract Background Frataxin is discussed as involved in the biogenesis of iron-sulfur clusters. Recently it was discovered that a frataxin homologue is a structural component of the respiratory NADH:ubiquinone oxidoreductase (complex I in Thermus thermophilus. It was not clear whether frataxin is in general a component of complex I from bacteria. The Escherichia coli homologue of frataxin is coined CyaY. Results We report that complex I is completely assembled to a stable and active enzyme complex equipped with all known iron-sulfur clusters in a cyaY mutant of E. coli. However, the amount of complex I is reduced by one third compared to the parental strain. Western blot analysis and live cell imaging of CyaY engineered with a GFP demonstrated that CyaY is located in the cytoplasm and not attached to the membrane as to be expected if it were a component of complex I. Conclusion CyaY plays a non-essential role in the assembly of complex I in E. coli. It is not a structural component but may transiently interact with the complex.

  19. Calcium signaling in brain mitochondria: interplay of malate aspartate NADH shuttle and calcium uniporter/mitochondrial dehydrogenase pathways.

    Science.gov (United States)

    Contreras, Laura; Satrústegui, Jorgina

    2009-03-13

    Ca2+ signaling in mitochondria has been mainly attributed to Ca2+ entry to the matrix through the Ca2+ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca2+ through a mechanism that involves the aspartate-glutamate carriers, extramitochondrial Ca2+ activation of the NADH malate-aspartate shuttle (MAS). Both pathways are linked through the shared substrate alpha-ketoglutarate (alphaKG). Here we have studied the interplay between the two pathways under conditions of Ca2+ activation. We show that alphaKG becomes limiting when Ca2+ enters in brain or heart mitochondria, but not liver mitochondria, resulting in a drop in alphaKG efflux through the oxoglutarate carrier and in a drop in MAS activity. Inhibition of alphaKG efflux and MAS activity by matrix Ca2+ in brain mitochondria was fully reversible upon Ca2+ efflux. Because of their differences in cytosolic calcium concentration requirements, the MAS and Ca2+ uniporter-mitochondrial dehydrogenase pathways are probably sequentially activated during a Ca2+ transient, and the inhibition of MAS at the center of the transient may provide an explanation for part of the increase in lactate observed in the stimulated brain in vivo.

  20. Subcellular localization of glycolytic enzymes and characterization of intermediary metabolism of Trypanosoma rangeli.

    Science.gov (United States)

    Rondón-Mercado, Rocío; Acosta, Héctor; Cáceres, Ana J; Quiñones, Wilfredo; Concepción, Juan Luis

    2017-09-01

    Trypanosoma rangeli is a hemoflagellate protist that infects wild and domestic mammals as well as humans in Central and South America. Although this parasite is not pathogenic for human, it is being studied because it shares with Trypanosoma cruzi, the etiological agent of Chagas' disease, biological characteristics, geographic distribution, vectors and vertebrate hosts. Several metabolic studies have been performed with T. cruzi epimastigotes, however little is known about the metabolism of T. rangeli. In this work we present the subcellular distribution of the T. rangeli enzymes responsible for the conversion of glucose to pyruvate, as determined by epifluorescense immunomicroscopy and subcellular fractionation involving either selective membrane permeabilization with digitonin or differential and isopycnic centrifugation. We found that in T. rangeli epimastigotes the first six enzymes of the glycolytic pathway, involved in the conversion of glucose to 1,3-bisphosphoglycerate are located within glycosomes, while the last four steps occur in the cytosol. In contrast with T. cruzi, where three isoenzymes (one cytosolic and two glycosomal) of phosphoglycerate kinase are expressed simultaneously, only one enzyme with this activity is detected in T. rangeli epimastigotes, in the cytosol. Consistent with this latter result, we found enzymes involved in auxiliary pathways to glycolysis needed to maintain adenine nucleotide and redox balances within glycosomes such as phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate reductase, pyruvate phosphate dikinase and glycerol-3-phosphate dehydrogenase. Glucokinase, galactokinase and the first enzyme of the pentose-phosphate pathway, glucose-6-phosphate dehydrogenase, were also located inside glycosomes. Furthermore, we demonstrate that T. rangeli epimastigotes growing in LIT medium only consume glucose and do not excrete ammonium; moreover, they are unable to survive in partially-depleted glucose medium. The

  1. 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.

  2. Mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation links the tricarboxylic acid (TCA) cycle with methionine metabolism and nuclear DNA methylation.

    Science.gov (United States)

    Lozoya, Oswaldo A; Martinez-Reyes, Inmaculada; Wang, Tianyuan; Grenet, Dagoberto; Bushel, Pierre; Li, Jianying; Chandel, Navdeep; Woychik, Richard P; Santos, Janine H

    2018-04-18

    Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechanistic link between mitochondrial function and epigenetic regulation of gene expression that involves polyamine and methionine metabolism responding to changes in the tricarboxylic acid (TCA) cycle. Given the implications of these findings, future studies across different physiological contexts and in vivo are warranted.

  3. Rhodium-Coordinated Poly(arylene-ethynylene)-alt-Poly(arylene-vinylene) Copolymer Acting as Photocatalyst for Visible-Light-Powered NAD+/NADH Reduction

    Science.gov (United States)

    2014-01-01

    A 2,2′-bipyridyl-containing poly(arylene-ethynylene)-alt-poly(arylene-vinylene) polymer, acting as a light-harvesting ligand system, was synthesized and coupled to an organometallic rhodium complex designed for photocatalytic NAD+/NADH reduction. The material, which absorbs over a wide spectral range, was characterized by using various analytical techniques, confirming its chemical structure and properties. The dielectric function of the material was determined from spectroscopic ellipsometry measurements. Photocatalytic reduction of nucleotide redox cofactors under visible light irradiation (390–650 nm) was performed and is discussed in detail. The new metal-containing polymer can be used to cover large surface areas (e.g. glass beads) and, due to this immobilization step, can be easily separated from the reaction solution after photolysis. Because of its high stability, the polymer-based catalyst system can be repeatedly used under different reaction conditions for (photo)chemical reduction of NAD+. With this concept, enzymatic, photo-biocatalytic systems for solar energy conversion can be facilitated, and the precious metal catalyst can be recycled. PMID:25130570

  4. Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

    Energy Technology Data Exchange (ETDEWEB)

    Sheard, Michael A., E-mail: msheard@chla.usc.edu [Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Ghent, Matthew V., E-mail: mattghent@gmail.com [Department of Pathology, Keck School of Medicine, University of Southern California, Health Sciences Campus, Los Angeles, CA 90089 (United States); Cabral, Daniel J., E-mail: dcabral14@gmail.com [Cancer Center and Departments of Cell Biology & Biochemistry, Pharmacology & Neuroscience, Internal Medicine and Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (United States); Lee, Joanne C., E-mail: joannebarnhart@gmail.com [Cancer Center and Departments of Cell Biology & Biochemistry, Pharmacology & Neuroscience, Internal Medicine and Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (United States); Khankaldyyan, Vazgen, E-mail: khangaldian@yahoo.com [Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Ji, Lingyun, E-mail: lingyun.ji@med.usc.edu [Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Wu, Samuel Q., E-mail: swu@chla.usc.edu [Medical Genetics, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Kang, Min H., E-mail: min.kang@ttuhsc.edu [Cancer Center and Departments of Cell Biology & Biochemistry, Pharmacology & Neuroscience, Internal Medicine and Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (United States); and others

    2015-05-15

    Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo. - Highlights: • Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function. • Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death. • The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent.

  5. Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

    International Nuclear Information System (INIS)

    Sheard, Michael A.; Ghent, Matthew V.; Cabral, Daniel J.; Lee, Joanne C.; Khankaldyyan, Vazgen; Ji, Lingyun; Wu, Samuel Q.; Kang, Min H.

    2015-01-01

    Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo. - Highlights: • Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function. • Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death. • The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent

  6. Testicular Metabolic Reprogramming in Neonatal Streptozotocin-Induced Type 2 Diabetic Rats Impairs Glycolytic Flux and Promotes Glycogen Synthesis

    Science.gov (United States)

    Rato, L.; Alves, M. G.; Dias, T. R.; Cavaco, J. E.; Oliveira, Pedro F.

    2015-01-01

    Defects in testicular metabolism are directly implicated with male infertility, but most of the mechanisms associated with type 2 diabetes- (T2DM) induced male infertility remain unknown. We aimed to evaluate the effects of T2DM on testicular glucose metabolism by using a neonatal-streptozotocin- (n-STZ) T2DM animal model. Plasma and testicular hormonal levels were evaluated using specific kits. mRNA and protein expression levels were assessed by real-time PCR and Western Blot, respectively. Testicular metabolic profile was assessed by 1H-NMR spectroscopy. T2DM rats showed increased glycemic levels, impaired glucose tolerance and hyperinsulinemia. Both testicular and serum testosterone levels were decreased, whereas those of 17β-estradiol were not altered. Testicular glycolytic flux was not favored in testicles of T2DM rats, since, despite the increased expression of both glucose transporters 1 and 3 and the enzyme phosphofructokinase 1, lactate dehydrogenase activity was severely decreased contributing to lower testicular lactate content. However, T2DM enhanced testicular glycogen accumulation, by modulating the availability of the precursors for its synthesis. T2DM also affected the reproductive sperm parameters. Taken together these results indicate that T2DM is able to reprogram testicular metabolism by enhancing alternative metabolic pathways, particularly glycogen synthesis, and such alterations are associated with impaired sperm parameters. PMID:26064993

  7. Improvement of H2 yield of Fermentative Bacteria by Gene Manipulation

    International Nuclear Information System (INIS)

    Makiko Harada; Takashi Kaneko; Shigeharu Tanisho

    2006-01-01

    Two method were proposed in this paper. One is to destroy the NADH dehydrogenase complex in the electron transport chain of a facultative anaerobic bacterium Enterobacter aerogenes and the other is to disrupt the butyrate producing pathway of a strict anaerobic bacterium Clostridium butyricum. In case of E. aerogenes, one of the 14 membrane-bound NADH dehydrogenases, nuoG, is targeted to destroy. In case of C. butyricum, function of thiolase is targeted to disrupt. (authors)

  8. Improvement of H2 yield of Fermentative Bacteria by Gene Manipulation

    Energy Technology Data Exchange (ETDEWEB)

    Makiko Harada; Takashi Kaneko; Shigeharu Tanisho [Department of Environmental and Information Sciences, Yokohama National University Hodogaya-Ku, Yokohama 240-8501, (Japan)

    2006-07-01

    Two method were proposed in this paper. One is to destroy the NADH dehydrogenase complex in the electron transport chain of a facultative anaerobic bacterium Enterobacter aerogenes and the other is to disrupt the butyrate producing pathway of a strict anaerobic bacterium Clostridium butyricum. In case of E. aerogenes, one of the 14 membrane-bound NADH dehydrogenases, nuoG, is targeted to destroy. In case of C. butyricum, function of thiolase is targeted to disrupt. (authors)

  9. NADH Oxidase of Streptococcus thermophilus 1131 is Required for the Effective Yogurt Fermentation with Lactobacillus delbrueckii subsp. bulgaricus 2038.

    Science.gov (United States)

    Sasaki, Yasuko; Horiuchi, Hiroshi; Kawashima, Hiroko; Mukai, Takao; Yamamoto, Yuji

    2014-01-01

    We previously reported that dissolved oxygen (DO) suppresses yogurt fermentation with an industrial starter culture composed of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) 2038 and Streptococcus thermophilus 1131, and also found that reducing the DO in the medium prior to fermentation (deoxygenated fermentation) shortens the fermentation time. In this study, we found that deoxygenated fermentation primarily increased the cell number of S. thermophilus 1131 rather than that of L. bulgaricus 2038, resulting in earlier l-lactate and formate accumulation. Measurement of the DO concentration and hydrogen peroxide generation in the milk medium suggested that DO is mainly removed by S. thermophilus 1131. The results using an H2O-forming NADH oxidase (Nox)-defective mutant of S. thermophilus 1131 revealed that Nox is the major oxygen-consuming enzyme of the bacterium. Yogurt fermentation with the S. thermophilus Δnox mutant and L. bulgaricus 2038 was significantly slower than with S. thermophilus 1131 and L. bulgaricus 2038, and the DO concentrations of the mixed culture did not decrease to less than 2 mg/kg within 3 hr. These observations suggest that Nox of S. thermophilus 1131 contributes greatly to yogurt fermentation, presumably by removing the DO in milk.

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

    Directory of Open Access Journals (Sweden)

    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

  11. Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

    Science.gov (United States)

    Gizak, Agnieszka; Grenda, Marcin; Mamczur, Piotr; Wisniewski, Janusz; Sucharski, Filip; Silberring, Jerzy; McCubrey, James A; Wisniewski, Jacek R; Rakus, Dariusz

    2015-07-10

    Phosphoglycerate mutase (PGAM), a conserved, glycolytic enzyme has been found in nucleoli of cancer cells. Here, we present evidence that accumulation of PGAM in the nucleolus is a universal phenomenon concerning not only neoplastically transformed but also non-malignant cells. Nucleolar localization of the enzyme is dependent on the presence of the PGAM2 (muscle) subunit and is regulated by insulin/IGF-1-PI3K signaling pathway as well as drugs influencing ribosomal biogenesis. We document that PGAM interacts with several 40S and 60S ribosomal proteins and that silencing of PGAM2 expression results in disturbance of nucleolar structure, inhibition of RNA synthesis and decrease of the mitotic index of squamous cell carcinoma cells. We conclude that presence of PGAM in the nucleolus is a prerequisite for synthesis and initial assembly of new pre-ribosome subunits.

  12. Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Lee, Sung-Haeng; Kodaki, Tsutomu; Park, Yong-Cheol; Seo, Jin-Ho

    2012-04-30

    Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Excess glycogen does not resolve high ultimate pH of oxidative muscle.

    Science.gov (United States)

    England, Eric M; Matarneh, Sulaiman K; Oliver, Emily M; Apaoblaza, Ariel; Scheffler, Tracy L; Shi, Hao; Gerrard, David E

    2016-04-01

    Skeletal muscle glycogen content can impact the extent of postmortem pH decline. Compared to glycolytic muscles, oxidative muscles contain lower glycogen levels antemortem which may contribute to the higher ultimate pH. In an effort to explore further the participation of glycogen in postmortem metabolism, we postulated that increasing the availability of glycogen would drive additional pH decline in oxidative muscles to equivalent pH values similar to the ultimate pH of glycolytic muscles. Glycolysis and pH declines were compared in porcine longissimus lumborum (glycolytic) and masseter (oxidative) muscles using an in vitro system in the presence of excess glycogen. The ultimate pH of the system containing longissimus lumborum reached a value similar to that observed in intact muscle. The pH decline of the system containing masseter samples stopped prematurely resulting in a higher ultimate pH which was similar to that of intact masseter muscle. To investigate further, we titrated powdered longissimus lumborum and masseter samples in the reaction buffer. As the percentage of glycolytic sample increased, the ultimate pH decreased. These data show that oxidative muscle produces meat with a high ultimate pH regardless of glycogen content and suggest that inherent muscle factors associated with glycolytic muscle control the extent of pH decline in pig muscles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Warburg effect increases steady-state ROS condition in cancer cells through decreasing their antioxidant capacities (anticancer effects of 3-bromopyruvate through antagonizing Warburg effect).

    Science.gov (United States)

    El Sayed, Salah Mohamed; Mahmoud, Ahmed Alamir; El Sawy, Samer Ahmed; Abdelaal, Esam Abdelrahim; Fouad, Amira Murad; Yousif, Reda Salah; Hashim, Marwa Shaban; Hemdan, Shima Badawy; Kadry, Zainab Mahmoud; Abdelmoaty, Mohamed Ahmed; Gabr, Adel Gomaa; Omran, Faten M; Nabo, Manal Mohamed Helmy; Ahmed, Nagwa Sayed

    2013-11-01

    Cancer cells undergo an increased steady-state ROS condition compared to normal cells. Among the major metabolic differences between cancer cells and normal cells is the dependence of cancer cells on glycolysis as a major source of energy even in the presence of oxygen (Warburg effect). In Warburg effect, glucose is catabolized to lactate that is extruded through monocarboxylate transporters to the microenvironment of cancer cells, while in normal cells, glucose is metabolized into pyruvate that is not extruded. Pyruvate is a potent antioxidant, while lactate has no antioxidant effect. Pyruvate in normal cells may be further metabolized to acetyl CoA and then through Krebs cycle with production of antioxidant intermediates e.g. citrate, malate and oxaloacetate together with the reducing equivalents (NADH.H+). Through activity of mitochondrial transhydrogenase, NADH.H+ replenishes NADPH.H+, coenzyme of glutathione reductase which replenishes reduced form of glutathione (potent antioxidant). This enhances antioxidant capacities of normal cells, while cancer cells exhibiting Warburg effect may be deprived of all that antioxidant capabilities due to loss of extruded lactate (substrate for Krebs cycle). Although intrinsic oxidative stress in cancer cells is high, it may be prevented from reaching progressively increasing levels that are cytotoxic to cancer cells. This may be due to some antioxidant effects exerted by hexokinase II (HK II) and NADPH.H+ produced through HMP shunt. Glycolytic phenotype in cancer cells maintains a high non-toxic oxidative stress in cancer cells and may be responsible for their malignant behavior. Through HK II, glycolysis fuels the energetic arm of malignancy, the mitotic arm of malignancy (DNA synthesis through HMP shunt pathway) and the metastatic arm of malignancy (hyaluronan synthesis through uronic acid pathway) in addition to the role of phosphohexose isomerase (autocrine motility factor). All those critical three arms start with the

  15. Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

    DEFF Research Database (Denmark)

    Olavarria, K.; De Ingeniis, J.; Zielinski, D. C.

