Sample records for b-nicotinamide adenine dinucleotide

  1. The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

    PubMed

    Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

    2002-04-01

    Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

  2. Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides.

    PubMed

    Sallin, Olivier; Reymond, Luc; Gondrand, Corentin; Raith, Fabio; Koch, Birgit; Johnsson, Kai

    2018-05-29

    We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD + ) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP + ) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP + are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP + ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action. © 2018, Sallin et al.

  3. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

    PubMed

    Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

    2017-04-15

    Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

    PubMed

    Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W; Davis, James G; Agarwal, Beamon; Baur, Joseph A

    2017-02-01

    The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630). © 2016 by the American Association for the Study of Liver Diseases.

  5. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration

    PubMed Central

    Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W.; Davis, James G.; Agarwal, Beamon; Baur, Joseph A.

    2016-01-01

    The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing Nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nampt overexpressing mice were mildly hyperglycemic at baseline and, similarly to the mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking Nampt in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. Conclusion NAD availability is limiting during liver regeneration and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. PMID:27809334

  6. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. © 2016 The Author. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

  7. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

    PubMed

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  8. Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system.

    PubMed

    Fujita, Akiko; Sato, Chihiro; Münster-Kühnel, Anja-K; Gerardy-Schahn, Rita; Kitajima, Ken

    2005-02-01

    A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay

  9. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

    PubMed

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

    2016-06-01

    The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

  10. Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide.

    PubMed

    Slama, J T; Simmons, A M

    1989-09-19

    Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    PubMed

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

  12. Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

    PubMed

    Loera-Muro, Abraham; Jacques, Mario; Avelar-González, Francisco J; Labrie, Josée; Tremblay, Yannick D N; Oropeza-Navarro, Ricardo; Guerrero-Barrera, Alma L

    2016-06-27

    Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by

  13. Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grissom, C.B.; Willeford, O.; Wedding, R.T.

    1987-05-05

    The /sup 13/C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on V/sub max/. This indicates a stepwise conversion of malate to pyruvate and CO/sub 2/ with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylatemore » oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean and acid metabolism while maintaining the catalytic events founds in malic enzymes from animal sources.« less

  14. Roles of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase in Angiogenesis: Isoform-Specific Effects

    PubMed Central

    Wang, Haibo; Hartnett, M. Elizabeth

    2017-01-01

    Angiogenesis is the formation of new blood vessels from preexisting ones and is implicated in physiologic vascular development, pathologic blood vessel growth, and vascular restoration. This is in contrast to vasculogenesis, which is de novo growth of vessels from vascular precursors, or from vascular repair that occurs when circulating endothelial progenitor cells home into an area and develop into blood vessels. The objective of this review is to discuss the isoform-specific role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in physiologic and pathologic angiogenesis and vascular repair, but will not specifically address vasculogenesis. As the major source of reactive oxygen species (ROS) in vascular endothelial cells (ECs), NOX has gained increasing attention in angiogenesis. Activation of NOX leads to events necessary for physiologic and pathologic angiogenesis, including EC migration, proliferation and tube formation. However, activation of different NOX isoforms has different effects in angiogenesis. Activation of NOX2 promotes pathologic angiogenesis and vascular inflammation, but may be beneficial in revascularization in the hindlimb ischemic model. In contrast, activation of NOX4 appears to promote physiologic angiogenesis mainly by protecting the vasculature during ischemia, hypoxia and inflammation and by restoring vascularization, except in models of oxygen-induced retinopathy and diabetes where NOX4 activation leads to pathologic angiogenesis. PMID:28587189

  15. Preclinical evidence of mitochondrial nicotinamide adenine dinucleotide as an effective alarm parameter under hypoxia

    NASA Astrophysics Data System (ADS)

    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.

  16. In vivo native fluorescence spectroscopy and nicotinamide adinine dinucleotide/flavin adenine dinucleotide reduction and oxidation states of oral submucous fibrosis for chemopreventive drug monitoring

    NASA Astrophysics Data System (ADS)

    Sivabalan, Shanmugam; Vedeswari, C. Ponranjini; Jayachandran, Sadaksharam; Koteeswaran, Dornadula; Pravda, Chidambaranathan; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2010-01-01

    Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically proven cases of OSF in the age group of 20 to 40 years are diagnosed using native fluorescence spectroscopy. The OSF patients are given dexamethasone sodium phosphate and hyaluronidase twice a week for 6 weeks, and the therapeutic response is monitored using fluorescence spectroscopy. The fluorescence emission spectra of normal and OSF cases of both pre- and post-treated conditions are recorded in the wavelength region of 350 to 600 nm at an excitation wavelength of 330 nm. The statistical significance is verified using discriminant analysis. The oxidation-reduction ratio of the tissue is also calculated using the fluorescence emission intensities of flavin adenine dinucleotide and nicotinamide adinine dinucleotide at 530 and 440 nm, respectively, and they are compared with conventional physical clinical examinations. This study suggests that native fluorescence spectroscopy could also be extended to OSF diagnosis and therapeutic prognosis.

  17. [Influence exogenous nicotinamide adenine dinucleotide (NAD+) on contractile and bioelectric activity of the rat heart].

    PubMed

    Pustovit, K B; Kuz'min, V S; Sukhova, G S

    2014-04-01

    This study is aimed to the investigation of the nicotinamide adenine dinucleotide (NAD+) effects and mechanisms of action in a heart. NAD+ (mcM) induces multiphase alternation of contractile activity of isolated rat heart: short positive inotropic action is followed by a negative inotropic phase. NAD+ (1-100 mcM) induces decreasing of action potential duration (APD) in rat atrial myocardium (from 45 +/- 0.82 ms in control experiments to 39 +/- 1.05 (n = 8) and 32 +/- 2 (n = 8) during application of 10 and 100 mcM of NAD+, respectively). Significant APD increase (from 45 +/- 0.82 ms to 74 +/- 1.89 (n = 8) ms) was observed during washing out of NAD+ (100 mcM). ATP or adenosine was unable to increase APD both during application or washing out. NAD+ induced APD decrease was not suppressed by P1-antagonist theophylline. P1-purinoreceptor and metabolite independent direct action of NAD+ in rat heart is suggested. Activation of P2X or P2Y receptors, cyclic ADP-ribose accumulation in cardiomyocytes is proposed as a main mechanism of NAD(+)-induced effects in the heart.

  18. Photosensitized oxidation of nicotinamide adenine dinucleotide by diethoxyphosphorus(V)tetraphenylporphyrin and its fluorinated derivative: Possibility of chain reaction.

    PubMed

    Hirakawa, Kazutaka; Murata, Atsushi

    2018-01-05

    Water-soluble porphyrins, diethoxyphosphorus(V)tetraphenylporphyrin (EtP(V)TPP) and its fluorinated analogue (FEtP(V)TPP), decreased the typical absorption around 340nm of nicotinamide adenine dinucleotide (NADH) under visible light irradiation, indicating oxidative decomposition. A singlet oxygen quencher, sodium azide, and a triplet quencher, potassium iodide, slightly inhibited photosensitized NADH oxidation. However, these inhibitory effects were very small. Furthermore, the fluorescence lifetime of these P(V)porphyrins was decreased by NADH, suggesting the contribution of electron transfer to the singlet excited (S 1 ) state of P(V)porphyrin. The redox potential measurement supports the electron transfer-mediated oxidation of NADH. The quantum yields of NADH photodecomposition by P(V)porphyrins could be estimated from the kinetic data and the effect of these quenchers on NADH oxidation. The obtained values suggest that the electron accepting by the S 1 states of P(V)porphyrins triggers a chain reaction of NADH oxidation. This photosensitized reaction may play an important role in the photocytotoxicity of P(V)porphyrins. The axial ligand fluorination of P(V)porphyrins improved electron accepting ability. However, fluorination slightly suppressed static interaction with NADH, resulting in decreased oxidation quantum yield. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

    PubMed Central

    Gariani, Karim; Menzies, Keir J.; Ryu, Dongryeol; Wegner, Casey J.; Wang, Xu; Ropelle, Eduardo R.; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young‐Ki; Piersigilli, Alessandra; Pham, Tho X.; Yang, Yue; Ku, Chai Siah; Koo, Sung I.; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A.

    2015-01-01

    With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe −/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204) PMID:26404765

  20. A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana

    PubMed Central

    Wang, Chenggang; Zhou, Mingqi; Zhang, Xudong; Yao, Jin; Zhang, Yanping; Mou, Zhonglin

    2017-01-01

    Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 ± 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants. DOI: http://dx.doi.org/10.7554/eLife.25474.001 PMID:28722654

  1. Flow-injection analysis with electrochemical detection of reduced nicotinamide adenine dinucleotide using 2,6-dichloroindophenol as a redox coupling agent.

    PubMed

    Tang, H T; Hajizadeh, K; Halsall, H B; Heineman, W R

    1991-01-01

    The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow-injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 microM and with a detection limit of 0.38 microM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.

  2. Comparison of oral nicotinamide adenine dinucleotide (NADH) versus conventional therapy for chronic fatigue syndrome.

    PubMed

    Santaella, María L; Font, Ivonne; Disdier, Orville M

    2004-06-01

    To compare effectiveness of oral therapy with reduced nicotinamide adenine dinucleotide (NADH) to conventional modalities of treatment in patients with chronic fatigue syndrome (CFS). CFS is a potentially disabling condition of unknown etiology. Although its clinical presentation is associated to a myriad of symptoms, fatigue is a universal and essential finding for its diagnosis. No therapeutic regimen has proven effective for this condition. A total of 31 patients fulfilling the Centers for Disease Control criteria for CFS, were randomly assigned to either NADH or nutritional supplements and psychological therapy for 24 months. A thorough medical history, physical examination and completion of a questionnaire on the severity of fatigue and other symptoms were performed each trimester of therapy. In addition, all of them underwent evaluation in terms of immunological parameters and viral antibody titers. Statistical analysis was applied to the demographic data, as well as to symptoms scores at baseline and at each trimester of therapy. The twelve patients who received NADH had a dramatic and statistically significant reduction of the mean symptom score in the first trimester (p < 0.001). However, symptom scores in the subsequent trimesters of therapy were similar in both treatment groups. Elevated IgG and Ig E antibody levels were found in a significant number of patients. Observed effectiveness of NADH over conventional treatment in the first trimester of the trial and the trend of improvement of that modality in the subsequent trimesters should be further assessed in a larger patient sample.

  3. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice.

    PubMed

    Gariani, Karim; Menzies, Keir J; Ryu, Dongryeol; Wegner, Casey J; Wang, Xu; Ropelle, Eduardo R; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young-Ki; Piersigilli, Alessandra; Pham, Tho X; Yang, Yue; Ku, Chai Siah; Koo, Sung I; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A; Lee, Ji-Young; Auwerx, Johan

    2016-04-01

    With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD(+) ) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD(+) biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1- and SIRT3-dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 knockout mice (Sirt1(hep-/-) ), whereas apolipoprotein E-deficient mice (Apoe(-/-) ) challenged with a high-fat high-cholesterol diet affirmed the use of NR in other independent models of NAFLD. Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD. © 2015 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.

  4. Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

    NASA Astrophysics Data System (ADS)

    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.

  5. The human amygdaloid complex: a cytologic and histochemical atlas using Nissl, myelin, acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase staining.

    PubMed

    Sims, K S; Williams, R S

    1990-01-01

    We examined the distribution of acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase enzyme activity in the human amygdala using histochemical techniques. Both methods revealed compartments of higher or lower enzyme activity, in cells or neuropil, which corresponded to the nuclear subdivisions of the amygdala as defined with classical Nissl and myelin methods. The boundaries between the histochemical compartments were usually so sharp that the identification of these nuclear subdivisions was enhanced. There was also variation of staining intensity within many of the nuclear subdivisions, such as the lateral and central nuclei, anterior amygdaloid area and the intercalated groups. This histochemical difference corresponded to more subtle differences in Nissl and myelin staining patterns, and suggests further structural subdivisions of potential functional significance. We present a revised scheme of anatomical parcellation of the human amygdala based upon serial analysis with all four techniques. Our expectation is that this will allow the delineation of a clearer homology between the cytoarchitectonic subdivisions of the human amygdala and those of experimental animals.

  6. Nicotinamide adenine dinucleotide is transported into mammalian mitochondria.

    PubMed

    Davila, Antonio; Liu, Ling; Chellappa, Karthikeyani; Redpath, Philip; Nakamaru-Ogiso, Eiko; Paolella, Lauren M; Zhang, Zhigang; Migaud, Marie E; Rabinowitz, Joshua D; Baur, Joseph A

    2018-06-12

    Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH) transporter. © 2018, Davila et al.