    2014-01-01

    In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Δpgi). In this work......PDH(R46E,Q47E). Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Δpgi E. coli strains were replaced by DNA encoding LmG6PDH(R46E,Q47E). Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect...

  16. Deep sequencing of the mitochondrial genome reveals common heteroplasmic sites in NADH dehydrogenase genes.

    Science.gov (United States)

    Liu, Chunyu; Fetterman, Jessica L; Liu, Poching; Luo, Yan; Larson, Martin G; Vasan, Ramachandran S; Zhu, Jun; Levy, Daniel

    2018-03-01

    Increasing evidence implicates mitochondrial dysfunction in aging and age-related conditions. But little is known about the molecular basis for this connection. A possible cause may be mutations in the mitochondrial DNA (mtDNA), which are often heteroplasmic-the joint presence of different alleles at a single locus in the same individual. However, the involvement of mtDNA heteroplasmy in aging and age-related conditions has not been investigated thoroughly. We deep-sequenced the complete mtDNA genomes of 356 Framingham Heart Study participants (52% women, mean age 43, mean coverage 4570-fold), identified 2880 unique mutations and comprehensively annotated them by MITOMAP and PolyPhen-2. We discovered 11 heteroplasmic "hot" spots [NADH dehydrogenase (ND) subunit 1, 4, 5 and 6 genes, n = 7; cytochrome c oxidase I (COI), n = 2; 16S rRNA, n = 1; D-loop, n = 1] for which the alternative-to-reference allele ratios significantly increased with advancing age (Bonferroni correction p < 0.001). Four of these heteroplasmic mutations in ND and COI genes were predicted to be deleterious nonsynonymous mutations which may have direct impact on ATP production. We confirmed previous findings that healthy individuals carry many low-frequency heteroplasmy mutations with potentially deleterious effects. We hypothesize that the effect of a single deleterious heteroplasmy may be minimal due to a low mutant-to-wildtype allele ratio, whereas the aggregate effects of many deleterious mutations may cause changes in mitochondrial function and contribute to age-related diseases. The identification of age-related mtDNA mutations is an important step to understand the genetic architecture of age-related diseases and may uncover novel therapeutic targets for such diseases.

  17. Natural spectroscopic hydrogen isotope transfer in alcohol dehydrogenase-catalysed reduction

    International Nuclear Information System (INIS)

    Ben-Li Zhang; Pionnier, S.

    2002-01-01

    The enantiomeric purity of natural α-mono deuterated enantiomers, (R) and (S)ethanol-1-d 1 , in the alcohol produced by sugar fermentation with yeast was studied by 2 H NMR using their esters derived from optical mandelic acid. The results of isotope tracing experiments show that the transfer pathways of the two eantiotopic hydrogens of the methylene group are different. It was observed that (S)-deuterium comes only from the medium water. The (R)-deuterium transferred by NADH in alcohol dehydrogenase reduction of the acetaldehyde is complex origin. Some of them originates from carbon bound hydrogen of the sugar, especially from C(4) position of glucose and most of them comes from water. Only a small portion of the NADH deuterium is incorporated indirectly from water through enzyme catalysed exchange between the pro-S site of NADH and flavin. When a carbonyl compound (ethyl acetoacetate) was reduced under the same conditions during the alcoholic fermentation, among the NADH-transferred deuterium, only a small portion comes from water while most comes from the unexchangeable positions of the glucose. (author)

  18. Metabolic engineering of Escherichia coli for ethanol production without foreign genes

    Science.gov (United States)

    Kim, Youngnyun

    Worldwide dependence on finite petroleum-based energy necessitates alternative energy sources that can be produced from renewable resources. A successful example of an alternative transportation fuel is bioethanol, produced by microorganisms, from corn starch that is blended with gasoline. However, corn, currently the main feedstock for bioethanol production, also occupies a significant role in human food and animal feed chains. As more corn is diverted to bioethanol, the cost of corn is expected to increase with an increase in the price of food, feed and ethanol. Using lignocellulosic biomass for ethanol production is considered to resolve this problem. However, this requires a microbial biocatalyst that can ferment hexoses and pentoses to ethanol. Escherichia coli is an efficient biocatalyst that can use all the monomeric sugars in lignocellulose, and recombinant derivatives of E. coli have been engineered to produce ethanol as the major fermentation product. In my study, ethanologenic E. coli strains were isolated from a ldhA-, pflB- derivative without introduction of foreign genes. These isolates grew anaerobically and produced ethanol as the main fermentation product. The mutation responsible for anaerobic growth and ethanol production was mapped in the lpdA gene and the mutation was identified as E354K in three of the isolates tested. Another three isolates carried an lpdA mutation, H352Y. Enzyme kinetic studies revealed that the mutated form of the dihydrolipoamide dehydrogenase (LPD) encoded by the lpdA was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity to NADH of the pyruvate dehydrogenase complex in strain SE2378. The net yield of 4 moles of NADH and 2 moles of acetyl-CoA per mole of glucose produced by a combination of glycolysis and PDH provided a logical basis to explain the production of 2 moles of ethanol per glucose. The development of E

  19. Diagnosis and epidemiology of red blood cell enzyme disorders

    Directory of Open Access Journals (Sweden)

    Richard Van Wijk

    2013-03-01

    Full Text Available The red blood cell possess an active metabolic machinery that provides the cell with energy to pump ions against electrochemical gradients, to maintain its shape, to keep hemoglobin iron in the reduced (ferrous form, and to maintain enzyme and hemoglobin sulfhydryl groups. The main source of metabolic energy comes from glucose. Glucose is metabolized through the glycolytic pathway and through the hexose monophosphate shunt. Glycolysis catabolizes glucose to pyruvate and lactate, which represent the end products of glucose metabolism in the erythrocyte. Adenosine diphosphate (ADP is phosphorylated to adenosine triphosphate (ATP, and nicotinamide adenine dinucleotide (NAD+ is reduced to NADH in glycolysis. 2,3- Bisphosphoglycerate, an important regulator of the oxygen affinity of hemoglobin, is generated during glycolysis by the Rapoport-Luebering shunt. The hexose monophosphate shunt oxidizes glucose-6-phosphate, reducing NADP+ to reduced nicotinamide adenine dinucleotide phosphate (NADPH. The red cell lacks the capacity for de novo purine synthesis but has a salvage pathway that permits synthesis of purine nucleotides from purine bases...

  20. Oxygen reduction activity of carbon fibers cathode, and application for current generation from the NAD+ and NADH recycling reaction

    Directory of Open Access Journals (Sweden)

    H. Maeda

    2012-03-01

    Full Text Available Carbon fibers treated at 700 oC for 10 min were found to have O2 reduction activity when being used as a cathode. The special type of partition combined with both cationic and anionic exchange membranes was applied between anode cell and cathode cell in order to use a highly acidic solution such as 0.5 M H2SO4 as an electrolyte of the cathode cell for increasing the efficiency of O2 reduction activity. The current generation from NAD+ and NADH recycling system combined with D-gluconolactone production from 500 mg of D-glucose was performed by applying only carbon fibers for both anode and cathode. The total current volume obtained was 81.4 mAh during the reaction for 10 h, and the current efficiency was 93%. One gram of carbon fibers was pressed with Nafion paste on a piece of carbon paper(area : 50 mm×50mm with heating to prepare the cathode, and this construct was combined with conventional fuel cell. The power density was 3.6 mW/cm2, and the total power volume was calculated to be 90 mW per 1 g of carbon fibers.

  1. The potentiation of radiation effects on the Meth-a and RIF tumors and hela s-3 cells by the glycolytic inhibitor, Lonidamine

    International Nuclear Information System (INIS)

    Kim, J.H.; Alfieri, A.A.; Kim, S.H.; He, S.Q.; Young, C.W.

    1984-01-01

    The indazole carboxylic acid Lonidamine (L) a glycolytic inhibitor and antispermatogenic agent previously found to have hyperthermia sensitization properties was evaluated as a radiopotentiating agent in vitro and in vivo. Tumoricidal effects of (L) i.p. and single dose x-irradiation (3.0 or 3.2 Gy) was evaluated on 300-1000 mm/sup 3/ i.m. Meth-A in BALB/c and the RIF fibrosarcoma in C/sub 3/H/He mice L preceded or proceeded the radiation event to 1 hr intervals with maximum separation between treatments of 4 hrs. L conc in sera was biphasic (t1/2 -- 1 hr.). Optimal treatment efficacy was found at minimal time intervals between L and radiation interaction. TCD /sub 50/ for RIF and Meth-A resulted in DMF's of 1.15 - 1.35 (L dose dependent). Chronic L (50mg/kg/dy x 30) + fractionated radiation was found to be well tolerated as concomitant L potentiated combination therapy. Cell culture studies on monolayer and spheroids demonstrate L as an effective inhibitor of potential lethal radiation damage with L potential to be dependent upon both dose/fraction, number of fractions and total radiation dose accumulated

  2. 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.

  3. SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

    International Nuclear Information System (INIS)

    Campos, D; Peeters, W; Nickel, K; Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M

    2015-01-01

    Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response

  4. SU-C-303-02: Correlating Metabolic Response to Radiation Therapy with HIF-1alpha Expression

    Energy Technology Data Exchange (ETDEWEB)

    Campos, D [University of Wisconsin Madison, Madison, WI (United States); Peeters, W [Radboud University Medical Center, Nijmegen, GA (United States); Nickel, K [University of Wisconsin, Madison, WI (United States); Eliceiri, K; Kimple, R; Van Der Kogel, A; Kissick, M [University of Wisconsin, Madison, Wisconsin (United States)

    2015-06-15

    Purpose: To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient-specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM-SCC-22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF-1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Measurements of metabolism and HIF-1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x-ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF-1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package. Results: FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF-1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles. Conclusion: Radiation induces significant changes in metabolic activity and HIF-1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

  5. WE-E-BRE-12: Tumor Microenvironment Dynamics Following Radiation

    International Nuclear Information System (INIS)

    Campos, D; Niles, D; Adamson, E; Torres, A; Kissick, M; Eliceiri, K; Kimple, R

    2014-01-01

    Purpose: This work aims to understand the radiation-induced interplay between tumor oxygenation and metabolic activity. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient-specific adaptive radiotherapy. Methods: Using patient-derived xenografts of head and neck cancer we assessed tumor oxygenation via fiber-optic probe monitored hemoglobin saturation and Blood Oxygen Level Dependent (BOLD) MRI. Measurements were taken before and after a 10 Gy dose of radiation. Changes in metabolic activity were measured via Fluorescence Lifetime IMaging (FLIM) with the appropriate controls following a 10 Gy dose of radiation. FLIM can non-invasively monitor changes in fluorescence in response to the microenvironment including being able to detect free and bound states of the intrinsically fluorescent metabolite NADH (Nicotinamide Adenine Dinucleotide). With this information FLIM can accurately quantify the metabolic state of cells that have been radiated. To model the observed changes, a two-compartment, source-sink simulation relating hemoglobin saturation and metabolic activity was performed using MATLAB. Results: Hemoglobin saturation as measured by interstitial probe and BOLD-MRI decreased by 30% within 15 minutes following radiation. FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways. Simulation of radiation-induced alterations in tumor oxygenation demonstrated that these changes can be the result of changes in either vasculature or metabolic activity. Conclusion: Radiation induces significant changes in hemoglobin saturation and metabolic activity. These alterations occur on time scales approximately the duration of common radiation treatments. Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response

  6. Primary Metabolic Pathways and Metabolic Flux Analysis

    DEFF Research Database (Denmark)

    Villadsen, John

    2015-01-01

    his chapter introduces the metabolic flux analysis (MFA) or stoichiometry-based MFA, and describes the quantitative basis for MFA. It discusses the catabolic pathways in which free energy is produced to drive the cell-building anabolic pathways. An overview of these primary pathways provides...... the reader who is primarily trained in the engineering sciences with atleast a preliminary introduction to biochemistry and also shows how carbon is drained off the catabolic pathways to provide precursors for cell mass building and sometimes for important industrial products. The primary pathways...... to be examined in the following are: glycolysis, primarily by the EMP pathway, but other glycolytic pathways is also mentioned; fermentative pathways in which the redox generated in the glycolytic reactions are consumed; reactions in the tricarboxylic acid (TCA) cycle, which produce biomass precursors and redox...

  7. NADH:ubiquinone reductase and succinate dehydrogenase activity in the liver of rats with acetaminophen-induced toxic hepatitis on the background of alimentary protein deficiency

    Directory of Open Access Journals (Sweden)

    G. P. Kopylchuk

    2015-02-01

    Full Text Available The ratio between the redox forms of the nicotinamide coenzymes and key enzymatic activity of the I and II respiratory chain complexes in the liver cells mitochondria of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. It was estimated, that under the conditions of acute acetaminophen-induced hepatitis of rats kept on a low-protein diet during 4 weeks a significant decrease of the NADH:ubiquinone reductase and succinate dehydrogenase activity with simultaneous increase of the ratio between redox forms of the nicotinamide coenzymes (NAD+/NADН is observed compared to the same indices in the liver cells of animals with experimental hepatitis kept on the ration balanced by all nutrients. Results of research may become basic ones for the biochemical rationale for the approaches directed to the correction and elimination of the consequences­ of energy exchange in the toxic hepatitis, induced on the background of protein deficiency.

  8. GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts.

    Science.gov (United States)

    Bernier, Michel; Catazaro, Jonathan; Singh, Nagendra S; Wnorowski, Artur; Boguszewska-Czubara, Anna; Jozwiak, Krzysztof; Powers, Robert; Wainer, Irving W

    2017-11-15

    The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to l-lactate and is associated with upregulated expression of HIF-1α and activation of the EGFR-MEK-ERK, Wnt-β-catenin, and PI3K-AKT signaling pathways. (R,R')-4'-methoxy-1-naphthylfenoterol ((R,R')-MNF) significantly reduces proliferation, survival, and motility of PANC-1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R')-MNF's effect on glycolysis in PANC-1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R')-MNF-treated cells. LC/MS analysis was used to quantify intracellular concentrations of β-hydroxybutyrate, carnitine, and l-lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC-1 tumor xenografts after administration of (R,R')-MNF. Metabolomics data indicate that (R,R')-MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of β-hydroxybutyrate and carnitine while reducing l-lactate content. The cellular content of phosphoinositide-dependent kinase-1 and hexokinase 2 was reduced consistent with diminished PI3K-AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R')-MNF. Mice treated with (R,R')-MNF had significant accumulation of l-lactate in tumor tissue relative to vehicle-treated mice, together with reduced levels of the selective l-lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, β-catenin, hexokinase 2, and p-glycoprotein were also observed. The data suggest that (R,R')-MNF reduces glycolysis in PANC-1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway. © 2017 UICC.

  9. Oxidative protein modification as predigestive mechanism of the carnivorous plant Dionaea muscipula: an hypothesis based on in vitro experiments.

    Science.gov (United States)

    Galek, H; Osswald, W F; Elstner, E F

    1990-01-01

    Aqueous leaf extracts from Dionaea muscipula contain quinones such as the naphthoquinone plumbagin that couple to different NADH-dependent diaphorases, producing superoxide and hydrogen peroxide upon autoxidation. Upon preincubation of Dionaea extracts with certain diaphorases and NADH in the presence of serumalbumin (SA), subsequent tryptic digestion of SA is facilitated. Since the secretroy glands of Droseracea contain proteases and possibly other degradative enzymes it is suggested that the presence of oxygen-activating redox cofactors in the extracts function as extracellular predigestive oxidants which render membrane-bound proteins of the prey (insects) more susceptible to proteolytic attacks.

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

    Science.gov (United States)

    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.

  11. Electroactive Properties of 1-propyl-3-methylimidazolium Ionic Liquid Covalently Bonded on Mesoporous Silica Surface: Development of an Electrochemical Sensor Probed for NADH, Dopamine and Uric Acid Detection

    International Nuclear Information System (INIS)

    Maroneze, Camila M.; Rahim, Abdur; Fattori, Natália; Costa, Luiz P. da; Sigoli, Fernando A.; Mazali, Italo O.; Custodio, Rogério; Gushikem, Yoshitaka

    2014-01-01

    Graphical abstract: - Abstract: A hybrid organic-inorganic porous material was successfully prepared through chemical modification of a non-ordered mesoporous silica, obtained by the sol-gel process, with 1-propyl-3-methylimidazolium groups. The porous material was evaluated as a platform for the development of electrochemical sensors, here probed toward the electrooxidation of NADH (β-nicotinamide adenine dinucleotide), uric acid (UA) and dopamine (DA). The presence of cationic imidazolium groups on the surface of the hybrid silica-based material allowed the electrochemical detection of these biomolecules without any other electron mediator or biomolecular recognition component. Such behavior highlights the potentiality of this material to be applied in the development of new electrochemical sensing devices. Theoretical calculations based on density functional theory emphasizes that the cationic character of imidazolium group provides better oxidation conditions if the solvent effect is minimized

  12. The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Keith S Wong

    Full Text Available MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In Escherichia coli, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37 °C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of ravA and viaA indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I. Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex.

  13. LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

    2015-05-08

    Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

  14. NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice.

    Directory of Open Access Journals (Sweden)

    Lena Muchnik

    Full Text Available The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX, which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.

  15. Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum*

    Science.gov (United States)

    Moxley, Michael A.; Beard, Daniel A.; Bazil, Jason N.

    2016-01-01

    Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD+/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD+ activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat. PMID:26644471

  16. WE-FG-BRA-08: Potential Role of the Glycolytic Oscillator in Acute Hypoxia in Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Che Fru, L [University of Wisconsin, Madison, Madison, WI (United States); Adamson, E; Campos, D; Song, C; Kimple, R [University of Wisconsin Madison, Madison, WI (United States); Fain, S; Kissick, M [University of Wisconsin, Madison, WI (United States); Jacques, S [Oregon Health and Science University, Portland OR USA, Portland, OR (United States); Kogel, A van der [University of Wisconsin - Madison, Madison, WI (United States); Nickel, K [University of Wisconsin Madison, Madison, Wisconsin (United States)

    2016-06-15

    Purpose: Oscillatory dynamics in acute hypoxia have been observed, but poorly understood. They have mostly been attributed to vascular perturbations, but no link has yet been made to metabolic causes. We set out to determine the fundamental frequencies and test for coherence in tumor oxygen dynamics and spatial properties. Methods: Severe combined immunodeficient (SCID) mice were inoculated onto bilateral flanks with human derived head and neck carcinoma (UW-SCC22) cell line xenografts. Oxygen dynamics were monitored in the tumor every minute for an hour using three modalities: blood oxygen level dependent - magnetic resonance imaging (BOLD-MRI), hemoglobin oxygen saturation photoacoustic, and locally manufactured optical probes for spectral fitting. A statistical test was used to separate fluctuating from non-fluctuating voxels and pixels in BOLD-MRI and photoacoustic data respectively. The power spectrum density (PSD) and the autocorrelation functions were calculated for the time series of each voxel, pixel and region, of the BOLD-MRI, photoacoustic or fiber optic data respectively. Results: Using all three techniques, intermittent oxygen dynamics with both coherent and incoherent signatures was observed in the tumors. Upon averaging the PSDs of fluctuating voxels and pixels, it was found that these oscillations occurred with periods of minutes to tens of minutes from all three approaches. Observations from the BOLD-MRI and photoacoustic data showed that clusters of voxels oscillated in a synchronized manner. Conclusion: We were able to use three different modalities to show that fluctuation in tumor oxygen is both coherent and incoherent, with periods of minutes to tens of minutes. These periods are very similar to those from the well-established metabolic, non-linear biomechanical phenomenon called the glycolytic oscillator. This may provide an additional explanation to the cause of cyclic hypoxia. Such dynamics could have profound implications in

  17. WE-FG-BRA-08: Potential Role of the Glycolytic Oscillator in Acute Hypoxia in Tumors

    International Nuclear Information System (INIS)

    Che Fru, L; Adamson, E; Campos, D; Song, C; Kimple, R; Fain, S; Kissick, M; Jacques, S; Kogel, A van der; Nickel, K

    2016-01-01

    Purpose: Oscillatory dynamics in acute hypoxia have been observed, but poorly understood. They have mostly been attributed to vascular perturbations, but no link has yet been made to metabolic causes. We set out to determine the fundamental frequencies and test for coherence in tumor oxygen dynamics and spatial properties. Methods: Severe combined immunodeficient (SCID) mice were inoculated onto bilateral flanks with human derived head and neck carcinoma (UW-SCC22) cell line xenografts. Oxygen dynamics were monitored in the tumor every minute for an hour using three modalities: blood oxygen level dependent - magnetic resonance imaging (BOLD-MRI), hemoglobin oxygen saturation photoacoustic, and locally manufactured optical probes for spectral fitting. A statistical test was used to separate fluctuating from non-fluctuating voxels and pixels in BOLD-MRI and photoacoustic data respectively. The power spectrum density (PSD) and the autocorrelation functions were calculated for the time series of each voxel, pixel and region, of the BOLD-MRI, photoacoustic or fiber optic data respectively. Results: Using all three techniques, intermittent oxygen dynamics with both coherent and incoherent signatures was observed in the tumors. Upon averaging the PSDs of fluctuating voxels and pixels, it was found that these oscillations occurred with periods of minutes to tens of minutes from all three approaches. Observations from the BOLD-MRI and photoacoustic data showed that clusters of voxels oscillated in a synchronized manner. Conclusion: We were able to use three different modalities to show that fluctuation in tumor oxygen is both coherent and incoherent, with periods of minutes to tens of minutes. These periods are very similar to those from the well-established metabolic, non-linear biomechanical phenomenon called the glycolytic oscillator. This may provide an additional explanation to the cause of cyclic hypoxia. Such dynamics could have profound implications in

  18. NCBI nr-aa BLAST: CBRC-TTRU-01-1020 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1020 ref|YP_271950.1| NADH dehydrogenase subunit 5 [Montastraea faveol...ata] ref|YP_271937.1| NADH dehydrogenase subunit 5 [Montastraea franksi] ref|YP_271924.1| NADH dehydrogenase subunit 5 [Montastrae...a annularis] dbj|BAE16177.1| NADH dehydrogenase subunit 5 [Montastraea annularis] dbj|...BAE16190.1| NADH dehydrogenase subunit 5 [Montastraea annularis] dbj|BAE16203.1| ...NADH dehydrogenase subunit 5 [Montastraea franksi] dbj|BAE16216.1| NADH dehydrogenase subunit 5 [Montastraea

  19. NCBI nr-aa BLAST: CBRC-ETEL-01-0265 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-0265 ref|YP_271953.1| NADH dehydrogenase subunit 2 [Montastraea faveol...ata] ref|YP_271940.1| NADH dehydrogenase subunit 2 [Montastraea franksi] ref|YP_271927.1| NADH dehydrogenase subunit 2 [Montastrae...a annularis] dbj|BAE16180.1| NADH dehydrogenase subunit 2 [Montastraea annularis] dbj|...BAE16193.1| NADH dehydrogenase subunit 2 [Montastraea annularis] dbj|BAE16206.1| ...NADH dehydrogenase subunit 2 [Montastraea franksi] dbj|BAE16219.1| NADH dehydrogenase subunit 2 [Montastraea

  20. NCBI nr-aa BLAST: CBRC-DNOV-01-0366 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0366 ref|YP_271950.1| NADH dehydrogenase subunit 5 [Montastraea faveol...ata] ref|YP_271937.1| NADH dehydrogenase subunit 5 [Montastraea franksi] ref|YP_271924.1| NADH dehydrogenase subunit 5 [Montastrae...a annularis] dbj|BAE16177.1| NADH dehydrogenase subunit 5 [Montastraea annularis] dbj|...BAE16190.1| NADH dehydrogenase subunit 5 [Montastraea annularis] dbj|BAE16203.1| ...NADH dehydrogenase subunit 5 [Montastraea franksi] dbj|BAE16216.1| NADH dehydrogenase subunit 5 [Montastraea

  1. Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis

    DEFF Research Database (Denmark)

    Liu, Jianming; Wang, Zhihao; Kandasamy, Vijayalakshmi

    2017-01-01

    on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)−2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD......+, and a high titer of 371 mM (32 g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin...... is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361 mM (32 g/L) R-BDO with a yield...

  2. 24-h organization of glycolysis and control by photoperiodism.

    Science.gov (United States)

    Pierre, J N; Celati, C; Queiroz, O

    1985-01-01

    A study of the temporal organization of glycolysis at diverse levels of activity of the pathway showed that consideration of the phases of the 24h oscillations in glycolytic pools affords a means of detecting modifications in regulatory mechanisms according to the level of carbon flow along the pathway. The work utilized Kalanchoe blossfeldiana, a plant with crassulacean acid metabolism (CAM) in which glycolytic activity is under the control of photoperiodism: after transfer from long days to short days carbon flow through the pathway increases drastically. Analysis of the glycolytic pools performed during the day/night cycle showed that: a. 24 h-period variations exist in the content of the glycolytic intermediates; b. time of the acrophase of these rhythms changes as a function of the photoperiodic treatment: in long days the pools of the intermediates preceding the phosphofructokinase (PFK) step oscillate in phase and the same holds for the intermediates after the PFK step but these two sequences of the pathway oscillate out of phase (phase-jump of about 10h); transfer to short days besides producing (after a lag) changes in the mean level and amplitude of the oscillations, modifies their phase: this temporal reorganization of glycolysis results in splitting the pathway into 3 sequences of synchronously-oscillating pools, phase-jumps between successive sequences occurring at the PFK (4h) and at the 3-phosphoglyceraldehyde dehydrogenase (12h) steps.

  3. Dicty_cDB: SHE462 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4 Homology vs Protein Score E Sequences producing significant alignments: (bits) Value AY125003_1( AY125003 |pid:none) Calathe...a foliosa NADH dehydrogenas... 32 7.2 AY656084_1( AY656084 |pid:none) Calathea metallica v

  4. Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

    NARCIS (Netherlands)

    Medina, V.G.; Almering, M.J.H.; Van Maris, A.J.A.; Pronk, J.T.

    2009-01-01

    In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in

  5. Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

    Science.gov (United States)

    Geiss, K T; Abbas, G M; Makaroff, C A

    1994-04-01

    The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

  6. Obesity induced by a pair-fed high fat sucrose diet: methylation and expression pattern of genes related to energy homeostasis

    Directory of Open Access Journals (Sweden)

    Campión Javier

    2010-06-01

    Full Text Available Abstract Background The expression of some genes controlling energy homeostasis could be regulated by epigenetic mechanisms that may play a role in body weight regulation. Thus, it is known that various nutritional factors affect DNA methylation. In order to assess whether the macronutrient composition of the diet could be related to the epigenetic regulation of gene expression and with obesity development, we investigated the effects on methylation and expression patterns of two pair-fed isocaloric diets in rats: control (rich in starch and HFS (rich in fat and sucrose. Results The pair-fed HFS diet induced higher weight gain and adiposity as compared to the controls as well as liver triglyceride accumulation and oxidative stress. Feeding the HFS diet impaired glucose tolerance and serum triglycerides and cholesterol. Liver glucokinase expression, a key glycolytic gene, remained unaltered, as well as the mRNA values of fatty acid synthase and NADH dehydrogenase (ubiquinone 1 beta subcomplex, 6 (NDUFB6 in liver and visceral adipocytes, which regulate lipogenesis and mitochondrial oxidative metabolism, respectively. Liver expression of hydroxyacyl-coenzyme A dehydrogenase (HADHB, a key gene of β-oxidation pathway, was higher in the HFS-fed animals. However, the methylation status of CpG islands in HADHB and glucokinase genes remained unchanged after feeding the HFS diet. Conclusions These results confirm that the distribution and type of macronutrients (starch vs. sucrose, and percent of fat influence obesity onset and the associated metabolic complications. HFS diets produce obesity independently of total energy intake, although apparently no epigenetic (DNA methylation changes accompanied the modifications observed in gene expression.

  7. Hydrogen storage and evolution catalysed by metal hydride complexes.

    Science.gov (United States)

    Fukuzumi, Shunichi; Suenobu, Tomoyoshi

    2013-01-07

    The storage and evolution of hydrogen are catalysed by appropriate metal hydride complexes. Hydrogenation of carbon dioxide by hydrogen is catalysed by a [C,N] cyclometalated organoiridium complex, [Ir(III)(Cp*)(4-(1H-pyrazol-1-yl-κN(2))benzoic acid-κC(3))(OH(2))](2)SO(4) [Ir-OH(2)](2)SO(4), under atmospheric pressure of H(2) and CO(2) in weakly basic water (pH 7.5) at room temperature. The reverse reaction, i.e., hydrogen evolution from formate, is also catalysed by [Ir-OH(2)](+) in acidic water (pH 2.8) at room temperature. Thus, interconversion between hydrogen and formic acid in water at ambient temperature and pressure has been achieved by using [Ir-OH(2)](+) as an efficient catalyst in both directions depending on pH. The Ir complex [Ir-OH(2)](+) also catalyses regioselective hydrogenation of the oxidised form of β-nicotinamide adenine dinucleotide (NAD(+)) to produce the 1,4-reduced form (NADH) under atmospheric pressure of H(2) at room temperature in weakly basic water. In weakly acidic water, the complex [Ir-OH(2)](+) also catalyses the reverse reaction, i.e., hydrogen evolution from NADH to produce NAD(+) at room temperature. Thus, interconversion between NADH (and H(+)) and NAD(+) (and H(2)) has also been achieved by using [Ir-OH(2)](+) as an efficient catalyst and by changing pH. The iridium hydride complex formed by the reduction of [Ir-OH(2)](+) by H(2) and NADH is responsible for the hydrogen evolution. Photoirradiation (λ > 330 nm) of an aqueous solution of the Ir-hydride complex produced by the reduction of [Ir-OH(2)](+) with alcohols resulted in the quantitative conversion to a unique [C,C] cyclometalated Ir-hydride complex, which can catalyse hydrogen evolution from alcohols in a basic aqueous solution (pH 11.9). The catalytic mechanisms of the hydrogen storage and evolution are discussed by focusing on the reactivity of Ir-hydride complexes.

  8. Expression profiles of glyceraldehyde-3-phosphate dehydrogenase from Clonorchis sinensis: a glycolytic enzyme with plasminogen binding capacity.

    Science.gov (United States)

    Hu, Yue; Zhang, Erhong; Huang, Lisi; Li, Wenfang; Liang, Pei; Wang, Xiaoyun; Xu, Jin; Huang, Yan; Yu, Xinbing

    2014-12-01

    Globally, 15-20 million people are infected with Clonorchis sinensis (C. sinensis) which results in clonorchiasis. In China, clonorchiasis is considered to be one of the fastest-growing food-borne parasitic diseases. That more key molecules of C. sinensis are characterized will be helpful to understand biology and pathogenesis of the carcinogenic liver fluke. Glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) from many species have functions other than their catalytic role in glycolysis. In the present study, we analyzed the sequence and structure of GAPDH from C. sinensis (CsGAPDH) by using bioinformatics tools and obtained its recombinant protein by prokaryotic expression system, to learn its expression profiles and molecular property. CsGAPDH could bind to human intrahepatic biliary epithelial cell in vivo and in vitro by the method of immunofluorescence assays. CsGAPDH also disturbed in lumen of biliary tract near to the parasite in the liver of infected rat. Western blotting analysis together with immunofluorescence assay indicated that CsGAPDH was a component of excretory/secretory proteins (CsESPs) and a surface-localized protein of C. sinensis. Quantitative real-time PCR (Q-PCR) and Western blotting demonstrated that CsGAPDHs are expressed at the life stages of adult worm, metacercaria, and egg, but the expression levels were different from each other. Recombinant CsGAPDH (rCsGAPDH) was confirmed to have the capacity to catalyze the conversion of glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate which was inhibited by AMP in a dose-dependent manner. In addition, rCsGAPDH was able to interact with human plasminogen in a dose-dependent manner by ELISA. The interaction could be inhibited by lysine. The plasminogen binding capacity of rCsGAPDH along with the distribution of CsGAPDH in vivo and in the liver of C. sinensis-infected rat hinted that surface-localized CsGAPDH might play an important role in host invasion of the worm besides its glycolytic

  9. Electrocatalytic oxidation behavior of NADH at Pt/Fe{sub 3}O{sub 4}/reduced-graphene oxide nanohybrids modified glassy carbon electrode and its determination

    Energy Technology Data Exchange (ETDEWEB)

    Roushani, Mahmoud, E-mail: mahmoudroushani@yahoo.com [Department of Chemistry, Faculty of Sciences, Ilam University, Ilam, 69315516 (Iran, Islamic Republic of); Hoseini, S. Jafar [Department of Chemistry, Faculty of Sciences, Yasouj University, Yasouj, 7591874831 (Iran, Islamic Republic of); Azadpour, Mitra [Department of Chemistry, Faculty of Sciences, Ilam University, Ilam, 69315516 (Iran, Islamic Republic of); Heidari, Vahid; Bahrami, Mehrangiz; Maddahfar, Mahnaz [Department of Chemistry, Faculty of Sciences, Yasouj University, Yasouj, 7591874831 (Iran, Islamic Republic of)

    2016-10-01

    We have developed Pt/Fe{sub 3}O{sub 4}/reduced-graphene oxide nanohybrids modified glassy carbon (Pt/Fe{sub 3}O{sub 4}/RGO/GC) electrode as a novel system for the preparation of electrochemical sensing platform. Characterization of as-made composite was determined using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM) and energy-dispersive analysis of X-ray (EDAX) where the Pt, Fe, Si, O and C elements were observed. The Pt/Fe{sub 3}O{sub 4}/RGO/GC electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Due to the synergistic effect between Pt, Fe{sub 3}O{sub 4} and RGO, the nanohybrid exhibited excellent performance toward dihydronicotinamide adenine dinucleotide (NADH) oxidation in 0.1 M phosphate buffer solution, pH 7.0, with a low detection limit of 5 nM. - Highlights: • Preparation of a novel electrochemical sensing platform system • Excellent performance of Pt/Fe{sub 3}O{sub 4}/reduced-graphene oxide nanohybrids • Dihydronicotinamide adenine dinucleotide oxidation with a low detection limit of 5 nM.