  7. Photosensitized oxidation of nicotinamide adenine dinucleotide by diethoxyphosphorus(V)tetraphenylporphyrin and its fluorinated derivative: Possibility of chain reaction

    NASA Astrophysics Data System (ADS)

    Hirakawa, Kazutaka; Murata, Atsushi

    2018-01-01

    Water-soluble porphyrins, diethoxyphosphorus(V)tetraphenylporphyrin (EtP(V)TPP) and its fluorinated analogue (FEtP(V)TPP), decreased the typical absorption around 340 nm of nicotinamide adenine dinucleotide (NADH) under visible light irradiation, indicating oxidative decomposition. A singlet oxygen quencher, sodium azide, and a triplet quencher, potassium iodide, slightly inhibited photosensitized NADH oxidation. However, these inhibitory effects were very small. Furthermore, the fluorescence lifetime of these P(V)porphyrins was decreased by NADH, suggesting the contribution of electron transfer to the singlet excited (S1) state of P(V)porphyrin. The redox potential measurement supports the electron transfer-mediated oxidation of NADH. The quantum yields of NADH photodecomposition by P(V)porphyrins could be estimated from the kinetic data and the effect of these quenchers on NADH oxidation. The obtained values suggest that the electron accepting by the S1 states of P(V)porphyrins triggers a chain reaction of NADH oxidation. This photosensitized reaction may play an important role in the photocytotoxicity of P(V)porphyrins. The axial ligand fluorination of P(V)porphyrins improved electron accepting ability. However, fluorination slightly suppressed static interaction with NADH, resulting in decreased oxidation quantum yield.

  8. Generation of Reduced Nicotinamide Adenine Dinucleotide for Nitrate Reduction in Green Leaves 1

    PubMed Central

    Klepper, Lowell; Flesher, Donna; Hageman, R. H.

    1971-01-01

    An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO3 (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals. By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase. It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction. This conclusion was supported by experiments that included: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue. The level of nitrate reductase activity obtained with the in vitro assay is higher (2.5- to 20-fold) than with the in vivo assay for most plant species. The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species. The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted. PMID:16657841

  9. Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

    PubMed Central

    Jamieson, D; Tung, A T Y; Knox, R J; Boddy, A V

    2006-01-01

    NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species. PMID:17031400

  10. Nicotinamide Adenine Dinucleotide (NAD+) and Nicotinamide: Sex Differences in Cerebral Ischemia

    PubMed Central

    Siegel, Chad S.; McCullough, Louise D.

    2013-01-01

    Background Previous literature suggests that cell death pathways activated after cerebral ischemia differ between the sexes. While caspase-dependent mechanisms predominate in the female brain, caspase-independent cell death induced by activation of Poly (ADP-ribose) polymerase (PARP) predominates in the male brain. PARP-1 gene deletion decreases infarction volume in the male brain, but paradoxically increases damage in PARP-1 knockout females. Purpose This study examined stroke induced changes in NAD+, a key energy molecule involved in PARP-1 activation in both sexes. Methods Mice were subjected to Middle Cerebral Artery Occlusion and NAD+ levels were assessed. Caspase-3 activity and nuclear translocation was assessed 6 hours after ischemia. In additional cohorts, Nicotinamide (500mg/kg i.p.) a precursor of NAD+ or vehicle was administered and infarction volume was measured 24 hours after ischemia. Results Males have higher baseline NAD+ levels than females. Significant stroke-induced NAD+ depletion occurred in males and ovariectomized females but not in intact females. PARP-1 deletion prevented the stroke induced loss in NAD+ in males, but worsened NAD+ loss in PARP-1 deficient females. Preventing NAD+ loss with nicotinamide reduced infarct in wild-type males and PARP-1 knockout mice of both sexes, with no effect in WT females. Caspase-3 activity was significantly increased in PARP-1 knockout females compared to males and wild-type females, this was reversed with nicotinamide. Conclusions Sex differences exist in baseline and stroke-induced NAD+ levels. Nicotinamide protected males and PARP knockout mice, but had minimal effects in the wild-type female brain. This may be secondary to differences in energy metabolism between the sexes. PMID:23403179

  11. Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes.

    PubMed

    Braidy, Nady; Berg, Jade; Clement, James; Khorshidi, Fatemeh; Poljak, Anne; Jayasena, Tharusha; Grant, Ross; Sachdev, Perminder

    2018-05-11

    Nicotinamide adenine dinucleotide (NAD + ) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD + depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD + , and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD + levels. Recent studies have shown that enhancing NAD + levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD + anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD + synthesis pathway in mammalian cells. NAD + can also be produced by the NAD + salvage pathway. Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD + precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD + decline in degenerative disease states and physiological aging. Results obtained in recent years have shown that NAD + precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD + synthesis via their location in the NAD + anabolic pathway. Increased synthesis of NAD + promotes protective cell responses, further demonstrating that NAD + is a regulatory molecule associated with several

  12. Transcriptional regulation of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase in murine hepatoma cells by 6-(methylsufinyl)hexyl isothiocyanate, an active principle of wasabi (Eutrema wasabi Maxim).

    PubMed

    Hou, D X; Fukuda, M; Fujii, M; Fuke, Y

    2000-12-20

    Wasabi is a very popular pungent spice in Japan. This study examined the ability of 6-(methylsufinyl)hexyl isothiocyanate (6-MITC), an active principle of wasabi, to induce the cellular expression of nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase (QR) in Hepa 1c1c7 cells. The cells were treated with various concentrations of 6-MITC, and were then assessed for cell growth, QR activity and QR mRNA expression. The induction of QR activity and QR mRNA expression was time- and dose-responsive over a narrow range of 0.1-5 microM, with declining induction at higher concentrations due to cell toxicity. Furthermore, transfection studies demonstrated that the induction of transcription of the QR gene by 6-MITC involved an antioxidant/electrophile-responsive element (ARE/EpRE) activation. Our results suggest a novel mechanism by which dietary wasabi 6-MITC may be implicated in cancer chemoprevention.

  13. Safety assessment of nicotinamide riboside, a form of vitamin B3.

    PubMed

    Conze, D B; Crespo-Barreto, J; Kruger, C L

    2016-01-20

    Nicotinamide riboside (NR) is a naturally occurring form of vitamin B 3 present in trace amounts in some foods. Like niacin, it has been shown to be a precursor in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). The safety of Niagen™, a synthetic form of NR, was determined using a bacterial reverse mutagenesis assay (Ames), an in vitro chromosome aberration assay, an in vivo micronucleus assay, and acute, 14-day and 90-day rat toxicology studies. NR was not genotoxic. There was no mortality at an oral dose of 5000 mg/kg. Based on the results of a 14-day study, a 90-day study was performed comparing NR at 300, 1000, and 3000 mg/kg/day to an equimolar dose of nicotinamide at 1260 mg/kg/day as a positive control. Results from the study show that NR had a similar toxicity profile to nicotinamide at the highest dose tested. Target organs of toxicity were liver, kidney, ovaries, and testes. The lowest observed adverse effect level for NR was 1000 mg/kg/day, and the no observed adverse effect level was 300 mg/kg/day. © The Author(s) 2016.

  14. β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

    PubMed

    Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin; Jurastow, Innokentij; Küllmar, Mira; Hecker, Andreas; Wilker, Sigrid; Fuchs-Moll, Gabriele; Manzini, Ivan; Schmalzing, Günther; Kummer, Wolfgang; Padberg, Winfried; McIntosh, J Michael; Damm, Jelena; Zakrzewicz, Anna; Grau, Veronika

    2018-04-10

    While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

  15. Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding.

    PubMed

    Drew, Janice E; Farquharson, Andrew J; Horgan, Graham W; Williams, Lynda M

    2016-11-01

    The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Protective effect of nicotinamide adenine dinucleotide (NAD+) against spinal cord ischemia-reperfusion injury via reducing oxidative stress-induced neuronal apoptosis.

    PubMed

    Xie, Lei; Wang, Zhenfei; Li, Changwei; Yang, Kai; Liang, Yu

    2017-02-01

    As previous studies demonstrate that oxidative stress and apoptosis play crucial roles in ischemic pathogenesis and nicotinamide adenine dinucleotide (NAD + ) treatment attenuates oxidative stress-induced cell death among primary neurons and astrocytes as well as significantly reduce cerebral ischemic injury in rats. We used a spinal cord ischemia injury (SCII) model in rats to verify our hypothesis that NAD + could ameliorate oxidative stress-induced neuronal apoptosis. Adult male rats were subjected to transient spinal cord ischemia for 60min, and different doses of NAD + were administered intraperitoneally immediately after the start of reperfusion. Neurological function was determined by Basso, Beattie, Bresnahan (BBB) scores. The oxidative stress level was assessed by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The degree of apoptosis was analyzed by deoxyuridinetriphosphate nick-end labeling (TUNEL) staining and protein levels of cleaved caspase-3 and AIF (apoptosis inducing factor). The results showed that NAD + at 50 or 100mg/kg significantly decreased the oxidative stress level and neuronal apoptosis in the spinal cord of ischemia-reperfusion rats compared with saline, as accompanied with the decreased oxidative stress, NAD + administration significantly restrained the neuronal apoptosis after ischemia injury while improved the neurological and motor function. These findings suggested that NAD + might protect against spinal cord ischemia-reperfusion via reducing oxidative stress-induced neuronal apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection.

    PubMed

    Chi, Yuling; Sauve, Anthony A

    2013-11-01

    This review focuses upon the biology and metabolism of a trace component in foods called nicotinamide riboside. Nicotinamide riboside is a precursor of nicotinamide adenine dinucleotide (NAD), and is a source of Vitamin B3. Evidence indicates that nicotinamide riboside has unique properties as a Vitamin B3. We review knowledge of the metabolism of this substance, as well as recent work suggesting novel health benefits that might be associated with nicotinamide riboside taken in larger quantities than is found naturally in foods. Recent work investigating the effects of nicotinamide riboside in yeast and mammals established that it is metabolized by at least two types of metabolic pathways. The first of these is degradative and produces nicotinamide. The second pathway involves kinases called nicotinamide riboside kinases (Nrk1 and Nrk2, in humans). The likely involvement of the kinase pathway is implicated in the unique effects of nicotinamide riboside in raising tissue NAD concentrations in rodents and for potent effects in eliciting insulin sensitivity, mitochondrial biogenesis, and enhancement of sirtuin functions. Additional studies with nicotinamide riboside in models of Alzheimer's disease indicate bioavailability to brain and protective effects, likely by stimulation of brain NAD synthesis. Initial studies have clarified the potential for a lesser-known Vitamin B3 called nicotinamide riboside that is available in selected foods, and possibly available to humans by supplements. It has properties that are insulin sensitizing, enhancing to exercise, resisting to negative effects of high-fat diet, and neuroprotecting.

  18. The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

    PubMed

    Jadhao, Arun G; Biswas, Saikat P; Bhoyar, Rahul C; Pinelli, Claudia

    2017-04-01

    Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

    PubMed

    Bogan, Katrina L; Brenner, Charles

    2008-01-01

    Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research.

  20. Effects of Nicotinamide Adenine Dinucleotide (NAD(+)) and Diadenosine Tetraphosphate (Ap4A) on Electrical Activity of Working and Pacemaker Atrial Myocardium in Guinea Pigs.

    PubMed

    Pustovit, K B; Abramochkin, D V

    2016-04-01

    Effects of nucleotide polyphosphate compounds (nicotinamide adenine dinucleotide, NAD(+); diadenosine tetraphosphate, Ap4A) on the confi guration of action potentials were studied in isolated preparations of guinea pig sinoatrial node and right atrial appendage (auricle). In the working myocardium, NAD(+) and Ap4A in concentrations of 10(-5) and 10(-4) M had no effect on resting potential, but significantly reduced the duration of action potentials; the most pronounced decrease was found at 25% repolarization. In the primary pacemaker of the sinoatrial node, both concentrations of NAD(+) and Ap4A induced hyperpolarization and reduction in the rate of slow diastolic depolarization, but significant slowing of the sinus rhythm was produced by these substances only in the concentration of 10(-4) M. Moreover, AP shortening and marked acceleration of AP upstroke were observed in the pacemaker myocardium after application of polyphosphates. Comparative analysis of the effects of NAD(+) and Ap4A in the working and pacemaker myocardium drove us to a hypothesis on inhibitory effects of these substances on L-type calcium current accompanied by stimulation of one or several potassium currents, which induce enhancement of repolarization and hyperpolarization of membranes probably mediated by the activation of purine receptors.

  1. Rho-kinase inhibitor and nicotinamide adenine dinucleotide phosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats.

    PubMed

    Iida, Hiroki; Iida, Mami; Takenaka, Motoyasu; Fukuoka, Naokazu; Dohi, Shuji

    2008-06-01

    We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

  2. Meat Intake and the Dose of Vitamin B3 – Nicotinamide: Cause of the Causes of Disease Transitions, Health Divides, and Health Futures?