  10. Proteomic changes in renal cancer and co-ordinate demonstration of both the glycolytic and mitochondrial aspects of the Warburg effect.

    Science.gov (United States)

    Unwin, Richard D; Craven, Rachel A; Harnden, Patricia; Hanrahan, Sarah; Totty, Nick; Knowles, Margaret; Eardley, Ian; Selby, Peter J; Banks, Rosamonde E

    2003-08-01

    Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill-defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two-dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient-matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (>/= 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC-associated changes such as increases in Mn-superoxide dismutase, lactate dehydrogenase-A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.

  11. Biochemical gas sensor (bio-sniffer) for ultrahigh-sensitive gaseous formaldehyde monitoring.

    Science.gov (United States)

    Kudo, Hiroyuki; Suzuki, Yuki; Gessei, Tomoko; Takahashi, Daishi; Arakawa, Takahiro; Mitsubayashi, Kohji

    2010-10-15

    An ultrahigh-sensitive fiber-optic biochemical gas sensor (bio-sniffer) for continuous monitoring of indoor formaldehyde was constructed and tested. The bio-sniffer measures gaseous formaldehyde as fluorescence of nicotinamide adenine dinucleotide (NADH), which is the product of formaldehyde dehydrogenase (FALDH) reaction. The bio-sniffer device was constructed by attaching a flow cell with a FALDH immobilized membrane onto a fiber-optic NADH measurement system. The NADH measurement system utilizes an ultraviolet-light emitting diode (UV-LED) with peak emission of 335 nm as an excitation light source. The excitation light was introduced to an optical fiber probe, and fluorescence emission of neighboring NADH, which was produced by applying formaldehyde vapor to the FALDH membrane, was concentrically measured with a photomultiplier tube. Assessment of the bio-sniffer was carried out using a standard gas generator. Response, calibration range and selectivity to other chemical substances were investigated. Circulating phosphate buffer, which contained NAD+, available for continuous monitoring of formaldehyde vapor. The calibration range of the bio-sniffer was 2.5 ppb to 10 ppm, which covers the guideline value of the World Health Organization (80 ppb). High selectivity to other gaseous substances due to specific activity of FALDH was also confirmed. Considering its high sensitivity, a possible application of the bio-sniffer is continuous indoor formaldehyde monitoring to provide healthy residential atmosphere. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Dicty_cDB: Contig-U12435-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available hromosome 2 map 4915084... 40 0.24 7 ( AY934834 ) Cotesia plutellae clone 501-510 NADH dehydrogenas... 40 0....25 2 ( AY934846 ) Cotesia plutellae clone 641-646 NADH dehydrogenas... 40 0.25 2 ( AY934845 ) Cotesia plutella...e clone 631-636 NADH dehydrogenas... 40 0.25 2 ( AY934844 ) Cotesia plutellae c...lone 611-620 NADH dehydrogenas... 40 0.25 2 ( AY934842 ) Cotesia plutellae clone 581-583 NADH dehydrogenas...... 40 0.25 2 ( AY934840 ) Cotesia plutellae clone 561-570 NADH dehydrogenas... 40 0.25 2 ( AY934839 ) Cotesia plutella

  13. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity

  14. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Feng; Miyakawa, Takuya [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kataoka, Michihiko [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Takeshita, Daijiro [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Kumashiro, Shoko [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Uzura, Atsuko [Research and Development Center, Nagase and Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241 (Japan); Urano, Nobuyuki [Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531 (Japan); Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Nagata, Koji [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shimizu, Sakayu [Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555 (Japan); Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  15. Elimination of glycerol production in anaerobic cultures of a Saccharomyces cerevisiae strain engineered to use acetic acid as an electron acceptor.

    Science.gov (United States)

    Guadalupe Medina, Víctor; Almering, Marinka J H; van Maris, Antonius J A; Pronk, Jack T

    2010-01-01

    In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde+NAD++coenzyme Aacetyl coenzyme A+NADH+H+), was expressed in the gpd1Delta gpd2Delta strain, anaerobic growth was restored by supplementation with 2.0 g liter(-1) acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).

  16. Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme

    International Nuclear Information System (INIS)

    Solomonson, L.P.; McCreery, M.J.; Kay, C.J.; Barber, M.J.

    1987-01-01

    Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain

  17. Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel

    Directory of Open Access Journals (Sweden)

    Lushchak V.I.

    2001-01-01

    Full Text Available The effects of short-term burst (5 min at 1.8 m/s swimming and long-term cruiser (60 min at 1.2 m/s swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK, pyruvate kinase (PK, fructose-1,6-bisphosphatase (FBPase, and phosphoglucomutase (PGM all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

  18. Untitled

    African Journals Online (AJOL)

    M Tris-acetate or phosphate buffer. Monitoring the decreasing NADH concentration after the enzymatic reaction produced peaks which formed the basis for measuring ammonia. Detection was made with a flow-through spectrophotometer (at 340 nm) or amperometric detector with a wall-jet phenoxazine-modified graphite ...

  19. Capsaicin Inhibits Multiple Bladder Cancer Cell Phenotypes by Inhibiting Tumor-Associated NADH Oxidase (tNOX and Sirtuin1 (SIRT1

    Directory of Open Access Journals (Sweden)

    Ming-Hung Lin

    2016-06-01

    Full Text Available Bladder cancer is one of the most frequent cancers among males, and its poor survival rate reflects problems with aggressiveness and chemo-resistance. Recent interest has focused on the use of chemopreventatives (nontoxic natural agents that may suppress cancer progression to induce targeted apoptosis for cancer therapy. Capsaicin, which has anti-cancer properties, is one such agent. It is known to preferentially inhibit a tumor-associated NADH oxidase (tNOX that is preferentially expressed in cancer/transformed cells. Here, we set out to elucidate the correlation between tNOX expression and the inhibitory effects of capsaicin in human bladder cancer cells. We showed that capsaicin downregulates tNOX expression and decreases bladder cancer cell growth by enhancing apoptosis. Moreover, capsaicin was found to reduce the expression levels of several proteins involved in cell cycle progression, in association with increases in the cell doubling time and enhanced cell cycle arrest. Capsaicin was also shown to inhibit the activation of ERK, thereby reducing the phosphorylation of paxillin and FAK, which leads to decreased cell migration. Finally, our results indicate that RNA interference-mediated tNOX depletion enhances spontaneous apoptosis, prolongs cell cycle progression, and reduces cell migration and the epithelial-mesenchymal transition. We also observed a downregulation of sirtuin 1 (SIRT1 in these tNOX-knockdown cells, a deacetylase that is important in multiple cellular functions. Taken together, our results indicate that capsaicin inhibits the growth of bladder cancer cells by inhibiting tNOX and SIRT1 and thereby reducing proliferation, attenuating migration, and prolonging cell cycle progression.

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

    Science.gov (United States)

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

    2017-01-01

    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 Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth

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

    Directory of Open Access Journals (Sweden)

    Khalid N. M. Abdelazeem

    2017-04-01

    Full Text Available 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 Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi, NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. Methods: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl-5-(and-6-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS from 2’,7’-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm/ fluorometric enzymatic assay. Results: A 48 hour treatment with Ellagic acid (20 µM significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Conclusion: Ellagic acid (20µM markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those

  2. Hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea: mechanisms for reductant disposal.

    Science.gov (United States)

    Verhaart, Marcel R A; Bielen, Abraham A M; van der Oost, John; Stams, Alfons J M; Kengen, Servé W M

    2010-01-01

    Hydrogen produced from biomass by bacteria and archaea is an attractive renewable energy source. However, to make its application more feasible, microorganisms are needed with high hydrogen productivities. For several reasons, hyperthermophilic and extremely thermophilic bacteria and archaea are promising is this respect. In addition to the high polysaccharide-hydrolysing capacities of many of these organisms, an important advantage is their ability to use most of the reducing equivalents (e.g. NADH, reduced ferredoxin) formed during glycolysis for the production of hydrogen, enabling H2/hexose ratios of between 3.0 and 4.0. So, despite the fact that the hydrogen-yielding reactions, especially the one from NADH, are thermodynamically unfavourable, high hydrogen yields are obtained. In this review we focus on three different mechanisms that are employed by a few model organisms, viz. Caldicellulosiruptor saccharolyticus and Thermoanaerobacter tengcongensis, Thermotoga maritima, and Pyrococcus furiosus, to efficiently produce hydrogen. In addition, recent developments to improve hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea are discussed.

  3. Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy

    Science.gov (United States)

    Li, Chao; Tao, Fei; Ni, Jun; Wang, Yu; Yao, Feng; Xu, Ping

    2015-05-01

    It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO2. However, cofactor bias of cyanobacteria is different from bacteria that prefer NADH, which hampers cyanobacterial strain engineering. In this study, the key enzyme D-lactate dehydrogenase (LdhD) from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH. Then, the engineered enzyme was introduced into Synechococcus elongatus PCC7942 to construct an efficient light-driven system that produces D-lactic acid from CO2. Mutation of LdhD drove a fundamental shift in cofactor preference towards NADPH, and increased D-lactate productivity by over 3.6-fold. We further demonstrated that introduction of a lactic acid transporter and bubbling CO2-enriched air also enhanced D-lactate productivity. Using this combinational strategy, increased D-lactate concentration and productivity were achieved. The present strategy may also be used to engineer cyanobacteria for producing other useful chemicals.

  4. Aqueous Molecular Dynamics Simulations of the M. tuberculosis Enoyl-ACP Reductase-NADH System and Its Complex with a Substrate Mimic or Diphenyl Ethers Inhibitors

    Directory of Open Access Journals (Sweden)

    Camilo Henrique da Silva Lima

    2015-10-01

    Full Text Available Molecular dynamics (MD simulations of 12 aqueous systems of the NADH-dependent enoyl-ACP reductase from Mycobacterium tuberculosis (InhA were carried out for up to 20–40 ns using the GROMACS 4.5 package. Simulations of the holoenzyme, holoenzyme-substrate, and 10 holoenzyme-inhibitor complexes were conducted in order to gain more insight about the secondary structure motifs of the InhA substrate-binding pocket. We monitored the lifetime of the main intermolecular interactions: hydrogen bonds and hydrophobic contacts. Our MD simulations demonstrate the importance of evaluating the conformational changes that occur close to the active site of the enzyme-cofactor complex before and after binding of the ligand and the influence of the water molecules. Moreover, the protein-inhibitor total steric (ELJ and electrostatic (EC interaction energies, related to Gly96 and Tyr158, are able to explain 80% of the biological response variance according to the best linear equation, pKi = 7.772 − 0.1885 × Gly96 + 0.0517 × Tyr158 (R2 = 0.80; n = 10, where interactions with Gly96, mainly electrostatic, increase the biological response, while those with Tyr158 decrease. These results will help to understand the structure-activity relationships and to design new and more potent anti-TB drugs.

  5. Slow-tonic muscle fibers and their potential innervation in the turtle, Pseudemys (Trachemys) scripta elegans.

    Science.gov (United States)

    Callister, Robert J; Pierce, Patricia A; McDonagh, Jennifer C; Stuart, Douglas G

    2005-04-01

    A description is provided of the ratio of slow-tonic vs. slow- and fast-twitch fibers for five muscles in the adult turtle, Pseudemys (Trachemys) scripta elegans. The cross-sectional area of each fiber type and an estimation of the relative (weighted) cross-sectional area occupied by the different fiber types are also provided. Two hindlimb muscles (flexor digitorum longus, FDL; external gastrocnemius, EG) were selected on the basis of their suitability for future motor-unit studies. Three neck muscles (the fourth head of testo-cervicis, TeC4; the fourth head of retrahens capitus collique, RCCQ4; transversalis cervicis, TrC) were chosen for their progressively decreasing oxidative capacity. Serial sections were stained for myosin adenosine triphosphatase (ATPase), NADH-diaphorase, and alpha-glycerophosphate dehydrogenase (alpha-GPDH). Conventional fiber-type classification was then performed using indirect markers for contraction speed and oxidative (aerobic) vs. glycolytic (anaerobic) metabolism: i.e., slow oxidative (SO, including slow-twitch and possibly slow-tonic fibers), fast-twitch, oxidative-glycolytic (FOG), and fast-twitch glycolytic (Fg) fibers. Slow-tonic fibers in the SO class were then revealed by directing the monoclonal antibody, ALD-58 (raised against the slow-tonic fiber myosin heavy chain of chicken anterior latissimus dorsi), to additional muscle cross sections. All five of the tested muscles contained the four fiber types, with the ATPase-stained fibers including both slow-tonic and slow-twitch fibers. The extreme distributions of SO fibers were in the predominately glycolytic TrC vs. the predominately oxidative TeC4 muscle (TrC-SO, 9%; FOG, 20%; Fg, 71% vs. TeC4-SO, 58%: FOG, 16%; Fg, 25%). Across the five muscles, the relative prevalence of slow-tonic fibers (4-47%) paralleled that of the SO fibers (9-58%). TeC4 had the highest prevalence of slow-tonic fibers (47%). The test muscles exhibited varying degrees of regional concentration of each

  6. Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine

    International Nuclear Information System (INIS)

    Goto, Tatsufumi; Sugawara, Kotomi; Nakamura, Shigeyoshi; Kidokoro, Shun-Ichi; Wakui, Hideki; Nunomura, Wataru

    2016-01-01

    Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD + . In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (H 2 M 2 ) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the K m values for H 2 M 2 –mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M 4 and H 4 whereas the V max values were similar; (2) the K m and V max values for H 2 M 2 –mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of H 2 M 2 were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of H 2 M 2 was closer to that for M 4 than for H 4 . We also show that HCQ had different affinities for various LDH isozymes. - Highlights: • Heterotetrameric (H 2 M 2 ) LDH isozyme was isolated from swine brain. • Kinetics of H 2 M 2 were intermediate between the two homotetramers. • Thermodynamics of H 2 M 2 were also intermediate between the two homotetramers. • Hydroxychloroquine inhibited more strongly H 2 M 2 than homotetramers.

  7. Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii.

    Science.gov (United States)

    Oliveira, Bruno M; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto

    2014-01-01

    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

  8. NCBI nr-aa BLAST: CBRC-DNOV-01-0655 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0655 gb|AAB41179.1| NADH dehydrogenase subunit 2 [Apis mellifera] gb|A...AB41181.1| NADH dehydrogenase subunit 2 [Apis mellifera] gb|AAB41183.1| NADH dehydrogenase subunit 2 [Apis mellife...ra] gb|AAB41186.1| NADH dehydrogenase subunit 2 [Apis mellifera] AAB41179.1 0.039 28% ...

  9. ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

    Science.gov (United States)

    Juaristi, Inés; García-Martín, María L; Rodrigues, Tiago B; Satrústegui, Jorgina; Llorente-Folch, Irene; Pardo, Beatriz

    2017-07-01

    ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells. © 2017 International Society for Neurochemistry.

  10. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Lages, Nuno; Oldiges, M.

    2009-01-01

    to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH...... oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol...

  11. Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

    Directory of Open Access Journals (Sweden)

    Michael Katzberg

    2010-04-01

    Full Text Available The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S-hexanediol and the γ-hydroxyketone (5S-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%. This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.

  12. Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

    Science.gov (United States)

    Matsushika, Akinori; Nagashima, Atsushi; Goshima, Tetsuya; Hoshino, Tamotsu

    2013-01-01

    In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4

  13. Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

    Science.gov (United States)

    Kim, Soo Rin; Skerker, Jeffrey M; Kong, In Iok; Kim, Heejin; Maurer, Matthew J; Zhang, Guo-Chang; Peng, Dairong; Wei, Na; Arkin, Adam P; Jin, Yong-Su

    2017-03-01

    Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A

  14. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

    Directory of Open Access Journals (Sweden)

    Wittmann Christoph

    2008-03-01

    Full Text Available Abstract Background Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS and phosphoenolpyruvate carboxylase (PEPC, whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood. Results In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose. Conclusion The metabolic

  15. Triple Quenching of a Novel Self-Enhanced Ru(II) Complex by Hemin/G-Quadruplex DNAzymes and Its Potential Application to Quantitative Protein Detection.

    Science.gov (United States)

    Zhao, Min; Liao, Ni; Zhuo, Ying; Chai, Ya-Qin; Wang, Ji-Peng; Yuan, Ruo

    2015-08-04

    Herein, a novel "on-off" electrochemiluminescence (ECL) aptasensor for highly sensitive determination of thrombin has been constructed based on the triple quenching of the effect of hemin/G-quadruplex DNAzymes upon the Ru(II) complex-based ECL system. First, a strong initial ECL signal was achieved by the dual amplification strategies of (i) intramolecular coreaction of a self-enhanced Ru(II)-based molecule (PTCA-PEI-Ru(II)) and (ii) intermolecular coreaction between PTCA-PEI-Ru(II) and nicotinamide adenine dinucleotide (NADH), which was named the signal-on state. Then, a novel triple quenching of the effect of multifunctional hemin/G-quadruplex DNAzymes upon the Ru(II) complex-based ECL system was designed to realize the desirable signal-off state, which was outlined as follows: (i) the hemin/G-quadruplex DNAzymes mimicked NADH oxidase to oxidize NADH and in situ generate the H2O2, consuming the coreactant of NADH; (ii) its active center of hemin could oxidize the excited state PTCA-PEI-Ru(II)* to PTCA-PEI-Ru(III), making the energy and electron transfer quench; (iii) it also acted as horseradish peroxidase (HRP) to catalyze the H2O2 for in situ producing the quencher of O2. Based on triple quenching of the effect of hemin/G-quadruplex DNAzymes, the highly sensitive "on-off" thrombin aptasensor was developed with a wide linear detection range of 1.0 × 10(-14) M to 1.0 × 10(-10) M and a detection limit down to the femtomolar level.

  16. Flavoprotein-mediated tellurite reduction: structural basis and applications to the synthesis of tellurium-containing nanostructures

    Directory of Open Access Journals (Sweden)

    Mauricio Arenas-Salinas

    2016-07-01

    Full Text Available The tellurium oxyanion tellurite (TeO32- is extremely harmful for most organisms. It has been suggested that a potential bacterial tellurite resistance mechanism would consist of an enzymatic, NAD(PH-dependent, reduction to the less toxic form elemental tellurium (Te0. To date, a number of enzymes such as catalase, type II NADH dehydrogenase and terminal oxidases from the electron transport chain, nitrate reductases, and dihydrolipoamide dehydrogenase (E3, among others, have been shown to display tellurite-reducing activity. This activity is generically referred to as tellurite reductase (TR. Bioinformatic data resting on some of the abovementioned enzymes enabled the identification of common structures involved in tellurite reduction including vicinal catalytic cysteine residues and the FAD/NAD(P+-binding domain, which is characteristic of some flavoproteins. Along this line, thioredoxin reductase (TrxB, alkyl hydroperoxide reductase (AhpF, glutathione reductase (GorA, mercuric reductase (MerA, NADH: flavorubredoxin reductase (NorW, dihydrolipoamide dehydrogenase, and the putative oxidoreductase YkgC from Escherichia coli or environmental bacteria were purified and assessed for TR activity. All of them displayed in vitro TR activity at the expense of NADH or NADPH oxidation. In general, optimal reducing conditions occurred around pH 9-10 and 37 °C.Enzymes exhibiting strong TR activity produced Te-containing nanostructures (TeNS. While GorA and AhpF generated TeNS of 75 nm average diameter, E3 and YkgC produced larger structures (> 100 nm. Electron-dense structures were observed in cells over-expressing genes encoding TrxB, GorA and YkgC.

  17. Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

    Science.gov (United States)

    Wang, Xiaoguang; Hargrove, Mark S.

    2013-01-01

    Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia. PMID:24376554

  18. Perfil do corpo celular de neurônios mioentéricos nadh-diaforase positivos do estômago de ratos (Rattus norvegicus submetidos ao alcoolismo crônico - DOI: 10.4025/actascibiolsci.v26i3.1599 Profile of the cell body of nadh-diaphorase positive myenteric neurons from the stomach of rats (Rattus norvegicus subjected to chronic alcoholism - DOI: 10.4025/actascibiolsci.v26i3.1599

    Directory of Open Access Journals (Sweden)

    Marcílio Hubner de Miranda Neto

    2004-04-01

    Full Text Available O objetivo do trabalho foi comparar o perfil dos corpos celulares dos neurônios mioentéricos NADH-diaforase positivos do estômago de ratos. Utilizou-se 10 animais (Rattus norvegicus, provenientes dos grupos: a controle (n=5, que durante 210 dias receberam, ad libitum, dieta com teor protéico normal (22% e água; e b experimental (n = 5, que durante 210 dias receberam, ad libitum, ração com teor protéico normal (22% e aguardente-de-cana diluída a 30 Gay Lussac (30º v/v. Os estômagos coletados foram submetidos à técnica de evidenciação neuronal. A mensuração do perfil dos corpos celulares dos neurônios (n = 1.000 foi através de um Sistema Computadorizado de Análise de Imagem. O perfil dos corpos celulares dos neurônios do grupo controle ficou entre 60,16 a 638,64 µm2. No grupo experimental variou de 40,84 a 599,15 µm2. Constatamos redução significante no tamanho do corpo celular, aumento de neurônios pequenos e diminuição de neurônios grandesThe purpose of this work was to compare the profile of the cell body of the NADH-diaphorase positive myenteric neurons from the stomach of rats. It was used 10 animals (Rattus norvegicus, from groups a control (n = 5, that during 210 days had ad libitum supply of chow with normal protein level (22% and water; and b experimental (n = 5, that during 210 days had ad libitum supply of chow with normal protein level (22% and sugar cane brandy diluted to 30 Gay Lussac (30o v/v. The stomachs collected and subjected to the technique for neuronal staining. The cell body of the neurons (n = 1.000 was measured through a computerized system of image analysis. The profile of the neurons from the control rats ranged from 60.16 and 638.64 µm2. In the experimental group the values ranged from 40.84 to 599.15 µm2. We observed a significant decrease on the cell body size, increase of the small neurons and decrease of the large neurons

  19. Metabolic and Proliferative State of Vascular Adventitial Fibroblasts in Pulmonary Hypertension Is Regulated Through a MicroRNA-124/PTBP1 (Polypyrimidine Tract Binding Protein 1)/Pyruvate Kinase Muscle Axis.

    Science.gov (United States)

    Zhang, Hui; Wang, Daren; Li, Min; Plecitá-Hlavatá, Lydie; D'Alessandro, Angelo; Tauber, Jan; Riddle, Suzette; Kumar, Sushil; Flockton, Amanda; McKeon, B Alexandre; Frid, Maria G; Reisz, Julie A; Caruso, Paola; El Kasmi, Karim C; Ježek, Petr; Morrell, Nicholas W; Hu, Cheng-Jun; Stenmark, Kurt R

    2017-12-19

    reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH. © 2017 American Heart Association, Inc.

  20. Glycolysis Is Dynamic and Relates Closely to Respiration Rate in Stored Sugarbeet Roots

    Directory of Open Access Journals (Sweden)

    Clarice A. Megguer

    2017-05-01

    Full Text Available Although respiration is the principal cause of the loss of sucrose in postharvest sugarbeet (Beta vulgaris L., the internal mechanisms that control root respiration rate are unknown. Available evidence, however, indicates that respiration rate is likely to be controlled by the availability of respiratory substrates, and glycolysis has a central role in generating these substrates. To determine glycolytic changes that occur in sugarbeet roots after harvest and to elucidate relationships between glycolysis and respiration, sugarbeet roots were stored for up to 60 days, during which activities of glycolytic enzymes and concentrations of glycolytic substrates, intermediates, cofactors, and products were determined. Respiration rate was also determined, and relationships between respiration rate and glycolytic enzymes and metabolites were evaluated. Glycolysis was highly variable during storage, with 10 of 14 glycolytic activities and 14 of 17 glycolytic metabolites significantly altered during storage. Changes in glycolytic enzyme activities and metabolites occurred throughout the 60 day storage period, but were greatest in the first 4 days after harvest. Positive relationships between changes in glycolytic enzyme activities and root respiration rate were abundant, with 10 of 14 enzyme activities elevated when root respiration was elevated and 9 glycolytic activities static during periods of unchanging respiration rate. Major roles for pyruvate kinase and phosphofructokinase in the regulation of postharvest sugarbeet root glycolysis were indicated based on changes in enzymatic activities and concentrations of their substrates and products. Additionally, a strong positive relationship between respiration rate and pyruvate kinase activity was found indicating that downstream TCA cycle enzymes were unlikely to regulate or restrict root respiration in a major way. Overall, these results establish that glycolysis is not static during sugarbeet root

  1. Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism.

    Science.gov (United States)

    Oh, Seh-Hoon; Witek, Rafal P; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E

    2009-01-01

    Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

  2. Detection of Transketolase in Bone Marrow—Derived Insulin-Producing Cells: Benfotiamine Enhances Insulin Synthesis and Glucose Metabolism

    Science.gov (United States)

    Witek, Rafal P.; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E.

    2009-01-01

    Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus. PMID:18393672

  3. Retinoblastoma treatment: impact of the glycolytic inhibitor 2-deoxy-d-glucose on molecular genomics expression in LHBETATAG retinal tumors

    Directory of Open Access Journals (Sweden)

    Piña Y

    2012-05-01

    involved in its in vitro and in vivo activity in inhibiting tumor cell growth.Keywords: retinoblastoma, hypoxia, genetic expression, glycolytic inhibitor, 2-DG

  4. Kinetic mechanism and isotope effects of Pseudomonas cepacia 3-hydroxybenzoate-t-hydroxylase

    International Nuclear Information System (INIS)

    Wang, L.H.; Yu, Y.; Hamzah, R.Y.; Tu, S.C.

    1986-01-01

    The kinetic mechanism of Pseudomonas cepacia 3-hydroxybenzoate-6-hydroxylase has been delineated. Double reciprocal plots of initial rate versus m-hydroxybenzoate concentration at a constant level of oxygen and several fixed concentrations of NADH yielded a set of converging lines. Similar reciprocal plots of velocity versus NADH concentration at a constant oxygen level and several fixed m-hydroxybenzoate concentrations also showed converging lines. In contrast, double reciprocal plots of initial rate versus NADH concentration at a fixed m-hydroxybenzoate level and several oxygen concentrations showed a series of parallel lines. Parallel lines were also obtained from double reciprocal plots of initial rate versus m-hydroxybenzoate concentration at a fixed NADH level and varying oxygen concentrations. These results suggest a sequential binding of m-hydroxybenzoate and NADH by the hydroxylase. The enzyme-bound FAD is reduced and NAD is released. The reduced enzyme subsequently reacts with oxygen leading to the formation of other products. This hydroxylase exhibited a primary isotope effect of /sup D/V = 3.5 for (4R)-[4- 2 H] NADH but no isotope effect was observed with (4S)-[4- 2 H]NADH. An isotope effect of /sup T/V/K = 5.0 was also observed using (4R)-[4- 3 H]NADH. This tritium isotope effect was apparently independent of m-hydroxybenzoate concentration

  5. Thermodynamic Driving Force of Hydrogen on Rumen Microbial Metabolism: A Theoretical Investigation.

    Science.gov (United States)

    van Lingen, Henk J; Plugge, Caroline M; Fadel, James G; Kebreab, Ermias; Bannink, André; Dijkstra, Jan

    2016-01-01

    Hydrogen is a key product of rumen fermentation and has been suggested to thermodynamically control the production of the various volatile fatty acids (VFA). Previous studies, however, have not accounted for the fact that only thermodynamic near-equilibrium conditions control the magnitude of reaction rate. Furthermore, the role of NAD, which is affected by hydrogen partial pressure (PH2), has often not been considered. The aim of this study was to quantify the control of PH2 on reaction rates of specific fermentation pathways, methanogenesis and NADH oxidation in rumen microbes. The control of PH2 was quantified using the thermodynamic potential factor (FT), which is a dimensionless factor that corrects a predicted kinetic reaction rate for the thermodynamic control exerted. Unity FT was calculated for all glucose fermentation pathways considered, indicating no inhibition of PH2 on the production of a specific type of VFA (e.g., acetate, propionate and butyrate) in the rumen. For NADH oxidation without ferredoxin oxidation, increasing PH2 within the rumen physiological range decreased FT from unity to zero for different NAD+ to NADH ratios and pH of 6.2 and 7.0, which indicates thermodynamic control of PH2. For NADH oxidation with ferredoxin oxidation, increasing PH2 within the rumen physiological range decreased FT from unity at pH of 7.0 only. For the acetate to propionate conversion, FT increased from 0.65 to unity with increasing PH2, which indicates thermodynamic control. For propionate to acetate and butyrate to acetate conversions, FT decreased to zero below the rumen range of PH2, indicating full thermodynamic suppression. For methanogenesis by archaea without cytochromes, FT differed from unity only below the rumen range of PH2, indicating no thermodynamic control. This theoretical investigation shows that thermodynamic control of PH2 on individual VFA produced and associated yield of hydrogen and methane cannot be explained without considering NADH

  6. Thermodynamic Driving Force of Hydrogen on Rumen Microbial Metabolism: A Theoretical Investigation.

    Directory of Open Access Journals (Sweden)

    Henk J van Lingen

    Full Text Available Hydrogen is a key product of rumen fermentation and has been suggested to thermodynamically control the production of the various volatile fatty acids (VFA. Previous studies, however, have not accounted for the fact that only thermodynamic near-equilibrium conditions control the magnitude of reaction rate. Furthermore, the role of NAD, which is affected by hydrogen partial pressure (PH2, has often not been considered. The aim of this study was to quantify the control of PH2 on reaction rates of specific fermentation pathways, methanogenesis and NADH oxidation in rumen microbes. The control of PH2 was quantified using the thermodynamic potential factor (FT, which is a dimensionless factor that corrects a predicted kinetic reaction rate for the thermodynamic control exerted. Unity FT was calculated for all glucose fermentation pathways considered, indicating no inhibition of PH2 on the production of a specific type of VFA (e.g., acetate, propionate and butyrate in the rumen. For NADH oxidation without ferredoxin oxidation, increasing PH2 within the rumen physiological range decreased FT from unity to zero for different NAD+ to NADH ratios and pH of 6.2 and 7.0, which indicates thermodynamic control of PH2. For NADH oxidation with ferredoxin oxidation, increasing PH2 within the rumen physiological range decreased FT from unity at pH of 7.0 only. For the acetate to propionate conversion, FT increased from 0.65 to unity with increasing PH2, which indicates thermodynamic control. For propionate to acetate and butyrate to acetate conversions, FT decreased to zero below the rumen range of PH2, indicating full thermodynamic suppression. For methanogenesis by archaea without cytochromes, FT differed from unity only below the rumen range of PH2, indicating no thermodynamic control. This theoretical investigation shows that thermodynamic control of PH2 on individual VFA produced and associated yield of hydrogen and methane cannot be explained without

  7. Structural and functional investigation of flavin binding center of the NqrC subunit of sodium-translocating NADH:quinone oxidoreductase from Vibrio harveyi.

    Directory of Open Access Journals (Sweden)

    Valentin Borshchevskiy

    Full Text Available Na+-translocating NADH:quinone oxidoreductase (NQR is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium.

  8. High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans.

    Science.gov (United States)

    Manfrini, Nicola; Ricciardi, Sara; Miluzio, Annarita; Fedeli, Maya; Scagliola, Alessandra; Gallo, Simone; Brina, Daniela; Adler, Thure; Busch, Dirk H; Gailus-Durner, Valerie; Fuchs, Helmut; Hrabě de Angelis, Martin; Biffo, Stefano

    2017-12-01

    Eukaryotic Initiation Factor 6 (eIF6) is required for 60S ribosomal subunit biogenesis and efficient initiation of translation. Intriguingly, in both mice and humans, endogenous levels of eIF6 are detrimental as they act as tumor and obesity facilitators, raising the question on the evolutionary pressure that maintains high eIF6 levels. Here we show that, in mice and humans, high levels of eIF6 are required for proper immune functions. First, eIF6 heterozygous (het) mice show an increased mortality during viral infection and a reduction of peripheral blood CD4 + Effector Memory T cells. In human CD4 + T cells, eIF6 levels rapidly increase upon T-cell receptor activation and drive the glycolytic switch and the acquisition of effector functions. Importantly, in CD4 + T cells, eIF6 levels control interferon-γ (IFN-γ) secretion without affecting proliferation. In conclusion, the immune system has a high evolutionary pressure for the maintenance of a dynamic and powerful regulation of the translational machinery. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

    Science.gov (United States)

    Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

    2017-05-05

    Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Preclinical evidence of mitochondrial nicotinamide adenine dinucleotide as an effective alarm parameter under hypoxia

    Science.gov (United States)

    Shi, Hua; Sun, Nannan; Mayevsky, Avraham; Zhang, Zhihong; Luo, Qingming

    2014-01-01

    Early detection of tissue hypoxia in the intensive care unit is essential for effective treatment. Reduced nicotinamide adenine dinucleotide (NADH) has been suggested to be the most sensitive indicator of tissue oxygenation at the mitochondrial level. However, no experimental evidence comparing the kinetics of changes in NADH and other physiological parameters has been provided. The aim of this study is to obtain the missing data in a systematic and reliable manner. We constructed four acute hypoxia models, including hypoxic hypoxia, hypemic hypoxia, circulatory hypoxia, and histogenous hypoxia, and measured NADH fluorescence, tissue reflectance, cerebral blood flow, respiration, and electrocardiography simultaneously from the induction of hypoxia until death. We found that NADH was not always the first onset parameter responding to hypoxia. The order of responses was mainly affected by the cause of hypoxia. However, NADH reached its alarm level earlier than the other monitored parameters, ranging from several seconds to >10 min. As such, we suggest that the NADH can be used as a hypoxia indicator, although the exact level that should be used must be further investigated. When the NADH alarm is detected, the body still has a chance to recover if appropriate and timely treatment is provided.

  11. Raman spectroscopic study of acute oxidative stress induced changes in mice skeletal muscles

    Science.gov (United States)

    Sriramoju, Vidyasagar; Alimova, Alexandra; Chakraverty, Rahul; Katz, A.; Gayen, S. K.; Larsson, L.; Savage, H. E.; Alfano, R. R.