    PubMed Central

    Hill, Lisa J; Williams, Adrian C

    2017-01-01

    Meat and vitamin B3 – nicotinamide – intake was high during hunter-gatherer times. Intake then fell and variances increased during and after the Neolithic agricultural revolution. Health, height, and IQ deteriorated. Low dietary doses are buffered by ‘welcoming’ gut symbionts and tuberculosis that can supply nicotinamide, but this co-evolved homeostatic metagenomic strategy risks dysbioses and impaired resistance to pathogens. Vitamin B3 deficiency may now be common among the poor billions on a low-meat diet. Disease transitions to non-communicable inflammatory disorders (but longer lives) may be driven by positive ‘meat transitions’. High doses of nicotinamide lead to reduced regulatory T cells and immune intolerance. Loss of no longer needed symbiotic ‘old friends’ compounds immunological over-reactivity to cause allergic and auto-immune diseases. Inhibition of nicotinamide adenine dinucleotide consumers and loss of methyl groups or production of toxins may cause cancers, metabolic toxicity, or neurodegeneration. An optimal dosage of vitamin B3 could lead to better health, but such a preventive approach needs more equitable meat distribution. Some people may require personalised doses depending on genetic make-up or, temporarily, when under stress. PMID:28579801

  3. The Emergence of the Nicotinamide Riboside Kinases in the regulation of NAD+ Metabolism.

    PubMed

    Fletcher, Rachel S; Lavery, Gareth

    2018-05-30

    The concept of replenishing or elevating nicotinamide adenine dinucleotide (NAD+) availability to combat metabolic disease and ageing (described extensively in recent reviews [1, 2]) is an area of intense research. This has led to a need to define the endogenous regulatory pathways and mechanisms cell and tissues utilise to maximise NAD+ availability such that strategies to intervene in the clinical setting are able to be fully realised. This review discusses the importance of different salvage pathways involved in metabolising the vitamin B3 class of NAD+ precursor molecules, with a particular focus on the recently identified nicotinamide riboside kinase (NRK) pathway at both a tissue-specific and systemic level.

  4. A Single Primary Blast-Induced Traumatic Brain Injury in a Rodent Model Causes Cell-Type Dependent Increase in Nicotinamide Adenine Dinucleotide Phosphate Oxidase Isoforms in Vulnerable Brain Regions.

    PubMed

    Rama Rao, Kakulavarapu V; Iring, Stephanie; Younger, Daniel; Kuriakose, Matthew; Skotak, Maciej; Alay, Eren; Gupta, Raj K; Chandra, Namas

    2018-06-12

    Blast-induced traumatic brain injury (bTBI) is a leading cause of morbidity in soldiers on the battlefield and in training sites with long-term neurological and psychological pathologies. Previous studies from our laboratory demonstrated activation of oxidative stress pathways after blast injury, but their distribution among different brain regions and their impact on the pathogenesis of bTBI have not been explored. The present study examined the protein expression of two isoforms: nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 and 2 (NOX1, NOX2), corresponding superoxide production, a downstream event of NOX activation, and the extent of lipid peroxidation adducts of 4-hydroxynonenal (4HNE) to a range of proteins. Brain injury was evaluated 4 h after the shock-wave exposure, and immunofluorescence signal quantification was performed in different brain regions. Expression of NOX isoforms displayed a differential increase in various brain regions: in hippocampus and thalamus, there was the highest increase of NOX1, whereas in the frontal cortex, there was the highest increase of NOX2 expression. Cell-specific analysis of changes in NOX expression with respect to corresponding controls revealed that blast resulted in a higher increase of NOX1 and NOX 2 levels in neurons compared with astrocytes and microglia. Blast exposure also resulted in increased superoxide levels in different brain regions, and such changes were reflected in 4HNE protein adduct formation. Collectively, this study demonstrates that primary blast TBI induces upregulation of NADPH oxidase isoforms in different regions of the brain parenchyma and that neurons appear to be at higher risk for oxidative damage compared with other neural cells.

  5. [Effect of flavin adenine dinucleotide on ultraviolet B induced damage in cultured human corneal epithelial cells].

    PubMed

    Sakamoto, Asuka; Nakamura, Masatsugu

    2012-01-01

    This study evaluated the effects of flavin adenine dinucleotide (FAD) on ultraviolet B (UV-B)-induced damage in cultured human corneal epithelial (HCE-T) cells. The cultured HCE-T cells were treated with 0.003125-0.05% FAD before exposure to 80 mJ/cm2 UV-B. Cell viability was measured 24 h after UV-B irradiation using the MTS assay. Reactive oxygen species (ROS) were detected 30 min after UV-B irradiation using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Apoptosis was evaluated 4 h after UV-B irradiation in the caspase-3/7 activity assay. UV-B irradiation reduced cell viability and stimulated ROS production and caspase-3/7 activity in HCE-T cells. Pretreatment of UV-B irradiated HCE-T cells with FAD significantly attenuated cell viability reduction and inhibited the stimulation of both ROS production and caspase-3/7 activity due to UV-B exposure compared with those with vehicle (0% FAD). These results clarified that FAD inhibits ROS-mediated apoptosis by UV-B irradiation in HCE-T cells and suggest that FAD may be effective as a radical scavenger in UV-B-induced corneal damage.

  6. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

    PubMed Central

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

    2015-01-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

  7. Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

    PubMed

    Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

    2007-12-27

    A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells.

  8. The Influence of Nicotinamide on Health and Disease in the Central Nervous System

    PubMed Central

    Fricker, Rosemary A; Green, Emma L; Jenkins, Stuart I; Griffin, Síle M

    2018-01-01

    Nicotinamide, the amide form of vitamin B3 (niacin), has long been associated with neuronal development, survival, and function in the central nervous system (CNS), being implicated in both neuronal death and neuroprotection. Here, we summarise a body of research investigating the role of nicotinamide in neuronal health within the CNS, with a focus on studies that have shown a neuroprotective effect. Nicotinamide appears to play a role in protecting neurons from traumatic injury, ischaemia, and stroke, as well as being implicated in 3 key neurodegenerative conditions: Alzheimer’s, Parkinson’s, and Huntington’s diseases. A key factor is the bioavailability of nicotinamide, with low concentrations leading to neurological deficits and dementia and high levels potentially causing neurotoxicity. Finally, nicotinamide’s potential mechanisms of action are discussed, including the general maintenance of cellular energy levels and the more specific inhibition of molecules such as the nicotinamide adenine dinucleotide-dependent deacetylase, sirtuin 1 (SIRT1). PMID:29844677

  9. Salicylate Treatment Improves Age-Associated Vascular Endothelial Dysfunction: Potential Role of Nuclear Factor κB and Forkhead Box O Phosphorylation

    PubMed Central

    Durrant, Jessica R.; Connell, Melanie L.; Folian, Brian J.; Donato, Anthony J.; Seals, Douglas R.

    2011-01-01

    We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased expression/activity of nicotinamide adenine dinucleotide phosphate oxidase and lower manganese superoxide dismutase expression. Salicylate lowered IKK phosphorylation and reversed age-associated changes in nitrotyrosine, endothelium-dependent dilation, NO bioavailability, endothelial NO synthase, nuclear factor kappa B and forkhead BoxO3a phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase, and manganese superoxide dismutase. Increased activation of IKK with advancing age stimulates nuclear factor kappa B and inactivates forkhead BoxO3a. This altered transcription factor activation contributes to a pro-inflammatory/pro-oxidative arterial phenotype that is characterized by increased cytokines and nicotinamide adenine dinucleotide phosphate oxidase and decreased manganese superoxide dismutase leading to oxidative stress-mediated endothelial dysfunction. PMID:21303813

  10. PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae.

    PubMed

    Sauer, Elizabeta; Merdanovic, Melisa; Mortimer, Anne Price; Bringmann, Gerhard; Reidl, Joachim

    2004-12-01

    The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.

  11. Meat and Nicotinamide: A Causal Role in Human Evolution, History, and Demographics

    PubMed Central

    Williams, Adrian C; Hill, Lisa J

    2017-01-01

    Hunting for meat was a critical step in all animal and human evolution. A key brain-trophic element in meat is vitamin B3 / nicotinamide. The supply of meat and nicotinamide steadily increased from the Cambrian origin of animal predators ratcheting ever larger brains. This culminated in the 3-million-year evolution of Homo sapiens and our overall demographic success. We view human evolution, recent history, and agricultural and demographic transitions in the light of meat and nicotinamide intake. A biochemical and immunological switch is highlighted that affects fertility in the ‘de novo’ tryptophan-to-kynurenine-nicotinamide ‘immune tolerance’ pathway. Longevity relates to nicotinamide adenine dinucleotide consumer pathways. High meat intake correlates with moderate fertility, high intelligence, good health, and longevity with consequent population stability, whereas low meat/high cereal intake (short of starvation) correlates with high fertility, disease, and population booms and busts. Too high a meat intake and fertility falls below replacement levels. Reducing variances in meat consumption might help stabilise population growth and improve human capital. PMID:28579800

  12. Navigating novel mechanisms of cellular plasticity with the NAD+ precursor and nutrient nicotinamide.

    PubMed

    Li, Faqi; Chong, Zhao Zhong; Maiese, Kenneth

    2004-09-01

    Interest in neuroprotectants for the central nervous system continues to garner significant attention. Nicotinamide, the amide form of niacin (vitamin B3), is the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD+) and is considered to be necessary for cellular function and metabolism. However, recent work has focused on the development of nicotinamide as a novel agent that is critical for modulating cellular plasticity, longevity, and inflammatory microglial function. The ability of nicotinamide to preserve both neuronal and vascular cell populations in the brain during injury is intriguing, but further knowledge of the specific cellular mechanisms that determine protection by this agent is required. The capacity of nicotinamide to govern not only intrinsic cellular integrity, but also extrinsic cellular inflammation rests with the modulation of a host of cellular targets that involve protein kinase B, glycogen synthase kinase-3 beta (GSK-3 beta), Forkhead transcription factors, mitochondrial dysfunction, poly(ADP-ribose) polymerase, cysteine proteases, and microglial activation. Intimately tied to the cytoprotection of nicotinamide is the modulation of an early and late phase of apoptotic injury that is triggered by the loss of membrane asymmetry. Identifying robust cytoprotective agents as nicotinamide in conjunction with the elucidation of the cellular mechanisms responsible for cell survival will continue to solidify the development of therapeutic strategies against neurodegenerative diseases

  13. Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells.

    PubMed

    Fletcher, Rachel S; Ratajczak, Joanna; Doig, Craig L; Oakey, Lucy A; Callingham, Rebecca; Da Silva Xavier, Gabriella; Garten, Antje; Elhassan, Yasir S; Redpath, Philip; Migaud, Marie E; Philp, Andrew; Brenner, Charles; Canto, Carles; Lavery, Gareth G

    2017-08-01

    Augmenting nicotinamide adenine dinucleotide (NAD + ) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD + precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD + . Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD + from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis. We exploited expression profiling of muscle NAD + biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD + recycling to evaluate NMN and NR utilization. Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD + . NAMPT inhibition depletes muscle NAD + availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD + in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD + . Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD + availability. These results identify skeletal muscle cells as requiring NAMPT to maintain NAD + availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD + availability.

  14. Application of a coupled enzyme assay to characterize nicotinamide riboside kinases.

    PubMed

    Dölle, Christian; Ziegler, Mathias

    2009-02-15

    The recently identified nicotinamide riboside kinases (Nrks) constitute a distinct pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. Here we present the combination of an established optical adenosine triphosphatase (ATPase) test, the pyruvate kinase/lactate dehydrogenase system, with the Nrk-catalyzed reaction to determine kinetic properties of these enzymes, in particular affinities for ATP. The assay allows variation of both nucleoside and phosphate donor substrates, thereby providing major advantages for the characterization of these enzymes. We confirm previously established kinetic parameters and identify differences in substrate selectivity between the two human Nrk isoforms. The proposed assay is inexpensive and may be applied for high-throughput screening.

  15. Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease.

    PubMed

    Yao, Zhiwen; Yang, Wenhao; Gao, Zhiqiang; Jia, Peng

    2017-04-24

    Amyloid-β (Aβ) oligomers have been accepted as major neurotoxic agents in the therapy of Alzheimer's disease (AD). It has been shown that the activity of nicotinamide adenine dinucleotide (NAD+) is related with the decline of Aβ toxicity in AD. Nicotinamide mononucleotide (NMN), the important precursor of NAD+, is produced during the reaction of nicotinamide phosphoribosyl transferase (Nampt). This study aimed to figure out the potential therapeutic effects of NMN and its underlying mechanisms in APPswe/PS1dE9 (AD-Tg) mice. We found that NMN gave rise to a substantial improvement in behavioral measures of cognitive impairments compared to control AD-Tg mice. In addition, NMN treatment significantly decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in transgenic animals. Mechanistically, NMN effectively controlled JNK activation. Furthermore, NMN potently progressed nonamyloidogenic amyloid precursor protein (APP) and suppressed amyloidogenic APP by mediating the expression of APP cleavage secretase in AD-Tg mice. Based on our findings, it was suggested that NMN substantially decreases multiple AD-associated pathological characteristically at least partially by the inhibition of JNK activation. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Distinctive Spectral Features of Exciton and Excimer States in the Ultrafast Electronic Deactivation of the Adenine Dinucleotide

    NASA Astrophysics Data System (ADS)

    Stuhldreier, Mayra C.; Röttger, Katharina; Temps, Friedrich

    We report the observation by transient absorption spectroscopy of distinctive spectro-temporal signatures of delocalized exciton versus relaxed, weakly bound excimer states in the ultrafast electronic deactivation after UV photoexcitation of the adenine dinucleotide.

  17. Nicotinamide riboside kinase structures reveal new pathways to NAD+.

    PubMed

    Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

    2007-10-02

    The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

  18. Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy.