    2008-02-01

    The oxidative stress due to free radicals is implicated in the pathogenesis of tissue damage in diseases such as muscular dystrophy, Alzheimer dementia, diabetes mellitus, and mitochrondrial myopathies. In this study, the acute oxidative stress induced changes in nicotinamide adenine dinucleotides in mouse skeletal muscles are studied in vitro using Raman spectroscopy. Mammalian skeletal muscles are rich in nicotinamide adenine dinucleotides in both reduced (NADH) and oxidized (NAD) states, as they are sites of aerobic and anaerobic respiration. The relative levels of NAD and NADH are altered in certain physiological and pathological conditions of skeletal muscles. In this study, near infrared Raman spectroscopy is used to identify the molecular fingerprints of NAD and NADH in five-week-old mice biceps femoris muscles. A Raman vibrational mode of NADH is identified in fresh skeletal muscle samples suspended in buffered normal saline. In the same samples, when treated with 1% H IIO II for 5 minutes and 15 minutes, the Raman spectrum shows molecular fingerprints specific to NAD and the disappearance of NADH vibrational bands. The NAD bands after 15 minutes were more intense than after 5 minutes. Since NADH fluoresces and NAD does not, fluorescence spectroscopy is used to confirm the results of the Raman measurements. Fluorescence spectra exhibit an emission peak at 460 nm, corresponding to NADH emission wavelength in fresh muscle samples; while the H IIO II treated muscle samples do not exhibit NADH fluorescence. Raman spectroscopy may be used to develop a minimally invasive, in vivo optical biopsy method to measure the relative NAD and NADH levels in muscle tissues. This may help to detect diseases of muscle, including mitochondrial myopathies and muscular dystrophies.

  12. Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

    Science.gov (United States)

    Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

    2014-04-01

    Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition.

    Science.gov (United States)

    Lawlis, V B; Roche, T E

    1981-04-28

    Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP

  14. Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering.

    Science.gov (United States)

    Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

    2015-01-01

    In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. © 2014 Wiley Periodicals, Inc.

  15. A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

    Directory of Open Access Journals (Sweden)

    Gordon Ng

    Full Text Available BACKGROUND: Nitrogen (N(2 fixation also yields hydrogen (H(2 at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2 as sole N-source bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase, has nevertheless been presumed responsible for recycling such endogenous hydrogen. METHODS AND FINDINGS: As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase. An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. CONCLUSIONS: Representative of aerobic N(2-fixing and H(2-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2 respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2, specifically that produced by N(2 fixation. To benefit human civilization, H(2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As

  16. A novel endo-hydrogenase activity recycles hydrogen produced by nitrogen fixation.

    Science.gov (United States)

    Ng, Gordon; Tom, Curtis G S; Park, Angela S; Zenad, Lounis; Ludwig, Robert A

    2009-01-01

    Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such

  17. Glycerol Production from Glucose and Fructose by 3T3-L1 Cells: A Mechanism of Adipocyte Defense from Excess Substrate.

    Directory of Open Access Journals (Sweden)

    María del Mar Romero

    Full Text Available Cultured adipocytes (3T3-L1 produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively sustained. Proportionally (with respect to lactate plus glycerol, more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of

  18. Environmentally Robust Rhodamine Reporters for Probe-based Cellular Detection of the Cancer-linked Oxidoreductase hNQO1.

    Science.gov (United States)

    Best, Quinn A; Johnson, Amanda E; Prasai, Bijeta; Rouillere, Alexandra; McCarley, Robin L

    2016-01-15

    We successfully synthesized a fluorescent probe capable of detecting the cancer-associated quinoneoxidoreductase isozyme-1 within human cells, based on results from an investigation of the stability of various rhodamines and seminaphthorhodamines toward the biological reductant NADH, present at ∼100-200 μM within cells. While rhodamines are generally known for their chemical stability, we observe that NADH causes significant and sometimes rapid modification of numerous rhodamine analogues, including those oftentimes used in imaging applications. Results from mechanistic studies lead us to rule out a radical-based reduction pathway, suggesting rhodamine reduction by NADH proceeds by a hydride transfer process to yield the reduced leuco form of the rhodamine and oxidized NAD(+). A relationship between the structural features of the rhodamines and their reactivity with NADH is observed. Rhodamines with increased alkylation on the N3- and N6-nitrogens, as well as the xanthene core, react the least with NADH; whereas, nonalkylated variants or analogues with electron-withdrawing substituents have the fastest rates of reaction. These outcomes allowed us to judiciously construct a seminaphthorhodamine-based, turn-on fluorescent probe that is capable of selectively detecting the cancer-associated, NADH-dependent enzyme quinoneoxidoreductase isozyme-1 in human cancer cells, without the issue of NADH-induced deactivation of the seminaphthorhodamine reporter.

  19. Optical imaging of oxidative stress in retinitis pigmentosa (RP) in rodent model

    Science.gov (United States)

    Ghanian, Zahra; Maleki, Sepideh; Gopalakrishnan, Sandeep; Sepehr, Reyhaneh; Eells, Janis T.; Ranji, Mahsa

    2013-02-01

    Oxidative stress (OS), which increases during retinal degenerative disorders, contributes to photoreceptor cell loss. The objective of this study was to investigate the changes in the metabolic state of the eye tissue in rodent models of retinitis pigmentosa by using the cryofluorescence imaging technique. The mitochondrial metabolic coenzymes NADH and FADH2 are autofluorescent and can be monitored without exogenous labels using optical techniques. The NADH redox ratio (RR), which is the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), was used as a quantitative diagnostic marker. The NADH RR was examined in an established rodent model of retinitis pigmentosa (RP), the P23H rat, and compared to that of control Sprague-Dawley (SD) rats and P23H NIR treated rats. Our results demonstrated 24% decrease in the mean NADH RR of the eyes from P23H transgenic rats compared to normal rats and 20% increase in the mean NADH RR of the eyes from the P23H NIR treated rats compared to P23H non-treated rats.

  20. Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae

    Science.gov (United States)

    Min Hyung Kang; Haiying Ni; Thomas W. Jeffries

    2003-01-01

    Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the...

  1. Multi-omic profiling of EPO-producing CHO cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we...... discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed...... a multi-omic physiological characterization including metabolic foot printing of amino acids, metabolite fingerprinting of glycolytic intermediates, NAD(P)H-/NAD(P)+ and adenosine nucleotide phosphates. We used qPCR, qRT-PCR, western blots and Affymetrix CHO microarrays to assess EPO gene copy numbers...

  2. Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

    CSIR Research Space (South Africa)

    Garny, S

    2014-02-01

    Full Text Available spectrophotometric quantification of NADH [19]. Kaya et al. (2004) [19] demonstrated the linearly proportional relationship between the oxidation of NADH to NAD+, with the release of NO from NOC-5, but did not develop a kinetic assay for NOR activity using... this principle, the primary aim of this study. Nakahara et al. (1993) [10] determined the stoichiometry of NO reduction by NOR as 2:1:1 for NO:NADH:N2O. Therefore, for each NADH oxidized to NAD+, two molecules of NO are converted to N2O (refer Equation 1). 2...

  3. Absence of sodA Increases the Levels of Oxidation of Key Metabolic Determinants of Borrelia burgdorferi.

    Directory of Open Access Journals (Sweden)

    Maria D Esteve-Gassent

    Full Text Available Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat. HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348 exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560, and outer surface protein A (OspA, BBA15 were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC, Decorin binding protein A (DbpA, fibronectin binding protein (BBK32, RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox-⁄- and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of

  4. Detection of triglyceride using an iridium nano-particle catalyst based amperometric biosensor.

    Science.gov (United States)

    Liao, Wei-Yin; Liu, Chung-Chiun; Chou, Tse-Chuan

    2008-12-01

    The detection and quantification of triglyceride (TG) using an iridium nano-particle modified carbon based biosensor was successfully carried out in this study. The detection procedures were based on the electrochemical detection of enzymatically produced NADH. TG was hydrolyzed by lipase and the glycerol produced was catalytically oxidized by NAD-dependent glycerol dehydrogenase producing NADH in a solution containing NAD(+). Glyceryl tributyrate, a short chain triglyceride, was chosen as the substrate for the evaluation of this TG biosensor in bovine serum and human serum. A linear response to glyceryl tributyrate in the concentration range of 0 to 10 mM and a sensitivity of 7.5 nA mM(-1) in bovine serum and 7.0 nA mM(-1) in human serum were observed experimentally. The potential interference of species such as uric acid (UA) and ascorbic acid (AA) was assessed. The incorporation of a selected surfactant and an increase in the incubation temperature appeared to enhance the performance of this biosensor. The conditions for the determination of TG levels in bovine serum using this biosensor were optimized, with sunflower seed oil being used as an analyte to simulate the detection of TG in blood. The experimental results demonstrated that this iridium nano-particle modified working electrode based biosensor provided a relatively simple means for the accurate determination of TG in serum.

  5. Emerging Glycolysis Targeting and Drug Discovery from Chinese Medicine in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Zhiyu Wang

    2012-01-01

    Full Text Available Molecular-targeted therapy has been developed for cancer chemoprevention and treatment. Cancer cells have different metabolic properties from normal cells. Normal cells mostly rely upon the process of mitochondrial oxidative phosphorylation to produce energy whereas cancer cells have developed an altered metabolism that allows them to sustain higher proliferation rates. Cancer cells could predominantly produce energy by glycolysis even in the presence of oxygen. This alternative metabolic characteristic is known as the “Warburg Effect.” Although the exact mechanisms underlying the Warburg effect are unclear, recent progress indicates that glycolytic pathway of cancer cells could be a critical target for drug discovery. With a long history in cancer treatment, traditional Chinese medicine (TCM is recognized as a valuable source for seeking bioactive anticancer compounds. A great progress has been made to identify active compounds from herbal medicine targeting on glycolysis for cancer treatment. Herein, we provide an overall picture of the current understanding of the molecular targets in the cancer glycolytic pathway and reviewed active compounds from Chinese herbal medicine with the potentials to inhibit the metabolic targets for cancer treatment. Combination of TCM with conventional therapies will provide an attractive strategy for improving clinical outcome in cancer treatment.

  6. Tight coupling of astrocyte energy metabolism to synaptic activity revealed by genetically encoded FRET nanosensors in hippocampal tissue.

    Science.gov (United States)

    Ruminot, Iván; Schmälzle, Jana; Leyton, Belén; Barros, L Felipe; Deitmer, Joachim W

    2017-01-01

    The potassium ion, K + , a neuronal signal that is released during excitatory synaptic activity, produces acute activation of glucose consumption in cultured astrocytes, a phenomenon mediated by the sodium bicarbonate cotransporter NBCe1 ( SLC4A4). We have explored here the relevance of this mechanism in brain tissue by imaging the effect of neuronal activity on pH, glucose, pyruvate and lactate dynamics in hippocampal astrocytes using BCECF and FRET nanosensors. Electrical stimulation of Schaffer collaterals produced fast activation of glucose consumption in astrocytes with a parallel increase in intracellular pyruvate and biphasic changes in lactate . These responses were blocked by TTX and were absent in tissue slices prepared from NBCe1-KO mice. Direct depolarization of astrocytes with elevated extracellular K + or Ba 2+ mimicked the metabolic effects of electrical stimulation. We conclude that the glycolytic pathway of astrocytes in situ is acutely sensitive to neuronal activity, and that extracellular K + and the NBCe1 cotransporter are involved in metabolic crosstalk between neurons and astrocytes. Glycolytic activation of astrocytes in response to neuronal K + helps to provide an adequate supply of lactate, a metabolite that is released by astrocytes and which acts as neuronal fuel and an intercellular signal.

  7. Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

    Science.gov (United States)

    Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

    1991-03-01

    It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

  8. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    Energy Technology Data Exchange (ETDEWEB)

    Belfort, Georges [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering; Grimaldi, Joseph J. [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

  9. Crystallization and preliminary X-ray diffraction analysis of phosphoglycerate kinase from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Bernardo-García, Noelia; Bartual, Sergio G.; Fulde, Marcus; Bergmann, Simone; Hermoso, Juan A.

    2011-01-01

    Phosphoglycerate kinase, a two-domain enzyme involved in the glycolytic pathway, has been crystallized. Both the N-terminal domain and a ternary complex of the full-length enzyme with substrates were crystallized by the sitting-drop vapour-diffusion method. Inclusion of the substrates was critical in order to obtain crystals of the full-length protein. Phosphoglycerate kinase (PGK) is a widespread two-domain enzyme that plays a critical role in the glycolytic pathway. Several glycolytic enzymes from streptococci have been identified as surface-exposed proteins that are involved in streptococcal virulence by their ability to bind host proteins. This binding allows pneumococcal cells to disseminate through the epithelial and endothelial layers. Crystallization of PGK from Streptococcus pneumoniae yielded orthorhombic crystals (space group I222, unit-cell parameters a = 62.73, b = 75.38, c = 83.63 Å). However, the unit cell of these crystals was not compatible with the presence of full-length PGK. Various analytical methods showed that only the N-terminal domain of PGK was present in the I222 crystals. The ternary complex of PGK with adenylyl imidodiphosphate (AMP-PNP) and 3-phospho-d-glycerate (3PGA) produced monoclinic crystals (space group P2 1 , unit-cell parameters a = 40.35, b = 78.23, c = 59.03 Å, β = 96.34°). Molecular replacement showed that this new crystal form contained full-length PGK, thereby indicating the relevance of including substrates in order to avoid proteolysis during the crystallization process

  10. Monocarboxylate transporters in the brain and in cancer.

    Science.gov (United States)

    Pérez-Escuredo, Jhudit; Van Hée, Vincent F; Sboarina, Martina; Falces, Jorge; Payen, Valéry L; Pellerin, Luc; Sonveaux, Pierre

    2016-10-01

    Monocarboxylate transporters (MCTs) constitute a family of 14 members among which MCT1-4 facilitate the passive transport of monocarboxylates such as lactate, pyruvate and ketone bodies together with protons across cell membranes. Their anchorage and activity at the plasma membrane requires interaction with chaperon protein such as basigin/CD147 and embigin/gp70. MCT1-4 are expressed in different tissues where they play important roles in physiological and pathological processes. This review focuses on the brain and on cancer. In the brain, MCTs control the delivery of lactate, produced by astrocytes, to neurons, where it is used as an oxidative fuel. Consequently, MCT dysfunctions are associated with pathologies of the central nervous system encompassing neurodegeneration and cognitive defects, epilepsy and metabolic disorders. In tumors, MCTs control the exchange of lactate and other monocarboxylates between glycolytic and oxidative cancer cells, between stromal and cancer cells and between glycolytic cells and endothelial cells. Lactate is not only a metabolic waste for glycolytic cells and a metabolic fuel for oxidative cells, but it also behaves as a signaling agent that promotes angiogenesis and as an immunosuppressive metabolite. Because MCTs gate the activities of lactate, drugs targeting these transporters have been developed that could constitute new anticancer treatments. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  11. 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.)

  12. 3-bromopyruvate ameliorate autoimmune arthritis by modulating Th17/Treg cell differentiation and suppressing dendritic cell activation

    OpenAIRE

    Okano, Takaichi; Saegusa, Jun; Nishimura, Keisuke; Takahashi, Soshi; Sendo, Sho; Ueda, Yo; Morinobu, Akio

    2017-01-01

    Recent studies have shown that cellular metabolism plays an important role in regulating immune cell functions. In immune cell differentiation, both interleukin-17-producing T (Th17) cells and dendritic cells (DCs) exhibit increased glycolysis through the upregulation of glycolytic enzymes, such as hexokinase-2 (HK2). Blocking glycolysis with 2-deoxyglucose was recently shown to inhibit Th17 cell differentiation while promoting regulatory T (Treg) cell generation. However, 2-DG inhibits all i...

  13. Glycophospholipid Formulation with NADH and CoQ10 Significantly Reduces Intractable Fatigue in Western Blot-Positive ‘Chronic Lyme Disease’ Patients: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2012-03-01

    Full Text Available Background: An open label 8-week preliminary study was conducted in a small number of patients to determine if a combination oral supplement containing a mixture of phosphoglycolipids, coenzyme Q10 and microencapsulated NADH and other nutrients could affect fatigue levels in long-term, Western blot-positive, multi-symptom ‘chronic Lyme disease’ patients (also called ‘post-treatment Lyme disease’ or ‘post Lyme syndrome’ with intractable fatigue. Methods: The subjects in this study were 6 males (mean age = 45.1 ± 12.4 years and 10 females (mean age = 54.6 ± 7.4 years with ‘chronic Lyme disease’ (determined by multiple symptoms and positive Western blot analysis that had been symptomatic with chronic fatigue for an average of 12.7 ± 6.6 years. They had been seen by multiple physicians (13.3 ± 7.6 and had used many other remedies, supplements and drugs (14.4 ± 7.4 without fatigue relief. Fatigue was monitored at 0, 7, 30 and 60 days using a validated instrument, the Piper Fatigue Scale.Results: Patients in this preliminary study responded to the combination test supplement, showing a 26% reduction in overall fatigue by the end of the 8-week trial (p< 0.0003. Analysis of subcategories of fatigue indicated that there were significant improvements in the ability to complete tasks and activities as well as significant improvements in mood and cognitive abilities. Regression analysis of the data indicated that reductions in fatigue were consistent and occurred with a high degree of confidence (R2= 0.998. Functional Foods in Health and Disease 2012, 2(3:35-47 Conclusions: The combination supplement was a safe and effective method to significantly reduce intractable fatigue in long-term patients with Western blot-positive ‘chronic Lyme disease.’

  14. Dicty_cDB: SSL464 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 59525.1 Graphium sarpedon mitochondrial ND5 gene for NADH dehydrogenase 5, partial cds, sample:Cameron Highland, Malaysia...ial ND5 gene for NADH dehydrogenase 5, partial cds, sample:Cameron Highland, Malaysia2,@101. 48 0.072 1 AB05...ND5 gene for NADH dehydrogenase 5, partial cds, sample:Cameron Highland, Malaysia1,@86. 48 0.072 1 AB059521

  15. Rewiring carbohydrate catabolism differentially affects survival of pancreatic cancer cell lines with diverse metabolic profiles

    Science.gov (United States)

    Tataranni, Tiziana; Agriesti, Francesca; Ruggieri, Vitalba; Mazzoccoli, Carmela; Simeon, Vittorio; Laurenzana, Ilaria; Scrima, Rosella; Pazienza, Valerio; Capitanio, Nazzareno; Piccoli, Claudia

    2017-01-01

    An increasing body of evidence suggests that targeting cellular metabolism represents a promising effective approach to treat pancreatic cancer, overcome chemoresistance and ameliorate patient's prognosis and survival. In this study, following whole-genome expression analysis, we selected two pancreatic cancer cell lines, PANC-1 and BXPC-3, hallmarked by distinct metabolic profiles with specific concern to carbohydrate metabolism. Functional comparative analysis showed that BXPC-3 displayed a marked deficit of the mitochondrial respiratory and oxidative phosphorylation activity and a higher production of reactive oxygen species and a reduced NAD+/NADH ratio, indicating their bioenergetic reliance on glycolysis and a different redox homeostasis as compared to PANC-1. Both cell lines were challenged to rewire their metabolism by substituting glucose with galactose as carbon source, a condition inhibiting the glycolytic flux and fostering full oxidation of the sugar carbons. The obtained data strikingly show that the mitochondrial respiration-impaired-BXPC-3 cell line was unable to sustain the metabolic adaptation required by glucose deprivation/substitution, thereby resulting in a G2\\M cell cycle shift, unbalance of the redox homeostasis, apoptosis induction. Conversely, the mitochondrial respiration-competent-PANC-1 cell line did not show clear evidence of cell sufferance. Our findings provide a strong rationale to candidate metabolism as a promising target for cancer therapy. Defining the metabolic features at time of pancreatic cancer diagnosis and likely of other tumors, appears to be crucial to predict the responsiveness to therapeutic approaches or coadjuvant interventions affecting metabolism. PMID:28476035

  16. A quantitative metabolomics study of high sodium response in Clostridium acetobutylicum ATCC 824 acetone-butanol-ethanol (ABE) fermentation

    Science.gov (United States)

    Zhao, Xinhe; Condruz, Stefan; Chen, Jingkui; Jolicoeur, Mario

    2016-01-01

    Hemicellulose hydrolysates, sugar-rich feedstocks used in biobutanol refinery, are normally obtained by adding sodium hydroxide in the hydrolyze process. However, the resulting high sodium concentration in the hydrolysate inhibits ABE (acetone-butanol-ethanol) fermentation, and thus limits the use of these low-cost feedstocks. We have thus studied the effect of high sodium on the metabolic behavior of Clostridium acetobutyricum ATCC 824, with xylose as the carbon source. At a threshold sodium concentration of 200 mM, a decrease of the maximum cell dry weight (−19.50 ± 0.85%) and of ABE yield (−35.14 ± 3.50% acetone, −33.37 ± 0.74% butanol, −22.95 ± 1.81% ethanol) were observed compared to control culture. However, solvents specific productivities were not affected by supplementing sodium. The main effects of high sodium on cell metabolism were observed in acidogenesis, during which we observed the accumulation of ATP and NADH, and the inhibition of the pentose phosphate (PPP) and the glycolytic pathways with up to 80.73 ± 1.47% and 68.84 ± 3.42% decrease of the associated metabolic intermediates, respectively. However, the NADP+-to-NADPH ratio was constant for the whole culture duration, a phenomenon explaining the robustness of solvents specific productivities. Therefore, high sodium, which inhibited biomass growth through coordinated metabolic effects, interestingly triggered cell robustness on solvents specific productivity. PMID:27321153

  17. 3D imaging of the mitochondrial redox state of rat hearts under normal and fasting conditions

    Directory of Open Access Journals (Sweden)

    He N. Xu

    2014-03-01

    Full Text Available The heart requires continuous ATP availability that is generated in the mitochondria. Although studies using the cell culture and perfused organ models have been carried out to investigate the biochemistry in the mitochondria in response to a change in substrate supply, mitochondrial bioenergetics of heart under normal feed or fasting conditions has not been studied at the tissue level with a sub-millimeter spatial resolution either in vivo or ex vivo. Oxidation of many food-derived metabolites to generate ATP in the mitochondria is realized through the NADH/NAD+ couple acting as a central electron carrier. We employed the Chance redox scanner — the low-temperature fluorescence scanner to image the three-dimensional (3D spatial distribution of the mitochondrial redox states in heart tissues of rats under normal feeding or an overnight starvation for 14.5 h. Multiple consecutive sections of each heart were imaged to map three redox indices, i.e., NADH, oxidized flavoproteins (Fp, including flavin adenine dinucleotide (FAD and the redox ratio NADH/Fp. The imaging results revealed the micro-heterogeneity and the spatial distribution of these redox indices. The quantitative analysis showed that in the fasted hearts the standard deviation of both NADH and Fp, i.e., SD_NADH and SD_Fp, significantly decreased with a p value of 0.032 and 0.045, respectively, indicating that the hearts become relatively more homogeneous after fasting. The fasted hearts contained 28.6% less NADH (p = 0.038. No significant change in Fp was found (p = 0.4. The NADH/Fp ratio decreased with a marginal p value (0.076. The decreased NADH in the fasted hearts is consistent with the cardiac cells' reliance of fatty acids consumption for energy metabolism when glucose becomes scarce. The experimental observation of NADH decrease induced by dietary restriction in the heart at tissue level has not been reported to our best knowledge. The Chance redox scanner demonstrated the

  18. 3D IMAGING OF THE MITOCHONDRIAL REDOX STATE OF RAT HEARTS UNDER NORMAL AND FASTING CONDITIONS.

    Science.gov (United States)

    Xu, He N; Zhou, Rong; Moon, Lily; Feng, Min; Li, Lin Z

    2014-03-01

    The heart requires continuous ATP availability that is generated in the mitochondria. Although studies using the cell culture and perfused organ models have been carried out to investigate the biochemistry in the mitochondria in response to a change in substrate supply, mitochondrial bioenergetics of heart under normal feed or fasting conditions has not been studied at the tissue level with a sub-millimeter spatial resolution either in vivo or ex vivo . Oxidation of many food-derived metabolites to generate ATP in the mitochondria is realized through the NADH/NAD + couple acting as a central electron carrier. We employed the Chance redox scanner - the low-temperature fluorescence scanner to image the three-dimensional (3D) spatial distribution of the mitochondrial redox states in heart tissues of rats under normal feeding or an overnight starvation for 14.5 h. Multiple consecutive sections of each heart were imaged to map three redox indices, i.e., NADH, oxidized flavoproteins (Fp, including flavin adenine dinucleotide (FAD)) and the redox ratio NADH/Fp. The imaging results revealed the micro-heterogeneity and the spatial distribution of these redox indices. The quantitative analysis showed that in the fasted hearts the standard deviation of both NADH and Fp, i.e., SD_NADH and SD_Fp, significantly decreased with a p value of 0.032 and 0.045, respectively, indicating that the hearts become relatively more homogeneous after fasting. The fasted hearts contained 28.6% less NADH ( p = 0.038). No significant change in Fp was found ( p = 0.4). The NADH/Fp ratio decreased with a marginal p value (0.076). The decreased NADH in the fasted hearts is consistent with the cardiac cells' reliance of fatty acids consumption for energy metabolism when glucose becomes scarce. The experimental observation of NADH decrease induced by dietary restriction in the heart at tissue level has not been reported to our best knowledge. The Chance redox scanner demonstrated the feasibility of 3D

  19. Phosphoglycerate Mutase Is a Highly Efficient Enzyme without Flux Control in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Petranovic, D.; Købmann, Brian

    2010-01-01

    The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15-465% ...

  20. Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans

    DEFF Research Database (Denmark)

    Welin, J; Wilkins, J C; Beighton, D

    2003-01-01

    . Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes...

  1. SU-E-J-102: Separation of Metabolic Supply and Demand: From Power Grid Economics to Cancer Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Epstein, T; Xu, L; Gillies, R; Gatenby, R [Moffitt Cancer Center and Research Institute, Tampa, FL (United States)

    2014-06-01

    Purpose: To study a new model of glucose metabolism which is primarily governed by the timescale of the energetic demand and not by the oxygen level, and its implication on cancer metabolism (Warburg effect) Methods: 1) Metabolic profiling of membrane transporters activity in several cell lines, which represent the spectrum from normal breast epithelium to aggressive, metastatic cancer, using Seahorse XF reader.2) Spatial localization of oxidative and non-oxidative metabolic components using immunocytochemical imaging of the glycolytic ATP-producing enzyme, pyruvate kinase and mitochondria. 3) Finite element simulations of coupled partial differential equations using COMSOL and MATLAB. Results: Inhibition or activation of pumps on the cell membrane led to reduction or increase in aerobic glycolysis, respectively, while oxidative phosphorylation remained unchanged. These results were consistent with computational simulations of changes in short-timescale demand for energy by cell membrane processes. 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. These predictions were confirmed experimentally. Conclusion: The results in this work support a new model for glucose metabolism in which glycolysis and oxidative phosphorylation supply different types of energy demand. Similar to power grid economics, optimal metabolic control requires the two pathways, even in normoxic conditions, to match two different types of energy demands. Cells use aerobic metabolism to meet baseline, steady energy demand and glycolytic metabolism to meet short-timescale energy demands, mainly from membrane transport activities, even in the presence of oxygen. This model provides a mechanism for the origin of the Warburg effect in cancer cells. Here, the Warburg effect emerges during carcinogenesis is a physiological

  2. SU-E-J-102: Separation of Metabolic Supply and Demand: From Power Grid Economics to Cancer Metabolism

    International Nuclear Information System (INIS)

    Epstein, T; Xu, L; Gillies, R; Gatenby, R

    2014-01-01

    Purpose: To study a new model of glucose metabolism which is primarily governed by the timescale of the energetic demand and not by the oxygen level, and its implication on cancer metabolism (Warburg effect) Methods: 1) Metabolic profiling of membrane transporters activity in several cell lines, which represent the spectrum from normal breast epithelium to aggressive, metastatic cancer, using Seahorse XF reader.2) Spatial localization of oxidative and non-oxidative metabolic components using immunocytochemical imaging of the glycolytic ATP-producing enzyme, pyruvate kinase and mitochondria. 3) Finite element simulations of coupled partial differential equations using COMSOL and MATLAB. Results: Inhibition or activation of pumps on the cell membrane led to reduction or increase in aerobic glycolysis, respectively, while oxidative phosphorylation remained unchanged. These results were consistent with computational simulations of changes in short-timescale demand for energy by cell membrane processes. 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. These predictions were confirmed experimentally. Conclusion: The results in this work support a new model for glucose metabolism in which glycolysis and oxidative phosphorylation supply different types of energy demand. Similar to power grid economics, optimal metabolic control requires the two pathways, even in normoxic conditions, to match two different types of energy demands. Cells use aerobic metabolism to meet baseline, steady energy demand and glycolytic metabolism to meet short-timescale energy demands, mainly from membrane transport activities, even in the presence of oxygen. This model provides a mechanism for the origin of the Warburg effect in cancer cells. Here, the Warburg effect emerges during carcinogenesis is a physiological

  3. Coenzyme protection of lactic dehydrogenase against inactivation by gamma-rays

    International Nuclear Information System (INIS)

    Saito, M.

    1978-01-01

    A comparison has been made of the radiation sensitivities of the ternary complexes, oxamate-LDH-NADH and pyruvate-LDH-NAD with those of free LDH molecules and the intermediate binary complexes LDH-NAD and LDH-NADH. The enzyme solutions were 60 Co γirradiated and the rate of pyruvate reduction then measured. At doses of more than 10 krad the coenzymes afforded considerable protection to LDH against inactivation, and the dose-effect curves deviated from the curve for the unprotected enzyme, implying very specific protection. Coenzyme protection for a 30 krad dose at various concentrations of NAD and NADH reached a saturation level at about 4.0 x 10 -4 M for both NAD and NADH; protection by pyruvate alone was slight in comparison. Pyruvate and NAD (or oxamate and NADH) together at 1.0 x 10 -3 M protected the enzyme in a cooperative way. The results suggest that the major events of protection occur on the substrate and coenzyme binding sites, and support the view that coenzyme binding protects the enzyme by altering its conformation. (U.K.)

  4. Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them

    Energy Technology Data Exchange (ETDEWEB)

    Bohmert-Tatarev, Karen; McAvoy, Susan; Peoples, Oliver P.; Snell, Kristi D.

    2015-08-04

    Transgenic plants that produce high levels of polyhydroxybutyrate and methods of producing them are provided. In a preferred embodiment the transgenic plants are produced using plastid transformation technologies and utilize genes which are codon optimized. Stably transformed plants able to produce greater than 10% dwt PHS in tissues are also provided.

  5. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

    Science.gov (United States)

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-01-01

    NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

  6. Dicty_cDB: Contig-U13501-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 054061 ) Phrynocephalus przewalskii isolate RM3939 NADH de... 44 1.7 1 ( AY054060 ) Phrynocephalus przewal...skii isolate 0790 NADH dehy... 44 1.7 1 ( AY054059 ) Phrynocephalus przewalskii iso

  7. NCBI nr-aa BLAST: CBRC-TTRU-01-1038 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1038 ref|YP_626438.1| NADH dehydrogenase subunit 1 [Schistosoma spindale...] gb|AAZ57325.1| NADH dehydrogenase subunit 1 [Schistosoma spindale] YP_626438.1 0.003 23% ...

  8. NCBI nr-aa BLAST: CBRC-BTAU-01-0109 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-0109 ref|YP_626443.1| NADH dehydrogenase subunit 2 [Schistosoma spindale...] gb|AAZ57327.1| NADH dehydrogenase subunit 2 [Schistosoma spindale] YP_626443.1 0.005 23% ...

  9. NCBI nr-aa BLAST: CBRC-TTRU-01-0271 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0271 ref|YP_626438.1| NADH dehydrogenase subunit 1 [Schistosoma spindale...] gb|AAZ57325.1| NADH dehydrogenase subunit 1 [Schistosoma spindale] YP_626438.1 0.067 25% ...

  10. NCBI nr-aa BLAST: CBRC-BTAU-01-1673 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1673 ref|YP_626443.1| NADH dehydrogenase subunit 2 [Schistosoma spindale...] gb|AAZ57327.1| NADH dehydrogenase subunit 2 [Schistosoma spindale] YP_626443.1 0.005 23% ...

  11. NCBI nr-aa BLAST: CBRC-TTRU-01-0535 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0535 ref|YP_626438.1| NADH dehydrogenase subunit 1 [Schistosoma spindale...] gb|AAZ57325.1| NADH dehydrogenase subunit 1 [Schistosoma spindale] YP_626438.1 0.001 25% ...

  12. Age affects the contraction-induced mitochondrial redox response in skeletal muscle

    Directory of Open Access Journals (Sweden)

    Dennis R Claflin

    2015-02-01

    Full Text Available Compromised mitochondrial respiratory function is associated with advancing age. Damage due to an increase in reactive oxygen species (ROS with age is thought to contribute to the mitochondrial deficits. The coenzyme nicotinamide adenine dinucleotide in its reduced (NADH and oxidized (NAD+ forms plays an essential role in the cyclic sequence of reactions that result in the regeneration of ATP by oxidative phosphorylation in mitochondria. Monitoring mitochondrial NADH/NAD+ redox status during recovery from an episode of high energy demand thus allows assessment of mitochondrial function. NADH fluoresces when excited with ultraviolet light in the UV-A band and NAD+ does not, allowing NADH/NAD+ to be monitored in real time using fluorescence microscopy. Our goal was to assess mitochondrial function by monitoring the NADH fluorescence response following a brief period of high energy demand in muscle from adult and old wild-type (WT mice. This was accomplished by isolating whole lumbrical muscles from the hind paws of 7- and 28-month-old WT mice and making simultaneous measurements of force and NADH fluorescence responses during and after a 5 s maximum isometric contraction. All muscles exhibited fluorescence oscillations that were qualitatively similar and consisted of a brief transient increase followed by a longer transient period of reduced fluorescence and, finally, an increase that included an overshoot before recovering to resting level. Compared with the adult WT mice, muscles from the 28 mo WT mice exhibited a delayed peak during the first fluorescence transient and an attenuated recovery following the second transient. These findings indicate an impaired mitochondrial capacity to maintain NADH/NAD+ redox homeostasis during contractile activity in skeletal muscles of old mice.