    PubMed

    Diguet, Nicolas; Trammell, Samuel A J; Tannous, Cynthia; Deloux, Robin; Piquereau, Jérôme; Mougenot, Nathalie; Gouge, Anne; Gressette, Mélanie; Manoury, Boris; Blanc, Jocelyne; Breton, Marie; Decaux, Jean-François; Lavery, Gareth G; Baczkó, István; Zoll, Joffrey; Garnier, Anne; Li, Zhenlin; Brenner, Charles; Mericskay, Mathias

    2018-05-22

    Myocardial metabolic impairment is a major feature in chronic heart failure. As the major coenzyme in fuel oxidation and oxidative phosphorylation and a substrate for enzymes signaling energy stress and oxidative stress response, nicotinamide adenine dinucleotide (NAD + ) is emerging as a metabolic target in a number of diseases including heart failure. Little is known on the mechanisms regulating homeostasis of NAD + in the failing heart. To explore possible alterations of NAD + homeostasis in the failing heart, we quantified the expression of NAD + biosynthetic enzymes in the human failing heart and in the heart of a mouse model of dilated cardiomyopathy (DCM) triggered by Serum Response Factor transcription factor depletion in the heart (SRF HKO ) or of cardiac hypertrophy triggered by transverse aorta constriction. We studied the impact of NAD + precursor supplementation on cardiac function in both mouse models. We observed a 30% loss in levels of NAD + in the murine failing heart of both DCM and transverse aorta constriction mice that was accompanied by a decrease in expression of the nicotinamide phosphoribosyltransferase enzyme that recycles the nicotinamide precursor, whereas the nicotinamide riboside kinase 2 (NMRK2) that phosphorylates the nicotinamide riboside precursor is increased, to a higher level in the DCM (40-fold) than in transverse aorta constriction (4-fold). This shift was also observed in human failing heart biopsies in comparison with nonfailing controls. We show that the Nmrk2 gene is an AMP-activated protein kinase and peroxisome proliferator-activated receptor α responsive gene that is activated by energy stress and NAD + depletion in isolated rat cardiomyocytes. Nicotinamide riboside efficiently rescues NAD + synthesis in response to FK866-mediated inhibition of nicotinamide phosphoribosyltransferase and stimulates glycolysis in cardiomyocytes. Accordingly, we show that nicotinamide riboside supplementation in food attenuates the

  19. Selectivity and activity of adenine dinucleotides at recombinant P2X2 and P2Y1 purinoceptors.

    PubMed Central

    Pintor, J.; King, B. F.; Miras-Portugal, M. T.; Burnstock, G.

    1996-01-01

    1. Adenine dinucleotides (Ap3A, x = 2-6) are naturally-occurring polyphosphated nucleotidic substances which are found in the CNS and are known to be released in a calcium-dependent manner from storage vesicles in brain synaptosomes. The selectivity and activity of adenine dinucleotides for neuronally-derived recombinant P2 purinoceptors were studied using P2X2 and P2Y1 subtypes expressed in Xenopus oocytes. 2. For the P2Y1 subtype derived from chick brain, Ap3A was equipotent and as active as ATP (EC50 values: 375 +/- 86 nM and 334 +/- 25 nM, respectively). Ap4A was a weak partial agonist and other dinucleotides were inactive as agonists. None of the inactive dinucleotides were antagonists nor modulated the activity of Ap3A and ATP. 3. For the P2X2 subtype derived from rat PC12 cells, Ap4A was as active as ATP but less potent (EC50 values: 15.2 +/- 1 microM and 3.7 +/- 0.7 microM, respectively). Other adenosine dinucleotides were inactive as either agonists or antagonists. 4. Ap5A (1-100 nM) potentiated ATP-responses at the P2X2 subtype, showing an EC50 of 2.95 +/- 0.7 nM for this modulatory effect. Ap5A (10 nM) shifted the concentration-response curves for ATP to the left by one-half log10 unit but did not alter the Hill co-efficient for ATP (nH = 2.1 +/- 0.1). Ap5A (10 nM) failed to potentiate Ap4A-responses but did enhance the efficacy of the P2 purinoceptor antagonist, suramin, by 12 fold at the P2X2 subtype. 5. In conclusion, the results show that ionotropic (P2X2) and metabotropic (P2Y1) ATP receptors which occur in the CNS are activated selectively by naturally-occurring adenine dinucleotides which are known to be released with nucleotides from storage vesicles. The observed potentiation of P2X2-responses by Ap5A, where co-released with ATP by brain synaptosomes, may have a functional bearing in purinergic signalling in the CNS. PMID:8922753

  20. In vitro evaluation of nicotinamide riboside analogs against Haemophilus influenzae.

    PubMed

    Godek, C P; Cynamon, M H

    1990-08-01

    Exogenous NAD, nicotinamide mononucleotide, or nicotinamide riboside is required for the growth of Haemophilus influenzae. These compounds have been defined as the V-factor growth requirement. We have previously shown that the internalization of nicotinamide riboside is energy dependent and carrier mediated with saturation kinetics. Thionicotinamide riboside, 3-pyridinealdehyde riboside, 3-acetylpyridine riboside, and 3-aminopyridine riboside were prepared from their corresponding NAD analogs. These compounds and several other nicotinamide riboside analogs were evaluated for their ability to support the growth of H. influenzae and for their ability to block the uptake of [carbonyl-14C]nicotinamide riboside by H. influenzae. 3-Aminopyridine riboside blocked the uptake of [carbonyl-14C]nicotinamide riboside and inhibited the growth of H. influenzae when NAD, nicotinamide mononucleotide, or nicotinamide riboside served as the V factor. The antibacterial activity of 3-aminopyridine riboside was found to be specific for H. influenzae but had no effect on the growth of Staphylococcus aureus or Escherichia coli. In additional experiments by reversed-phase high-performance liquid chromatography, it was determined that whole cells of H. influenzae degrade 3-aminopyridine adenine dinucleotide to 3-aminopyridine riboside, which is then internalized. Inside the cell, 3-aminopyridine riboside has the ability to interfere with the growth of H. influenzae by an undetermined mechanism.

  1. The NAD(+) precursor nicotinamide riboside decreases exercise performance in rats.

    PubMed

    Kourtzidis, Ioannis A; Stoupas, Andreas T; Gioris, Ioannis S; Veskoukis, Aristidis S; Margaritelis, Nikos V; Tsantarliotou, Maria; Taitzoglou, Ioannis; Vrabas, Ioannis S; Paschalis, Vassilis; Kyparos, Antonios; Nikolaidis, Michalis G

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) and its phosphorylated form (NADP(+)) are key molecules in ubiquitous bioenergetic and cellular signaling pathways, regulating cellular metabolism and homeostasis. Thus, supplementation with NAD(+) and NADP(+) precursors emerged as a promising strategy to gain many and multifaceted health benefits. In this proof-of-concept study, we sought to investigate whether chronic nicotinamide riboside administration (an NAD(+) precursor) affects exercise performance. Eighteen Wistar rats were equally divided in two groups that received either saline vehicle or nicotinamide riboside at a dose of 300 mg/kg body weight/day for 21 days via gavage. At the end of the 21-day administration protocol, both groups performed an incremental swimming performance test. The nicotinamide riboside group showed a tendency towards worse physical performance by 35 % compared to the control group at the final 10 % load (94 ± 53 s for the nicotinamide riboside group and 145 ± 59 s for the control group; P = 0.071). Our results do not confirm the previously reported ergogenic effect of nicotinamide riboside. The potentially negative effect of nicotinamide riboside administration on physical performance may be attributed to the pleiotropic metabolic and redox properties of NAD(+) and NADP(+).

  2. In vitro evaluation of nicotinamide riboside analogs against Haemophilus influenzae.

    PubMed Central

    Godek, C P; Cynamon, M H

    1990-01-01

    Exogenous NAD, nicotinamide mononucleotide, or nicotinamide riboside is required for the growth of Haemophilus influenzae. These compounds have been defined as the V-factor growth requirement. We have previously shown that the internalization of nicotinamide riboside is energy dependent and carrier mediated with saturation kinetics. Thionicotinamide riboside, 3-pyridinealdehyde riboside, 3-acetylpyridine riboside, and 3-aminopyridine riboside were prepared from their corresponding NAD analogs. These compounds and several other nicotinamide riboside analogs were evaluated for their ability to support the growth of H. influenzae and for their ability to block the uptake of [carbonyl-14C]nicotinamide riboside by H. influenzae. 3-Aminopyridine riboside blocked the uptake of [carbonyl-14C]nicotinamide riboside and inhibited the growth of H. influenzae when NAD, nicotinamide mononucleotide, or nicotinamide riboside served as the V factor. The antibacterial activity of 3-aminopyridine riboside was found to be specific for H. influenzae but had no effect on the growth of Staphylococcus aureus or Escherichia coli. In additional experiments by reversed-phase high-performance liquid chromatography, it was determined that whole cells of H. influenzae degrade 3-aminopyridine adenine dinucleotide to 3-aminopyridine riboside, which is then internalized. Inside the cell, 3-aminopyridine riboside has the ability to interfere with the growth of H. influenzae by an undetermined mechanism. Images PMID:2145800

  3. African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

    PubMed Central

    Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

    2006-01-01

    Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584

  4. African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

    PubMed

    Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

    2006-04-01

    Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

  5. Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light

    NASA Astrophysics Data System (ADS)

    Emmanuel, Megan A.; Greenberg, Norman R.; Oblinsky, Daniel G.; Hyster, Todd K.

    2016-12-01

    Enzymes are ideal for use in asymmetric catalysis by the chemical industry, because their chemical compositions can be tailored to a specific substrate and selectivity pattern while providing efficiencies and selectivities that surpass those of classical synthetic methods. However, enzymes are limited to reactions that are found in nature and, as such, facilitate fewer types of transformation than do other forms of catalysis. Thus, a longstanding challenge in the field of biologically mediated catalysis has been to develop enzymes with new catalytic functions. Here we describe a method for achieving catalytic promiscuity that uses the photoexcited state of nicotinamide co-factors (molecules that assist enzyme-mediated catalysis). Under irradiation with visible light, the nicotinamide-dependent enzyme known as ketoreductase can be transformed from a carbonyl reductase into an initiator of radical species and a chiral source of hydrogen atoms. We demonstrate this new reactivity through a highly enantioselective radical dehalogenation of lactones—a challenging transformation for small-molecule catalysts. Mechanistic experiments support the theory that a radical species acts as an intermediate in this reaction, with NADH and NADPH (the reduced forms of nicotinamide adenine nucleotide and nicotinamide adenine dinucleotide phosphate, respectively) serving as both a photoreductant and the source of hydrogen atoms. To our knowledge, this method represents the first example of photo-induced enzyme promiscuity, and highlights the potential for accessing new reactivity from existing enzymes simply by using the excited states of common biological co-factors. This represents a departure from existing light-driven biocatalytic techniques, which are typically explored in the context of co-factor regeneration.

  6. Nicotinamide: a vitamin able to shift macrophage differentiation toward macrophages with restricted inflammatory features.

    PubMed

    Weiss, Ronald; Schilling, Erik; Grahnert, Anja; Kölling, Valeen; Dorow, Juliane; Ceglarek, Uta; Sack, Ulrich; Hauschildt, Sunna

    2015-11-01

    The differentiation of human monocytes into macrophages is influenced by environmental signals. Here we asked in how far nicotinamide (NAM), a vitamin B3 derivative known to play a major role in nicotinamide adenine dinucleotide (NAD)-mediated signaling events, is able to modulate monocyte differentiation into macrophages developed in the presence of granulocyte macrophage colony-stimulating factor (GM-MØ) or macrophage colony-stimulating factor (M-MØ). We found that GM-MØ undergo biochemical, morphological and functional modifications in response to NAM, whereas M-MØ were hardly affected. GM-MØ exposed to NAM acquired an M-MØ-like structure while the LPS-induced production of pro-inflammatory cytokines and COX-derived eicosanoids were down-regulated. In contrast, NAM had no effect on the production of IL-10 or the cytochrome P450-derived eicosanoids. Administration of NAM enhanced intracellular NAD concentrations; however, it did not prevent the LPS-mediated drain on NAD pools. In search of intracellular molecular targets of NAM known to be involved in LPS-induced cytokine and eicosanoid synthesis, we found NF-κB activity to be diminished. In conclusion, our data show that vitamin B3, when present during the differentiation of monocytes into GM-MØ, interferes with biochemical pathways resulting in strongly reduced pro-inflammatory features. © The Author(s) 2015.