  13. Electrochemical oxidation of dihydronicotinamide adenine dinucleotide at nitrogen-doped carbon nanotube electrodes.

    Science.gov (United States)

    Goran, Jacob M; Favela, Carlos A; Stevenson, Keith J

    2013-10-01

    Nitrogen-doped carbon nanotubes (N-CNTs) substantially lower the overpotential necessary for dihydronicotinamide adenine dinucleotide (NADH) oxidation compared to nondoped CNTs or traditional carbon electrodes such as glassy carbon (GC). We observe a 370 mV shift in the peak potential (Ep) from GC to CNTs and another 170 mV shift from CNTs to 7.4 atom % N-CNTs in a sodium phosphate buffer solution (pH 7.0) with 2.0 mM NADH (scan rate 10 mV/s). The sensitivity of 7.4 atom % N-CNTs to NADH was measured at 0.30 ± 0.04 A M(-1) cm(-2), with a limit of detection at 1.1 ± 0.3 μM and a linear range of 70 ± 10 μM poised at a low potential of -0.32 V (vs Hg/Hg2SO4). NADH fouling, known to occur to the electrode surface during NADH oxidation, was investigated by measuring both the change in Ep and the resulting loss of electrode sensitivity. NADH degradation, known to occur in phosphate buffer, was characterized by absorbance at 340 nm and correlated with the loss of NADH electroactivity. N-CNTs are further demonstrated to be an effective platform for dehydrogenase-based biosensing by allowing glucose dehydrogenase to spontaneously adsorb onto the N-CNT surface and measuring the resulting electrode's sensitivity to glucose. The glucose biosensor had a sensitivity of 0.032 ± 0.003 A M(-1) cm(-2), a limit of detection at 6 ± 1 μM, and a linear range of 440 ± 50 μM.

  14. Evidence for Divergent Evolution of Growth Temperature Preference in Sympatric Saccharomyces Species

    Science.gov (United States)

    Gonçalves, Paula; Valério, Elisabete; Correia, Cláudia; de Almeida, João M. G. C. F.; Sampaio, José Paulo

    2011-01-01

    The genus Saccharomyces currently includes eight species in addition to the model yeast Saccharomyces cerevisiae, most of which can be consistently isolated from tree bark and soil. We recently found sympatric pairs of Saccharomyces species, composed of one cryotolerant and one thermotolerant species in oak bark samples of various geographic origins. In order to contribute to explain the occurrence in sympatry of Saccharomyces species, we screened Saccharomyces genomic data for protein divergence that might be correlated to distinct growth temperature preferences of the species, using the dN/dS ratio as a measure of protein evolution rates and pair-wise species comparisons. In addition to proteins previously implicated in growth at suboptimal temperatures, we found that glycolytic enzymes were among the proteins exhibiting higher than expected divergence when one cryotolerant and one thermotolerant species are compared. By measuring glycolytic fluxes and glycolytic enzymatic activities in different species and at different temperatures, we subsequently show that the unusual divergence of glycolytic genes may be related to divergent evolution of the glycolytic pathway aligning its performance to the growth temperature profiles of the different species. In general, our results support the view that growth temperature preference is a trait that may have undergone divergent selection in the course of ecological speciation in Saccharomyces. PMID:21674061

  15. Transfer of deuterium from [1R-2H]- and [1S-2H] ethanol to reduced metabolites formed in vivo

    International Nuclear Information System (INIS)

    Cronholm, T.; Fors, C.

    1977-01-01

    Since alcohol dehydrogenase is stereospecific and only removes the 1-pro-R hydrogen of ethanol, it is possible to label selectively NADH formed at the alcohol dehydrogenase by using [1R- 2 H]-ethanol. In contrast, [1S- 2 H]ethanol may be used to label NADH formed in the aldehyde dehydrogenase reaction. The present investigation is an attempt to study the relationship between the NADH pools at these two dehydrogenases, with special reference to subcellular localization, by using chiral monodeuteroethanols

  16. Dicty_cDB: Contig-U08397-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6 |pid:none) Polaromonas sp. JS666, complete... 43 1e-06 U40031_3( U40031 |pid:none) Reithrodontomys megalot...ogena... 53 1e-06 U83832_3( U83832 |pid:none) Reithrodontomys fulvescens NADH dehydr... 52 1e-06 U83822_3( U...83822 |pid:none) Sigmodon ochrognathus NADH dehydrogena... 51 1e-06 U83831_3( U83831 |pid:none) Reithrodon...tomys megalotis NADH dehydro... 52 1e-06 AP009385_2172( AP009385 |pid:none) Burkhol

  17. NCBI nr-aa BLAST: CBRC-XTRO-01-0692 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0692 ref|YP_063385.1| NADH dehydrogenase subunit 1 [Pteronarcys prince...ps] gb|AAT76660.1| NADH dehydrogenase subunit 1 [Pteronarcys princeps] YP_063385.1 0.33 33% ...

  18. NCBI nr-aa BLAST: CBRC-OPRI-01-0507 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0507 ref|YP_054491.1| NADH dehydrogenase subunit 4 [Periplaneta fuligi...nosa] dbj|BAD32637.1| NADH dehydrogenase subunit 4 [Periplaneta fuliginosa] YP_054491.1 0.19 34% ...

  19. NCBI nr-aa BLAST: CBRC-TTRU-01-1187 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1187 ref|YP_002970996.1| NADH dehydrogenase subunit 6 [Loligo opalesce...ns] gb|ACS12932.1| NADH dehydrogenase subunit 6 [Loligo opalescens] YP_002970996.1 0.12 23% ...

  20. NCBI nr-aa BLAST: CBRC-PCAP-01-0672 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PCAP-01-0672 ref|YP_003097087.1| NADH dehydrogenase subunit 1 [Rhopalomyia pom...um] gb|ACT80216.1| NADH dehydrogenase subunit 1 [Rhopalomyia pomum] YP_003097087.1 0.098 26% ...

  1. NCBI nr-aa BLAST: CBRC-TTRU-01-1203 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1203 ref|YP_003097082.1| NADH dehydrogenase subunit 5 [Rhopalomyia pom...um] gb|ACT80211.1| NADH dehydrogenase subunit 5 [Rhopalomyia pomum] YP_003097082.1 0.002 23% ...

  2. NCBI nr-aa BLAST: CBRC-TTRU-01-1013 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1013 ref|YP_003097082.1| NADH dehydrogenase subunit 5 [Rhopalomyia pom...um] gb|ACT80211.1| NADH dehydrogenase subunit 5 [Rhopalomyia pomum] YP_003097082.1 0.006 23% ...

  3. NCBI nr-aa BLAST: CBRC-DNOV-01-1200 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1200 ref|YP_784045.1| NADH dehydrogenase subunit 1 [Scutigerella cause...yae] gb|ABF93312.1| NADH dehydrogenase subunit 1 [Scutigerella causeyae] YP_784045.1 1.6 23% ...

  4. NCBI nr-aa BLAST: CBRC-TBEL-01-0894 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TBEL-01-0894 ref|YP_784041.1| NADH dehydrogenase subunit 4 [Scutigerella cause...yae] gb|ABF93309.1| NADH dehydrogenase subunit 4 [Scutigerella causeyae] YP_784041.1 0.050 28% ...

  5. NCBI nr-aa BLAST: CBRC-TTRU-01-1275 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1275 ref|YP_214003.1| NADH dehydrogenase subunit 4 [Macrobrachium rosenberg...ii] gb|AAT52176.1| NADH dehydrogenase subunit 4 [Macrobrachium rosenbergii] YP_214003.1 0.015 24% ...

  6. NCBI nr-aa BLAST: CBRC-TTRU-01-0825 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0825 ref|YP_187598.1| NADH dehydrogenase subunit 5 [Astropecten polyac...anthus] dbj|BAD86706.1| NADH dehydrogenase subunit 5 [Astropecten polyacanthus] YP_187598.1 0.040 28% ...

  7. NCBI nr-aa BLAST: CBRC-TTRU-01-0424 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0424 ref|YP_187598.1| NADH dehydrogenase subunit 5 [Astropecten polyac...anthus] dbj|BAD86706.1| NADH dehydrogenase subunit 5 [Astropecten polyacanthus] YP_187598.1 0.003 27% ...

  8. NCBI nr-aa BLAST: CBRC-DNOV-01-0458 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0458 ref|YP_001382296.1| NADH dehydrogenase subunit 4 [Argopecten irra...dians] gb|ABS17679.1| NADH dehydrogenase subunit 4 [Argopecten irradians] YP_001382296.1 0.79 24% ...

  9. NCBI nr-aa BLAST: CBRC-DYAK-08-0043 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-08-0043 ref|YP_026048.1| NADH dehydrogenase subunit 4 [Speleonectes tulum...ensis] gb|AAS00889.1| NADH dehydrogenase subunit 4 [Speleonectes tulumensis] YP_026048.1 0.41 37% ...

  10. NCBI nr-aa BLAST: CBRC-FRUB-02-0644 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0644 ref|YP_001083073.1| NADH dehydrogenase subunit 4 [Epiperipatus bi...olleyi] gb|ABF93291.1| NADH dehydrogenase subunit 4 [Epiperipatus biolleyi] YP_001083073.1 0.001 22% ...

  11. NCBI nr-aa BLAST: CBRC-DNOV-01-0350 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0350 ref|YP_026081.1| NADH dehydrogenase subunit 1 [Steinernema carpoc...apsae] gb|AAT00524.1| NADH dehydrogenase subunit 1 [Steinernema carpocapsae] YP_026081.1 0.41 25% ...

  12. NCBI nr-aa BLAST: CBRC-TTRU-01-0375 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0375 ref|YP_026086.1| NADH dehydrogenase subunit 4 [Steinernema carpoc...apsae] gb|AAT00529.1| NADH dehydrogenase subunit 4 [Steinernema carpocapsae] YP_026086.1 0.009 26% ...

  13. NCBI nr-aa BLAST: CBRC-DNOV-01-3062 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-3062 ref|YP_026086.1| NADH dehydrogenase subunit 4 [Steinernema carpoc...apsae] gb|AAT00529.1| NADH dehydrogenase subunit 4 [Steinernema carpocapsae] YP_026086.1 0.065 22% ...

  14. NCBI nr-aa BLAST: CBRC-DNOV-01-0383 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0383 ref|YP_026083.1| NADH dehydrogenase subunit 2 [Steinernema carpoc...apsae] gb|AAT00526.1| NADH dehydrogenase subunit 2 [Steinernema carpocapsae] YP_026083.1 0.044 26% ...

  15. NCBI nr-aa BLAST: CBRC-DNOV-01-1657 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1657 ref|YP_026086.1| NADH dehydrogenase subunit 4 [Steinernema carpoc...apsae] gb|AAT00529.1| NADH dehydrogenase subunit 4 [Steinernema carpocapsae] YP_026086.1 0.32 21% ...

  16. NCBI nr-aa BLAST: CBRC-DNOV-01-1079 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1079 ref|YP_026081.1| NADH dehydrogenase subunit 1 [Steinernema carpoc...apsae] gb|AAT00524.1| NADH dehydrogenase subunit 1 [Steinernema carpocapsae] YP_026081.1 0.43 25% ...

  17. NCBI nr-aa BLAST: CBRC-LAFR-01-2596 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-2596 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 0.11 29% ...

  18. NCBI nr-aa BLAST: CBRC-TTRU-01-1180 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1180 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 1.4 30% ...

  19. NCBI nr-aa BLAST: CBRC-ETEL-01-0585 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-0585 ref|YP_026086.1| NADH dehydrogenase subunit 4 [Steinernema carpoc...apsae] gb|AAT00529.1| NADH dehydrogenase subunit 4 [Steinernema carpocapsae] YP_026086.1 0.39 22% ...

  20. NCBI nr-aa BLAST: CBRC-XTRO-01-3943 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3943 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 0.42 30% ...

  1. NCBI nr-aa BLAST: CBRC-CFAM-22-0005 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CFAM-22-0005 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 0.009 29% ...

  2. NCBI nr-aa BLAST: CBRC-ACAR-01-1152 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-1152 ref|YP_026083.1| NADH dehydrogenase subunit 2 [Steinernema carpoc...apsae] gb|AAT00526.1| NADH dehydrogenase subunit 2 [Steinernema carpocapsae] YP_026083.1 0.14 32% ...

  3. NCBI nr-aa BLAST: CBRC-BTAU-01-1871 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1871 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 2.4 28% ...

  4. NCBI nr-aa BLAST: CBRC-TTRU-01-0179 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0179 ref|YP_026086.1| NADH dehydrogenase subunit 4 [Steinernema carpoc...apsae] gb|AAT00529.1| NADH dehydrogenase subunit 4 [Steinernema carpocapsae] YP_026086.1 0.011 23% ...

  5. NCBI nr-aa BLAST: CBRC-MDOM-01-0409 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-01-0409 ref|YP_026081.1| NADH dehydrogenase subunit 1 [Steinernema carpoc...apsae] gb|AAT00524.1| NADH dehydrogenase subunit 1 [Steinernema carpocapsae] YP_026081.1 0.022 26% ...

  6. NCBI nr-aa BLAST: CBRC-TTRU-01-0587 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0587 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 2.0 22% ...

  7. NCBI nr-aa BLAST: CBRC-OPRI-01-0823 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0823 ref|YP_003204946.1| NADH dehydrogenase subunit 5 [Ditaxis biseria...ta] gb|ACO92610.1| NADH dehydrogenase subunit 5 [Ditaxis biseriata] YP_003204946.1 0.82 27% ...

  8. NCBI nr-aa BLAST: CBRC-DNOV-01-2150 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-2150 ref|YP_073317.1| NADH dehydrogenase subunit 6 [Neomaskellia andro...pogonis] gb|AAS75443.1| NADH dehydrogenase subunit 6 [Neomaskellia andropogonis] YP_073317.1 0.86 27% ...

  9. NCBI nr-aa BLAST: CBRC-BTAU-01-1417 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1417 ref|YP_073322.1| NADH dehydrogenase subunit 2 [Neomaskellia andro...pogonis] gb|AAS75448.1| NADH dehydrogenase subunit 2 [Neomaskellia andropogonis] YP_073322.1 0.025 25% ...

  10. NCBI nr-aa BLAST: CBRC-BTAU-01-2897 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-2897 ref|YP_740836.1| NADH dehydrogenase subunit 5 [Tetrahymena pigmento...sa] gb|ABI51745.1| NADH dehydrogenase subunit 5 [Tetrahymena pigmentosa] YP_740836.1 0.019 21% ...

  11. NCBI nr-aa BLAST: CBRC-MDOM-05-0629 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-05-0629 ref|YP_025950.1| NADH dehydrogenase subunit 2 [Strigops habroptil...us] gb|AAP47794.1| NADH dehydrogenase subunit 2 [Strigops habroptilus] YP_025950.1 0.28 24% ...

  12. NCBI nr-aa BLAST: CBRC-ETEL-01-1447 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-1447 ref|YP_026033.1| NADH dehydrogenase subunit 3 [Hutchinsoniella ma...cracantha] gb|AAS00874.1| NADH dehydrogenase subunit 3 [Hutchinsoniella macracantha] YP_026033.1 0.92 36% ...

  13. Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases

    Directory of Open Access Journals (Sweden)

    Raynald eCossard

    2015-06-01

    Full Text Available Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function.The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabitis elegans with notable improvements in reproduction, whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression.We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals and without affecting their respiration rate and ATP content.We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g. NDUFV1, NDUFS1, NDUFS6, NDUFS8 or GRIM-19 human orthologs in wild type animals is significantly reduced in the Ndi1p expressing worm.All together these results open up the perspective to identify new genes involved in complex I function, assembly or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression.

  14. NCBI nr-aa BLAST: CBRC-PHAM-01-0643 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0643 ref|YP_026083.1| NADH dehydrogenase subunit 2 [Steinernema carpoc...apsae] gb|AAT00526.1| NADH dehydrogenase subunit 2 [Steinernema carpocapsae] YP_026083.1 5e-05 28% ...

  15. NCBI nr-aa BLAST: CBRC-CJAC-01-1253 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CJAC-01-1253 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 6e-06 30% ...

  16. NCBI nr-aa BLAST: CBRC-RMAC-09-0022 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RMAC-09-0022 ref|YP_026083.1| NADH dehydrogenase subunit 2 [Steinernema carpoc...apsae] gb|AAT00526.1| NADH dehydrogenase subunit 2 [Steinernema carpocapsae] YP_026083.1 1e-05 30% ...

  17. NCBI nr-aa BLAST: CBRC-CBRI-08-0176 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CBRI-08-0176 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 1e-123 72% ...

  18. NCBI nr-aa BLAST: CBRC-GGOR-01-0211 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GGOR-01-0211 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 2e-04 34% ...

  19. NCBI nr-aa BLAST: CBRC-CFAM-33-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CFAM-33-0017 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 9e-07 27% ...

  20. NCBI nr-aa BLAST: CBRC-PTRO-27-0292 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-27-0292 ref|YP_026090.1| NADH dehydrogenase subunit 5 [Steinernema carpoc...apsae] gb|AAT00533.1| NADH dehydrogenase subunit 5 [Steinernema carpocapsae] YP_026090.1 3e-10 31% ...