  7. Glaucoma as a Metabolic Optic Neuropathy: Making the Case for Nicotinamide Treatment in Glaucoma.

    PubMed

    Williams, Pete A; Harder, Jeffrey M; John, Simon W M

    2017-12-01

    Mitochondrial dysfunction may be an important, if not essential, component of human glaucoma. Using transcriptomics followed by molecular and neurobiological techniques, we have recently demonstrated that mitochondrial dysfunction within retinal ganglion cells is an early feature in the DBA/2J mouse model of inherited glaucoma. Guided by these findings, we discovered that the retinal level of nicotinamide adenine dinucleotide (NAD, a key molecule for mitochondrial health) declines in an age-dependent manner. We hypothesized that this decline in NAD renders retinal ganglion cells susceptible to damage during periods of elevated intraocular pressure. To replete NAD levels in this glaucoma, we administered nicotinamide (the amide of vitamin B3). At the lowest dose tested, nicotinamide robustly protected from glaucoma (~70% of eyes had no detectable glaucomatous neurodegeneration). At this dose, nicotinamide had no influence on intraocular pressure and so its effect was neuroprotective. At the highest dose tested, 93% of eyes had no detectable glaucoma. This represents a ~10-fold decrease in the risk of developing glaucoma. At this dose, intraocular pressure still became elevated but there was a reduction in the degree of elevation showing an additional benefit. Thus, nicotinamide is unexpectedly potent at preventing this glaucoma and is an attractive option for glaucoma therapeutics. Our findings demonstrate the promise for both preventing and treating glaucoma by interventions that bolster metabolism during increasing age and during periods of elevated intraocular pressure. Nicotinamide prevents age-related declines in NAD (a decline that occurs in different genetic contexts and species). NAD precursors are reported to protect from a variety of neurodegenerative conditions. Thus, nicotinamide may provide a much needed neuroprotective treatment against human glaucoma. This manuscript summarizes human data implicating mitochondria in glaucoma, and argues for studies to

  8. Metabolomics Analysis of Metabolic Effects of Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibition on Human Cancer Cells

    PubMed Central

    Tolstikov, Vladimir; Nikolayev, Alexander; Dong, Sucai; Zhao, Genshi; Kuo, Ming-Shang

    2014-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide–consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry–based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level. PMID:25486521

  9. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans.

    PubMed

    Trammell, Samuel A J; Schmidt, Mark S; Weidemann, Benjamin J; Redpath, Philip; Jaksch, Frank; Dellinger, Ryan W; Li, Zhonggang; Abel, E Dale; Migaud, Marie E; Brenner, Charles

    2016-10-10

    Nicotinamide riboside (NR) is in wide use as an NAD + precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD + metabolism in humans. We report that human blood NAD + can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD + with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD + metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD + , is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD + repletion.

  10. Big brains, meat, tuberculosis and the nicotinamide switches: co-evolutionary relationships with modern repercussions on longevity and disease?

    PubMed

    Williams, Adrian C; Dunbar, Robin I M

    2014-07-01

    Meat eating has been an important trigger for human evolution however the responsible component in meat has not been clearly identified. Here we propose that the limiting factors for expanding brains and increasing longevity were the micronutrient nicotinamide (vitamin B3) and the metabolically related essential amino-acid, tryptophan. Meat offers significant sourcing challenges and lack causes a deficiency of nicotinamide and tryptophan and consequently the energy carrier nicotinamide adenine dinucleotide (NAD) that gets consumed in regulatory circuits important for survival, resulting in premature ageing, poor cognition and brain atrophy. If a trophic supply of dietary nicotinamide/tryptophan is so essential for building brains, constraining their size and connectivity, we hypothesise that back-up mechanisms to ensure the supply evolved. One strategy may be increasing the reliance on gut symbionts to break down celluloses that produces NADH and only nicotinamide indirectly, and may cause diarrhoea. We suggest that a direct supplier was the chronic mycobacterial infection tuberculosis (TB) that is a surprise candidate but it co-evolved early, does not inevitably cause disease (90-95% of those infected are healthy), and secretes (and is inhibited by) nicotinamide. We hypothesise that TB evolved first as a symbiont that enabled humans to cope with short-lived shortages of meat and only later behaved as a pathogen when the supply deteriorated chronically, for those in poverty. (TB immunology and epidemiology is riddled with paradoxes for a conventional pathogen). We test this in pilot data showing that sharp declines in TB (and diarrhoea) - `environmental enteropathy' strongly correlate with increasing meat consumption and therefore nicotinamide exposure, unlike later onset cancers and Parkinson's disease that increased in incidence, perhaps - as we propose a hypothetical hypervitaminosis B3 (to include obesity and the metabolic syndrome) - as the trade-off for

  11. Relationships between laser powers and photoacoustic signal intensities of flavin adenine dinucleotide and beta-carotene dissolved in solutions

    NASA Astrophysics Data System (ADS)

    Imakubo, Keiichi

    1994-10-01

    Ar ion laser-induced photoacoustic spectroscopy has been performed on 0.01 mu M flavin adenine dinucleotide in H2O and 0.01 mu M beta-carotene in n-hexane where the optical absorption spectroscopy is not applicable. On the basis of the linear relationships between laser powers and photoacoustic signal intensities up to 500 mW, it may be concluded that laser power ranging from 10 to 50 mW is required for the successful observation of photoacoustic signals without any photochemical or photobiological effects.

  12. Nicotinamide pre-treatment ameliorates NAD(H) hyperoxidation and improves neuronal function after severe hypoxia

    PubMed Central

    Shetty, Pavan K; Galeffi, Francesca; Turner, Dennis A.

    2014-01-01

    Prolonged hypoxia leads to irreversible loss of neuronal function and metabolic impairment of nicotinamide adenine dinucleotide recycling (between NAD+ and NADH) immediately after reoxygenation, resulting in NADH hyperoxidation. We test whether addition of nicotinamide (to enhance NAD+ levels) or PARP-1 inhibition (to prevent consumption of NAD+) can be effective in improving either loss of neuronal function or hyperoxidation following severe hypoxic injury in hippocampal slices. After severe, prolonged hypoxia (maintained for 3 min after spreading depression) there was hyperoxidation of NADH following reoxygenation, an increased soluble NAD+/NADH ratio, loss of neuronal field excitatory post-synaptic potential (fEPSP) and decreased ATP content. Nicotinamide incubation (5 mM) 2 hr prior to hypoxia significantly increased total NAD(H) content, improved neuronal recovery, enhanced ATP content, and prevented NADH hyperoxidation. The nicotinamide-induced increase in total soluble NAD(H) was more significant in the cytosolic compartment than within mitochondria. Prolonged incubation with PJ-34 (>1hr) led to enhanced baseline NADH fluorescence prior to hypoxia, as well as improved neuronal recovery, NADH hyperoxidation and ATP content on recovery from severe hypoxia and reoxygenation. In this acute model of severe neuronal dysfunction prolonged incubation with either nicotinamide or PJ-34 prior to hypoxia improved recovery of neuronal function, enhanced NADH reduction and ATP content, but neither treatment restored function when administered during or after prolonged hypoxia and reoxygenation. PMID:24184921

  13. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F., E-mail: merrillg@onid.orst.ed

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involvedmore » in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.« less

  14. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

    PubMed

    Long, C M; Rohrmann, G F; Merrill, G F

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  15. Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults.

    PubMed

    Martens, Christopher R; Denman, Blair A; Mazzo, Melissa R; Armstrong, Michael L; Reisdorph, Nichole; McQueen, Matthew B; Chonchol, Michel; Seals, Douglas R

    2018-03-29

    Nicotinamide adenine dinucleotide (NAD + ) has emerged as a critical co-substrate for enzymes involved in the beneficial effects of regular calorie restriction on healthspan. As such, the use of NAD + precursors to augment NAD + bioavailability has been proposed as a strategy for improving cardiovascular and other physiological functions with aging in humans. Here we provide the evidence in a 2 × 6-week randomized, double-blind, placebo-controlled, crossover clinical trial that chronic supplementation with the NAD + precursor vitamin, nicotinamide riboside (NR), is well tolerated and effectively stimulates NAD + metabolism in healthy middle-aged and older adults. Our results also provide initial insight into the effects of chronic NR supplementation on physiological function in humans, and suggest that, in particular, future clinical trials should further assess the potential benefits of NR for reducing blood pressure and arterial stiffness in this group.

  16. Glutamate Dehydrogenase from Apodachlya (Oomycetes) 1

    PubMed Central

    Price, Jeffrey S.; Gleason, Frank H.

    1972-01-01

    A glutamate dehydrogenase specific for nicotinamide-adenine-dinucleotide has been purified 50-fold from Apodachlya brachynema (Leptomitales). Certain physical, chemical, and kinetic properties of this enzyme have been studied, particularly specificity for coenzymes and substrates. With glucose as the sole carbon source, the synthesis of glutamate dehydrogenase was repressed, whereas glutamate, proline, alanine, or ornithine plus aspartate as sole carbon sources induced synthesis of the enzyme. These data indicate that the function of this enzyme is primarily degradative, although there is no evidence for a nicotinamide-adenine-dinucleotide-phosphate-specific biosynthetic glutamate dehydrogenase in Apodachlya. PMID:16657902

  17. Preparation and storage of isotopically labeled reduced nicotinamide adenine dinucleotide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Northrop, D.B.; Duggleby, R.G.

    1987-09-01

    A method for obtaining highly purified NADH in a dry, solid, and stable form is described. The method involves improvements of the ion-exchange and reversed-phase chromatographic procedures of C. J. Newton and S. M. Faynor, and D. B. Northrop. The necessary time to prepare pure NADH has been reduced to a few hours. The final product, obtained by drying the nucleotide from absolute ethanol, shows no detectable decomposition either during the drying procedure or during storage under nitrogen gas at -20 degrees C for several months. Using dry product prepared from fixed volumes of ethanolic solution, standardized solutions of knownmore » amounts of the highly purified and stored NADH can be obtained in a few seconds.« less

  18. Activation of SIRT3 by the NAD⁺ precursor nicotinamide riboside protects from noise-induced hearing loss.

    PubMed

    Brown, Kevin D; Maqsood, Sadia; Huang, Jing-Yi; Pan, Yong; Harkcom, William; Li, Wei; Sauve, Anthony; Verdin, Eric; Jaffrey, Samie R

    2014-12-02

    Intense noise exposure causes hearing loss by inducing degeneration of spiral ganglia neurites that innervate cochlear hair cells. Nicotinamide adenine dinucleotide (NAD(+)) exhibits axon-protective effects in cultured neurons; however, its ability to block degeneration in vivo has been difficult to establish due to its poor cell permeability and serum instability. Here, we describe a strategy to increase cochlear NAD(+) levels in mice by administering nicotinamide riboside (NR), a recently described NAD(+) precursor. We find that administration of NR, even after noise exposure, prevents noise-induced hearing loss (NIHL) and spiral ganglia neurite degeneration. These effects are mediated by the NAD(+)-dependent mitochondrial sirtuin, SIRT3, since SIRT3-overexpressing mice are resistant to NIHL and SIRT3 deletion abrogates the protective effects of NR and expression of NAD(+) biosynthetic enzymes. These findings reveal that administration of NR activates a NAD(+)-SIRT3 pathway that reduces neurite degeneration caused by noise exposure. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Identification of a nicotinamide/nicotinate mononucleotide adenylyltransferase in Giardia lamblia (GlNMNAT).

    PubMed

    Forero-Baena, Nicolás; Sánchez-Lancheros, Diana; Buitrago, July Constanza; Bustos, Victor; Ramírez-Hernández, María Helena

    2015-01-01

    Giardia lamblia is an intestinal protozoan parasite that causes giardiasis, a disease of high prevalence in Latin America, Asia and Africa. Giardiasis leads to poor absorption of nutrients, severe electrolyte loss and growth retardation. In addition to its clinical importance, this parasite is of special biological interest due to its basal evolutionary position and simplified metabolism, which has not been studied thoroughly. One of the most important and conserved metabolic pathways is the biosynthesis of nicotinamide adenine dinucleotide (NAD). This molecule is widely known as a coenzyme in multiple redox reactions and as a substrate in cellular processes such as synthesis of Ca 2+ mobilizing agents, DNA repair and gene expression regulation. There are two pathways for NAD biosynthesis, which converge at the step catalyzed by nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18). Using bioinformatics tools, we found two NMNAT sequences in Giardia lamblia ( glnmnat-a and glnmnat-b ). We first verified the identity of the sequences in silico . Subsequently, glnmnat-a was cloned into an expression vector. The recombinant protein (His-GlNMNAT) was purified by nickel-affinity binding and was used in direct in vitro enzyme assays assessed by C18-HPLC, verifying adenylyltransferase activity with both nicotinamide (NMN) and nicotinic acid (NAMN) mononucleotides. Optimal reaction pH and temperature were 7.3 and 26 °C. Michaelis-Menten kinetics were observed for NMN and ATP, but saturation was not accomplished with NAMN, implying low affinity yet detectable activity with this substrate. Double-reciprocal plots showed no cooperativity for this enzyme. This represents an advance in the study of NAD metabolism in Giardia spp.

  20. Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

    PubMed Central

    Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

    2015-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

  1. Nicotinamide riboside, a form of vitamin B3 and NAD+ precursor, relieves the nociceptive and aversive dimensions of paclitaxel-induced peripheral neuropathy in female rats.

    PubMed

    Hamity, Marta V; White, Stephanie R; Walder, Roxanne Y; Schmidt, Mark S; Brenner, Charles; Hammond, Donna L

    2017-05-01

    Injury to sensory afferents may contribute to the peripheral neuropathies that develop after administration of chemotherapeutic agents. Manipulations that increase levels of nicotinamide adenine dinucleotide (NAD) can protect against neuronal injury. This study examined whether nicotinamide riboside (NR), a third form of vitamin B3 and precursor of NAD, diminishes tactile hypersensitivity and place escape-avoidance behaviors in a rodent model of paclitaxel-induced peripheral neuropathy. Female Sprague-Dawley rats received 3 intravenous injections of 6.6 mg/kg paclitaxel over 5 days. Daily oral administration of 200 mg/kg NR beginning 7 days before paclitaxel treatment and continuing for another 24 days prevented the development of tactile hypersensitivity and blunted place escape-avoidance behaviors. These effects were sustained after a 2-week washout period. This dose of NR increased blood levels of NAD by 50%, did not interfere with the myelosuppressive effects of paclitaxel, and did not produce adverse locomotor effects. Treatment with 200 mg/kg NR for 3 weeks after paclitaxel reversed the well-established tactile hypersensitivity in a subset of rats and blunted escape-avoidance behaviors. Pretreatment with 100 mg/kg oral acetyl-L-carnitine (ALCAR) did not prevent paclitaxel-induced tactile hypersensitivity or blunt escape-avoidance behaviors. ALCAR by itself produced tactile hypersensitivity. These findings suggest that agents that increase NAD, a critical cofactor for mitochondrial oxidative phosphorylation systems and cellular redox systems involved with fuel utilization and energy metabolism, represent a novel therapeutic approach for relief of chemotherapy-induced peripheral neuropathies. Because NR is a vitamin B3 precursor of NAD and a nutritional supplement, clinical tests of this hypothesis may be accelerated.

  2. Absorption and luminescence spectroscopy of mass-selected flavin adenine dinucleotide mono-anions

    NASA Astrophysics Data System (ADS)

    Giacomozzi, L.; Kjær, C.; Langeland Knudsen, J.; Andersen, L. H.; Brøndsted Nielsen, S.; Stockett, M. H.

    2018-06-01

    We report the absorption profile of isolated Flavin Adenine Dinucleotide (FAD) mono-anions recorded using photo-induced dissociation action spectroscopy. In this charge state, one of the phosphoric acid groups is deprotonated and the chromophore itself is in its neutral oxidized state. These measurements cover the first four optical transitions of FAD with excitation energies from 2.3 to 6.0 eV (210-550 nm). The S0 → S2 transition is strongly blue shifted relative to aqueous solution, supporting the view that this transition has a significant charge-transfer character. The remaining bands are close to their solution-phase positions. This confirms that the large discrepancy between quantum chemical calculations of vertical transition energies and solution-phase band maxima cannot be explained by solvent effects. We also report the luminescence spectrum of FAD mono-anions in vacuo. The gas-phase Stokes shift for S1 is 3000 cm-1, which is considerably larger than any previously reported for other molecular ions and consistent with a significant displacement of the ground and excited state potential energy surfaces. Consideration of the vibronic structure is thus essential for simulating the absorption and luminescence spectra of flavins.

  3. Oxidation of ethane by an Acremonium species.

    PubMed Central

    Davies, J S; Wellman, A M; Zajic, J E

    1976-01-01

    Ethane oxidation was studied in ethane-grown resting cells (mycelia) of an Acremonium sp. and in cell-free preparations of such mycelia. From resting cell experiments evidence was found for a pathway of ethane oxidation via ethanol, acetaldehyde, and acetic acid. In vitro studies indicated that ethane-oxidizing activity in such mycelia occurred predominantly in the microsomal fraction of crude homogenates. Microsomal preparations were inactive in the absence of added coenzyme. Marked stimulation of activity was obtained in such preparations with reduced nicotinamide adenine dinucleotide phosphate and to a much lesser degree with nicotinamide adenine dinucleotide phosphate. Ethane oxidation was inhibited by sodium azide and carbon monoxide. PMID:9900

  4. Combined adrenal failure and testicular adrenal rest tumor in a patient with nicotinamide nucleotide transhydrogenase deficiency.

    PubMed

    Hershkovitz, Eli; Arafat, Maram; Loewenthal, Neta; Haim, Alon; Parvari, Ruti

    2015-09-01

    The nicotinamide nucleotide transhydrogenase (NNT) enzyme is the main generator of nicotinamide adenine dinucleotide phosphate-oxidase in the mitochondrion. Mutations of the NNT gene have been recently implicated in familial glucocorticoid deficiency. We describe the long-term clinical course of a NNT-deficient 20-year-old patient with combined adrenal failure who had developed a testicular adrenal rest tumor and precocious puberty. The patient's medical records were reviewed. Whole-exome sequencing was performed on DNA obtained from the patient and family members. The patient experienced Addisonian crisis at 10 months of age. Enlarged testicular volume and precocious puberty, accompanied by increased testosterone levels, were noted at 6 years. Testicular biopsy revealed a adrenal rest tumor, which regressed after intensification of glucocorticoid treatment. Genetic studies disclosed a c.1163A>C, p.Tyr388Ser substitution on the NNT gene. This mutation is predicted to be damaging to NNT function. We demonstrated for the first time that the clinical spectrum of NNT deficiency may consist of mineralocorticoid deficiency and testicular involvement as well.

  5. Eco-synthesis of graphene and its use in dihydronicotinamide adenine dinucleotide sensing.

    PubMed

    Amouzadeh Tabrizi, Mahmoud; Jalilzadeh Azar, Somayeh; Nadali Varkani, Javad

    2014-09-01

    In this paper, we report a green and eco-friendly approach to synthesize reduced graphene oxide (rGO) via a mild hydrothermal process using malt as a reduced agent. The proposed method is based on the reduction of graphene oxide (GO) in malt solution by making use of the reducing capability of phenolic compounds contained in malt solution. The obtained rGO was characterized by atomic force microscopy (AFM), ultraviolet-visible (UV-vis) absorption spectroscopy, X-ray diffraction spectroscopy (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Electrochemical impedance spectroscopy analysis revealed that the charge transfer resistance of rGO modified glassy carbon (GC) electrode was much lower than that of the GC electrode. The electrochemical behavior of dihydronicotinamide adenine dinucleotide (NADH) on rGO modified GC electrode was investigated by cyclic voltammetry and amperometry. Electrochemical experiments indicated that rGO/GC electrode exhibited excellent electrocatalytic activity toward the NADH, which can be attributed to excellent electrical conductivity and high specific surface area of the rGO composite. The resulting biosensor showed highly sensitive amperometric response to NADH with a low detection limit (0.33μM). Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Profiling Redox and Energy Coenzymes in Whole Blood, Tissue and Cells Using NMR Spectroscopy.

    PubMed

    Gowda, G A Nagana

    2018-05-14

    Coenzymes of cellular redox reactions and cellular energy, as well as antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. Conventional analysis methods lack the opportunity to evaluate these important redox and energy coenzymes and antioxidants in a single step. Major coenzymes include redox coenzymes: NAD⁺ (oxidized nicotinamide adenine dinucleotide), NADH (reduced nicotinamide adenine dinucleotide), NADP⁺ (oxidized nicotinamide adenine dinucleotide phosphate) and NADPH (reduced nicotinamide adenine dinucleotide phosphate); energy coenzymes: ATP (adenosine triphosphate), ADP (adenosine diphosphate) and AMP (adenosine monophosphate); and antioxidants: GSSG (oxidized glutathione) and GSH (reduced glutathione). We show here that a simple ¹H NMR experiment can measure these coenzymes and antioxidants in tissue and whole blood apart from a vast pool of other metabolites. In addition, focused on the goal of identification of coenzymes in subcellular fractions, we demonstrate analysis of coenzymes in the cytoplasm using breast cancer cells. Owing to their unstable nature, or low concentrations, most of the coenzymes either evade detection or lose their integrity when established sample preparation and analysis methods are used. To overcome this challenge, here we describe the development of new methods to detect these molecules without affecting the integrity of other metabolites. We used an array of 1D and 2D NMR methods, chemical shift databases, pH measurements and spiking with authentic compounds to establish the identity of peaks for the coenzymes and antioxidants in NMR spectra. Interestingly, while none of the coenzymes and antioxidants were detected in plasma, they were abundant in whole blood. Considering that the coenzymes and antioxidants represent a sensitive measure of human health and risk for numerous diseases, the presented NMR methods to measure them in one step potentially open new opportunities in the

  7. Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae*

    PubMed Central

    Lu, Shu-Ping; Kato, Michiko; Lin, Su-Ju

    2009-01-01

    NAD+ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD+ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD+ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD+-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD+ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD+ precursors. Together, our studies provide a molecular basis for how NAD+ homeostasis factors confer metabolic flexibility. PMID:19416965

  8. Assimilation of endogenous nicotinamide riboside is essential for calorie restriction-mediated life span extension in Saccharomyces cerevisiae.

    PubMed

    Lu, Shu-Ping; Kato, Michiko; Lin, Su-Ju

    2009-06-19

    NAD(+) (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD(+) metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD(+) metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD(+)-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD(+) metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD(+) precursors. Together, our studies provide a molecular basis for how NAD(+) homeostasis factors confer metabolic flexibility.

  9. Biomarkers and Biological Spectral Imaging

    DTIC Science & Technology

    2001-01-23

    Image t iZ ~ Rotator SSteping r De m oo ontroler Frmegabe (single ams) ... Software -= • . ... PCCm ue Data acquisition Rotator control PC Co puterImage...the depth of bum injury", Bums, 7, pp. 197-202, 1981. 7. R. A. De Blasi, M. Cope, C. Elwell, F. Safoue and M. Ferrari, "Noninvasive measurement of...nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD) and porphyrins. A number of studies have shown that the measured

  10. Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weiss, P.M.; Urbauer, J.L.; Cleland, W.W.

    1991-06-11

    Deuterium isotope effects and {sup 13}C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the {sup 13}C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C effect increases when deuterated malate is the substrate comparedmore » to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the {sup 13}C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a {beta}-secondary {sup 13}C isotope effect accompanies hydride transfer as a result of hyperconjugation of the {beta}-carboxyl of malate as the transition state for the hydride transfer step is approached.« less

  11. Effective immobilization of alcohol dehydrogenase on carbon nanoscaffolds for ethanol biofuel cell.

    PubMed

    Umasankar, Yogeswaran; Adhikari, Bal-Ram; Chen, Aicheng

    2017-12-01

    An efficient approach for immobilizing alcohol dehydrogenase (ADH) while enhancing its electron transfer ability has been developed using poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT) cationic polymer and carbon nanoscaffolds. The carbon nanoscaffolds were comprised of single-walled carbon nanotubes (SWCNTs) wrapped with reduced graphene oxide (rGO). The ADH entrapped within the MADQUAT that was present on the carbon nanoscaffolds exhibited a high electron exchange capability with the electrode through its cofactor β-nicotinamide adenine dinucleotide hydrate and β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NAD + /NADH) redox reaction. The advantages of the carbon nanoscaffolds used as the support matrix and the MADQUAT employed for the entrapment of ADH versus physisorption were demonstrated via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Our experimental results showed a higher electron transfer, electrocatalytic activity, and rate constant for MADQUAT entrapped ADH on the carbon nanoscaffolds. The immobilization of ADH using both MADQUAT and carbon nanoscaffolds exhibited strong potential for the development of an efficient bio-anode for ethanol powered biofuel cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Nicotinamide mononucleotide inhibits post-ischemic NAD(+) degradation and dramatically ameliorates brain damage following global cerebral ischemia.

    PubMed

    Park, Ji H; Long, Aaron; Owens, Katrina; Kristian, Tibor

    2016-11-01

    Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor for multiple cellular metabolic reactions and has a central role in energy production. Brain ischemia depletes NAD(+) pools leading to bioenergetics failure and cell death. Nicotinamide mononucleotide (NMN) is utilized by the NAD(+) salvage pathway enzyme, nicotinamide adenylyltransferase (Nmnat) to generate NAD(+). Therefore, we examined whether NMN could protect against ischemic brain damage. Mice were subjected to transient forebrain ischemia and treated with NMN or vehicle at the start of reperfusion or 30min after the ischemic insult. At 2, 4, and 24h of recovery, the proteins poly-ADP-ribosylation (PAR), hippocampal NAD(+) levels, and expression levels of NAD(+) salvage pathway enzymes were determined. Furthermore, animal's neurologic outcome and hippocampal CA1 neuronal death was assessed after six days of reperfusion. NMN (62.5mg/kg) dramatically ameliorated the hippocampal CA1 injury and significantly improved the neurological outcome. Additionally, the post-ischemic NMN treatment prevented the increase in PAR formation and NAD(+) catabolism. Since the NMN administration did not affect animal's temperature, blood gases or regional cerebral blood flow during recovery, the protective effect was not a result of altered reperfusion conditions. These data suggest that administration of NMN at a proper dosage has a strong protective effect against ischemic brain injury. Published by Elsevier Inc.

  13. Sensitive colorimetric visualization of dihydronicotinamide adenine dinucleotide based on anti-aggregation of gold nanoparticles via boronic acid-diol binding.

    PubMed

    Liu, Shufeng; Du, Zongfeng; Li, Peng; Li, Feng

    2012-05-15

    A facile, highly sensitive colorimetric strategy for dihydronicotinamide adenine dinucleotide (NADH) detection is proposed based on anti-aggregation of gold nanoparticles (AuNPs) via boronic acid-diol binding chemistry. The aggregation agent, 4-mercaptophenylboronic acid (MPBA), has specific affinity for AuNPs through Au-S interaction, leading to the aggregation of AuNPs by self-dehydration condensation at a certain concentration, which is responsible for a visible color change of AuNPs from wine red to blue. With the addition of NADH, MPBA would prefer reacting with NADH to form stable borate ester via boronic acid-diol binding dependent on the pH and solvent, revealing an obvious color change from blue to red with increasing the concentration of NADH. The anti-aggregation effect of NADH on AuNPs was seen by the naked eye and monitored by UV-vis extinction spectra. The linear range of the colorimetric sensor for NADH is from 8.0 × 10(-9)M to 8.0 × 10(-6)M, with a low detection limit of 2.0 nM. The as-established colorimetric strategy opened a new avenue for NADH determination. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Synthesis, conformational analysis, and biological activity of new analogues of thiazole-4-carboxamide adenine dinucleotide (TAD) as IMP dehydrogenase inhibitors.

    PubMed

    Franchetti, Palmarisa; Cappellacci, Loredana; Pasqualini, Michela; Petrelli, Riccardo; Jayaprakasan, Vetrichelvan; Jayaram, Hiremagalur N; Boyd, Donald B; Jain, Manojkumar D; Grifantini, Mario

    2005-03-15

    Thiazole-4-carboxamide adenine dinucleotide (TAD) analogues T-2'-MeAD (1) and T-3'-MeAD (2) containing, respectively, a methyl group at the ribose 2'-C-, and 3'-C-position of the adenosine moiety, were prepared as potential selective human inosine monophosphate dehydrogenase (IMPDH) type II inhibitors. The synthesis of heterodinucleotides was carried out by CDI-catalyzed coupling reaction of unprotected 2'-C-methyl- or 3'-C-methyl-adenosine 5'-monophosphate with 2',3'-O-isopropylidene-tiazofurin 5'-monophosphate, and then deisopropylidenation. Biological evaluation of dinucleotides 1 and 2 as inhibitors of recombinant human IMPDH type I and type II resulted in a good activity. Inhibition of both isoenzymes by T-2'-MeAD and T-3'-MeAD was noncompetitive with respect to NAD substrate. Binding of T-3'-MeAD was comparable to that of parent compound TAD, while T-2'-MeAD proved to be a weaker inhibitor. However, no significant difference was found in inhibition of the IMPDH isoenzymes. T-2'-MeAD and T-3'-MeAD were found to inhibit the growth of K562 cells (IC(50) 30.7 and 65.0muM, respectively).

  15. Death by Protein Damage in Irradiated Cells

    DTIC Science & Technology

    2011-01-01

    tardigrades, J. Exp. Biol. 212 (2009) 4033– 4039. [63] A. Oren, N. Gunde-Cimerman, Mycosporines and mycosporine - like amino acids : UV protectants or...catalytically remove superoxide via a disproportionation mechanism [29,46]; and amino acids and peptides, which scav- enge hydroxyl radicals very efficiently...most radiation resistant mutants of B. pumilus displayed mul- tiple amino acid auxotrophies and a requirement for nicotinamide adenine dinucleotide

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

    PubMed

    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.

  17. Orofacial inflammatory pain affects the expression of MT1 and NADPH-d in rat caudal spinal trigeminal nucleus and trigeminal ganglion

    PubMed Central

    Huang, Fang; He, Hongwen; Fan, Wenguo; Liu, Yongliang; Zhou, Hongyu; Cheng, Bin

    2013-01-01

    Very little is known about the role of melatonin in the trigeminal system, including the function of melatonin receptor 1. In the present study, adult rats were injected with formaldehyde into the right vibrissae pad to establish a model of orofacial inflammatory pain. The distribution of melatonin receptor 1 and nicotinamide adenine dinucleotide phosphate diaphorase in the caudal spinal trigeminal nucleus and trigeminal ganglion was determined with immunohistochemistry and histochemistry. The results show that there are significant differences in melatonin receptor 1 expression and nicotinamide adenine dinucleotide phosphate diaphorase expression in the trigeminal ganglia and caudal spinal nucleus during the early stage of orofacial inflammatory pain. Our findings suggest that when melatonin receptor 1 expression in the caudal spinal nucleus is significantly reduced, melatonin's regulatory effect on pain is attenuated. PMID:25206619

  18. Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

    PubMed

    Rösner, J; Liotta, A; Angamo, E A; Spies, C; Heinemann, U; Kovács, R

    2016-11-01

    Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO 2 , field potentials and changes in extracellular potassium concentration ([K + ] o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K + ] o and pO 2 changes as compared to continuous illumination. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  19. Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

    PubMed Central

    Škodová, Ingrid; Verner, Zdeněk; Bringaud, Fréderic; Fabian, Peter

    2013-01-01

    Glycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD+ reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion. PMID:24142106

  20. Nicotinamide mononucleotide (NMN) supplementation ameliorates the impact of maternal obesity in mice: comparison with exercise.

    PubMed

    Uddin, Golam Mezbah; Youngson, Neil A; Doyle, Bronte M; Sinclair, David A; Morris, Margaret J

    2017-11-08

    Maternal overnutrition increases the risk of long-term metabolic dysfunction in offspring. Exercise improves metabolism partly by upregulating mitochondrial biogenesis or function, via increased levels of nicotinamide adenine dinucleotide (NAD + ). We have shown that the NAD + precursor, nicotinamide mononucleotide (NMN) can reverse some of the negative consequences of high fat diet (HFD) consumption. To investigate whether NMN can impact developmentally-set metabolic deficits, we compared treadmill exercise and NMN injection in offspring of obese mothers. Five week old lean and obese female C57BL6/J mice were mated with chow fed males. Female offspring weaned onto HFD were given treadmill exercise for 9 weeks, or NMN injection daily for 18 days. Maternal obesity programmed increased adiposity and liver triglycerides, with decreased glucose tolerance, liver NAD + levels and citrate synthase activity in offspring. Both interventions reduced adiposity, and showed a modest improvement in glucose tolerance and improved markers of mitochondrial function. NMN appeared to have stronger effects on liver fat catabolism (Hadh) and synthesis (Fasn) than exercise. The interventions appeared to exert the most global benefit in mice that were most metabolically challenged (HFD-consuming offspring of obese mothers). This work encourages further study to confirm the suitability of NMN for use in reversing metabolic dysfunction linked to programming by maternal obesity.

  1. Role of nicotinamide (vitamin B3) in acetaminophen-induced changes in rat liver: Nicotinamide effect in acetaminophen-damged liver.

    PubMed

    Mahmoud, Yomna I; Mahmoud, Asmaa A

    2016-06-01

    Acetaminophen is a widely used analgesic and antipyretic agent, which is safe at therapeutic doses. However, overdoses of acetaminophen induce severe oxidative stress, which leads to acute liver failure. Nicotinamide has proven effective in ameliorating many pathological conditions that occur due to oxidative stress. This study verifies the prophylactic and therapeutic effects of nicotinamide against the hepatic pathophysiological and ultrastructural alterations induced by acetaminophen. Wistar rats intoxicated with an acute overdose of acetaminophen (5g/kg b.wt) were given a single dose of nicotinamide (500mg/kg b.wt) either before or after intoxication. Acetaminophen caused significant elevation in the liver functions and lipid peroxidation marker, and decline in the activities of the hepatic antioxidant enzymes. This oxidative injury was associated with hepatic centrilobular necrosis, hemorrage, vacuolar degeneration, lipid accumulation and mitochondrial alterations. Treating intoxicated rats with nicotinamide (500mg/kg) significantly ameliorated acetaminophen-induced biochemical changes and pathological injuries. However, administering the same dose of nicotinamide to healthy animals or prior to acetaminophen-intoxication induced hepatotoxicity. Caution should be taken when administering high doses of NAM because of its possible hepatotoxicity. Considering the wide use of nicotinamide, there is an important need for monitoring nicotinamide tolerance, safety and efficacy in healthy and diseased subjects. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. Visualization of Nicotine Adenine Dinucleotide Redox Homeostasis with Genetically Encoded Fluorescent Sensors.

    PubMed

    Zhao, Yuzheng; Zhang, Zhuo; Zou, Yejun; Yang, Yi

    2018-01-20

    Beyond their roles as redox currency in living organisms, pyridine dinucleotides (NAD + /NADH and NADP + /NADPH) are also precursors or cosubstrates of great significance in various physiologic and pathologic processes. Recent Advances: For many years, it was challenging to develop methodologies for monitoring pyridine dinucleotides in situ or in vivo. Recent advances in fluorescent protein-based sensors provide a rapid, sensitive, specific, and real-time readout of pyridine dinucleotide dynamics in single cells or in vivo, thereby opening a new era of pyridine dinucleotide bioimaging. In this article, we summarize the developments in genetically encoded fluorescent sensors for NAD + /NADH and NADP + /NADPH redox states, as well as their applications in life sciences and drug discovery. The strengths and weaknesses of individual sensors are also discussed. These sensors have the advantages of being specific and organelle targetable, enabling real-time monitoring and subcellular-level quantification of targeted molecules in living cells and in vivo. NAD + /NADH and NADP + /NADPH have distinct functions in metabolic and redox regulation, and thus, a comprehensive evaluation of metabolic and redox states must be multiplexed with a combination of various metabolite sensors in a single cell. Antioxid. Redox Signal. 28, 213-229.

  3. Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes*

    PubMed Central

    Unciti-Broceta, Juan D.; Maceira, José; Morales, Sonia; García-Pérez, Angélica; Muñóz-Torres, Manuel E.; Garcia-Salcedo, Jose A.

    2013-01-01

    Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease. PMID:23443665

  4. Mannitol and Mannitol Dehydrogenases in Conidia of Aspergillus oryzae

    PubMed Central

    Horikoshi, Koki; Iida, Shigeji; Ikeda, Yonosuke

    1965-01-01

    Horikoshi, Koki (The Institute of Physical and Chemical Research, Tokyo, Japan), Shigeji Iida, and Yonosuke Ikeda. Mannitol and mannitol dehydrogenases in conidia of Aspergillus oryzae. J. Bacteriol. 89:326–330. 1965.—A sugar alcohol was isolated from the conidia of Aspergillus oryzae and identified as d-mannitol. Two types of d-mannitol dehydrogenases, nicotinamide adenine dinucleotide phosphate-linked and nicotinamide adenine dinucleotide-linked, were found in the conidia. Substrate specificities, pH optima, Michaelis-Menton constants, and the effects of inhibitors were studied. d-Mannitol was converted to fructose by the dehydrogenases. Synthesis of d-mannitol dehydrogenases was not observed during germination; the content of d-mannitol decreased at an early stage of germination. It was assumed, therefore, that d-mannitol might be used as the source of endogenous respiration and provide energy for the germination. PMID:14255698

  5. Visfatin and cardio-cerebro-vascular disease.

    PubMed

    Wang, Pei; Vanhoutte, Paul M; Miao, Chao-Yu

    2012-01-01

    Nicotinamide phosphoribosyltransferase is the rate-limiting enzyme that catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide from nicotinamide. This protein was originally cloned as a putative pre-B cell colony-enhancing factor and also found to be a visceral fat-derived adipokine (visfatin). As a multifunctional protein, visfatin plays an important role in immunity, metabolism, aging, inflammation, and responses to stress. Visfatin also participates in several pathophysiological processes contributing to cardio-cerebro-vascular diseases, including hypertension, atherosclerosis, ischemic heart disease, and ischemic stroke. However, whether visfatin is a friend or a foe in these diseases remains uncertain. This brief review focuses on the current understanding of the complex role of visfatin in the cardio-cerebro-vascular system under normal and pathophysiological conditions.

  6. Pleiotropic effects of the sirtuin inhibitor sirtinol involves concentration-dependent modulation of multiple nuclear receptor-mediated pathways in the androgen-responsive prostate cancer cell LNCaP

    USDA-ARS?s Scientific Manuscript database

    Sirtinol, a purported specific inhibitor of the nicotinamide adenine dinucleotide (NAD)-dependent type III histone deacetylase (also known as sirtuin), has been used extensively to identify chemopreventive/chemotherapeutic agents that modulate the activity of this group of enzymes. However, the mole...

  7. Singlet Oxygen Generation by UVA Light Exposure of Endogenous Photosensitizers

    PubMed Central

    Baier, Jürgen; Maisch, Tim; Maier, Max; Engel, Eva; Landthaler, Michael; Bäumler, Wolfgang

    2006-01-01

    UVA light (320–400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), β-nicotinamide adenine dinucleotide (NAD), and β-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (ΦΔ = 0.54 ± 0.07), FMN (ΦΔ = 0.51 ± 0.07), and FAD (ΦΔ = 0.07 ± 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue). PMID:16751234

  8. Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells.

    PubMed

    Yang, Ming-Yeh; Chang, Chih-Jui; Chen, Liang-Yü

    2017-08-01

    Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study. Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462nm). Under blue light irradiation at 3.744J/cm 2 (=0.52mW/cm 2 irradiated for 2h), the minimal lethal dose is 3.125μM and the median lethal doses (LD 50 ) for FMN and FAD are 6.5μM and 7.2μM, respectively. Individual exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic photosensitizer for cancer research. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Paper microfluidic-based enzyme catalyzed double microreactor.

    PubMed

    Ferrer, Ivonne M; Valadez, Hector; Estala, Lissette; Gomez, Frank A

    2014-08-01

    We describe a paper microfluidic-based enzyme catalyzed double microreactor assay using fluorescent detection. Here, solutions of lactate dehydrogenase (LDH) and diaphorase (DI) were directly spotted onto the microfluidic paper-based analytical device (μPAD). Samples containing lactic acid, resazurin, and nicotinamide adenine dinucleotide oxidized form (NAD(+) ), potassium chloride (KCl), and BSA, in MES buffer were separately spotted onto the μPAD and MES buffer flowed through the device. A cascade reaction occurs upon the sample spot overlapping with LDH to form pyruvate and nicotinamide adenine dinucleotide reduced form (NADH). Subsequently, NADH is used in the conversion of resazurin to fluorescent resorufin by DI. The μPAD avoids the need of surface functionalization or enzyme immobilization steps. These microreactor devices are low cost and easy to fabricate and effect reaction based solely on buffer capillary action. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH

    DOE PAGES

    Brinkmann-Chen, Sabine; Flock, Tilman; Cahn, Jackson K. B.; ...

    2013-06-17

    To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymesmore » having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. As a result, high-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.« less

  11. Inhibitor of Nicotinamide Phosphoribosyltransferase Sensitizes Glioblastoma Cells to Temozolomide via Activating ROS/JNK Signaling Pathway.

    PubMed

    Feng, Jun; Yan, Peng-Fei; Zhao, Hong-Yang; Zhang, Fang-Cheng; Zhao, Wo-Hua; Feng, Min

    2016-01-01

    Overcoming temozolomide (TMZ) resistance is a great challenge in glioblastoma (GBM) treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide and has a crucial role in cancer cell metabolism. In this study, we investigated whether FK866 and CHS828, two specific NAMPT inhibitors, could sensitize GBM cells to TMZ. Low doses of FK866 and CHS828 (5 nM and 10 nM, resp.) alone did not significantly decrease cell viability in U251-MG and T98 GBM cells. However, they significantly increased the antitumor action of TMZ in these cells. In U251-MG cells, administration of NAMPT inhibitors increased the TMZ (100  μ M)-induced apoptosis and LDH release from GBM cells. NAMPT inhibitors remarkably enhanced the activities of caspase-1, caspase-3, and caspase-9. Moreover, NAMPT inhibitors increased reactive oxygen species (ROS) production and superoxide anion level but reduced the SOD activity and total antioxidative capacity in GBM cells. Treatment of NAMPT inhibitors increased phosphorylation of c-Jun and JNK. Administration of JNK inhibitor SP600125 or ROS scavenger tocopherol with TMZ and NAMPT inhibitors substantially attenuated the sensitization of NAMPT inhibitor on TMZ antitumor action. Our data indicate a potential value of NAMPT inhibitors in combined use with TMZ for GBM treatment.

  12. Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs.

    PubMed Central

    Perez-Terzic, C M; Chini, E N; Shen, S S; Dousa, T P; Clapham, D E

    1995-01-01

    Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells. Images Figure 2 PMID:8554544

  13. NAD(P)H-Independent Asymmetric C=C Bond Reduction Catalyzed by Ene Reductases by Using Artificial Co-substrates as the Hydrogen Donor

    PubMed Central

    Winkler, Christoph K; Clay, Dorina; Entner, Marcello; Plank, Markus; Faber, Kurt

    2014-01-01

    To develop a nicotinamide-independent single flavoenzyme system for the asymmetric bioreduction of C=C bonds, four types of hydrogen donor, encompassing more than 50 candidates, were investigated. Six highly potent, cheap, and commercially available co-substrates were identified that (under the optimized conditions) resulted in conversions and enantioselectivities comparable with, or even superior to, those obtained with traditional two-enzyme nicotinamide adenine dinucleotide phosphate (NAD(P)H)-recycling systems. PMID:24382795

  14. Improved Ethanol Production from Xylose by Candida shehatae Induced by Dielectric Barrier Discharge Air Plasma

    NASA Astrophysics Data System (ADS)

    Chen, Huixia; Xiu, Zhilong; Bai, Fengwu

    2014-06-01

    Xylose fermentation is essential for ethanol production from lignocellulosic biomass. Exposure of the xylose-fermenting yeast Candida shehatae (C. shehatae) CICC1766 to atmospheric pressure dielectric barrier discharge (DBD) air plasma yields a clone (designated as C81015) with stability, which exhibits a higher ethanol fermentation rate from xylose, giving a maximal enhancement in ethanol production of 36.2% compared to the control (untreated). However, the biomass production of C81015 is lower than that of the control. Analysis of the NADH (nicotinamide adenine dinucleotide)- and NADPH (nicotinamide adenine dinucleotide phosphate)-linked xylose reductases and NAD+-linked xylitol dehydrogenase indicates that their activities are enhanced by 34.1%, 61.5% and 66.3%, respectively, suggesting that the activities of these three enzymes are responsible for improving ethanol fermentation in C81015 with xylose as a substrate. The results of this study show that DBD air plasma could serve as a novel and effective means of generating microbial strains that can better use xylose for ethanol fermentation.

  15. Purification and Characterization of Ferredoxin-Nicotinamide Adenine Dinucleotide Phosphate Reductase from a Nitrogen-Fixing Bacterium

    PubMed Central

    Yoch, Duane C.

    1973-01-01

    Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP+ reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP+ reductase in the photochemical reduction of NADP+ by blue-green algal particles. The ferredoxin-NADP+ reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP+ was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD+ transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (Km = 5.0 × 10−3M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase. PMID:4147648

  16. The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

    PubMed Central

    Jackson, J. F.; Atkinson, M. R.

    1966-01-01

    1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd2+, Mn2+, Mg2+, Zn2+, Co2+ and Ni2+ were approximately a linear function of heats of hydration of the corresponding ions. Ba2+, Sr2+, Ca2+, Cu2+ and Be2+ did not activate the enzyme; Be2+ inhibited the reaction in the presence of Mg2+ and, to a greater extent, in the presence of Ni2+. 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8·0 and 25°, in the presence of 3mm concentrations of the unvaried reactants, were 88±7μm-ATP, 42±4μm-NMN and 85±4μm-Mg2+. The results at this pH and at pH7·5 were consistent with mechanisms in which Mg2+–ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide–hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni2+ and Co2+ than with Mg2+. PMID:4291356

  17. Curcumin and resveratrol rescue cortical-hippocampal system from chronic fluoride-induced neurodegeneration and enhance memory retrieval.

    PubMed

    Sharma, Chhavi; Suhalka, Pooja; Bhatnagar, Maheep

    2018-04-13

    The aim of this study was: (1) to evaluate the neuroprotective effect of resveratrol and curcumin on nicotinamide adenine dinucleotide phosphate diaphorase activity in neuronal cell in subregions of mice brain, (2) to evaluate the effects on antioxidant status and (3) to evaluate the protective effects of phytochemicals on learning and memory following fluoride exposure. Young mice (one month old, body weight (BW) 30 ± 5 mg) were provided with 120 ppm sodium fluoride dissolved in drinking water. They were given curcumin (30 mg/kg BW) or resveratrol (30 mg/kg BW) orally once in a day up to 30 days. Effects of resveratrol and curcumin on spatial learning and memory were studied using Morris water maze and classic maze test. Effects on brain antioxidants' (lactose dehydrogenase (LDH), malondialdehyde and reactive oxygen species) status were also studied in vitro. Histochemistry was done to assess the effect of treatments on nitric oxide neurotransmitter. Our study showed that in fluoride-treated animals, the number of nicotinamide adenine dinucleotide phosphate diaphorase positive neurons, intracellular Ca 2+ , reactive oxygen species level, LDH and malondialdehyde concentration increased significantly. Interestingly, after treatment with curcumin or resveratrol, a significant decrease in the number of nicotinamide adenine dinucleotide phosphate diaphorase positive neurons and antioxidant status was observed. This decrease was more considerable in resveratrol-treated group. Our study indicates that both antioxidants, curcumin and resveratrol, are useful in reducing neurodegeneration in selective areas of cornus ammonis 1 (CA1), CA3, dentate gyrus (DG) and the cortex of mice brain and in recuperating the loss of memory and learning caused due to fluoride exposure.

  18. Protective effect of dietary potassium against vascular injury in salt-sensitive hypertension.

    PubMed

    Kido, Makiko; Ando, Katsuyuki; Onozato, Maristela L; Tojo, Akihiro; Yoshikawa, Masahiro; Ogita, Teruhiko; Fujita, Toshiro

    2008-02-01

    Hypertensive cardiovascular damage is accelerated by salt loading but counteracted by dietary potassium supplementation. We suggested recently that antioxidant actions of potassium contribute to protection against salt-induced cardiac dysfunction. Therefore, we examined whether potassium supplementation ameliorated cuff-induced vascular injury in salt-sensitive hypertension via suppression of oxidative stress. Four-week-old Dahl salt-sensitive rats were fed a normal-salt (0.3% NaCl), high-salt (8% NaCl), or high-salt plus high-potassium (8% KCl) diet for 5 weeks, and some of the rats fed a high-salt diet were also given antioxidants. One week after the start of the treatments, a silicone cuff was implanted around the femoral artery. Examination revealed increased cuff-induced neointimal proliferation with adventitial macrophage infiltration in arteries from salt-loaded Dahl salt-sensitive rats compared with that in arteries from non-salt-loaded animals (intima/media ratio: 0.471+/-0.070 versus 0.302+/-0.037; P<0.05), associated with regional superoxide overproduction and reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and mRNA overexpression. On the other hand, simultaneous potassium supplementation attenuated salt-induced neointimal hyperplasia (intima/media ratio: 0.205+/-0.012; P<0.001), adventitial macrophage infiltration, superoxide overproduction, and reduced nicotinamide-adenine dinucleotide phosphate oxidase activation and overexpression. Antioxidants, which decrease vascular oxidative stress, also reduced neointima formation induced by salt excess. In conclusion, high-potassium diets seems to have a protective effect against the development of vascular damage induced by salt loading mediated, at least in part, through suppression of the production of reactive oxygen species probably generated by reduced nicotinamide-adenine dinucleotide phosphate oxidase.

  19. Localization and phosphorylation of Plasmodium falciparum nicotinamide/nicotinate mononucleotide adenylyltransferase (PfNMNAT) in intraerythrocytic stages.

    PubMed

    Nieto, Carlos A; Sánchez, Lina M; Sánchez, Diana M; Díaz, Gonzalo J; Ramírez, María H

    2018-04-11

    Nicotinamide adenine dinucleotide (NAD+) is an essential molecule in the energy metabolism of living beings, and it has various cellular functions. The main enzyme in the biosynthesis of this nucleotide is nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) because it is the convergence point for all known biosynthetic pathways. NMNATs have divergences in both the number of isoforms detected and their distribution, depending on the organism. In the laboratory of basic research in biochemistry (LIBBIQ: acronym in Spanish) the NMNATs of protozoan parasites (Leishmania braziliensis, Plasmodium falciparum, Trypanosoma cruzi, and Giardia duodenalis) have been studied, analysing their catalytic properties through the use of proteins. Recombinants and their cellular distribution essentially. In 2014, O'Hara et al. determined the cytoplasmic localization of NMNAT of P. falciparum, using a transgene coupled to GFP, however, the addition of labels to the study protein can modify several of its characteristics, including its sub-cellular localization. This study confirms the cytoplasmic localization of this protein in the parasite through recognition of the endogenous protein in the different stages of the asexual life cycle. Additionally, the study found that PfNMNAT could be a phosphorylation target at serine, tyrosine and threonine residues, and it shows variations during the asexual life cycle. These experiments confirmed that the parasite is situated in the cytoplasm, fulfilling the required functions of NAD+ in this compartment, the PfNMNAT is regulated in post-transcription processes, and can be regulated by phosphorylation in its residues.

  20. Progress in utilisation of graphene for electrochemical biosensors.

    PubMed

    Lawal, Abdulazeez T

    2018-05-30

    This review discusses recent graphene (GR) electrochemical biosensor for accurate detection of biomolecules, including glucose, hydrogen peroxide, dopamine, ascorbic acid, uric acid, nicotinamide adenine dinucleotide, DNA, metals and immunosensor through effective immobilization of enzymes, including glucose oxidase, horseradish peroxidase, and haemoglobin. GR-based biosensors exhibited remarkable performance with high sensitivities, wide linear detection ranges, low detection limits, and long-term stabilities. Future challenges for the field include miniaturising biosensors and simplifying mass production are discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Nicotinamide phosphoribosyltransferase protects against ischemic stroke through SIRT1-dependent adenosine monophosphate-activated kinase pathway.

    PubMed

    Wang, Pei; Xu, Tian-Ying; Guan, Yun-Feng; Tian, Wei-Wei; Viollet, Benoit; Rui, Yao-Cheng; Zhai, Qi-Wei; Su, Ding-Feng; Miao, Chao-Yu

    2011-02-01

    Stroke is a leading cause of mortality and disability. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in mammalian nicotinamide adenine dinucleotide (NAD)(+) biosynthesis and contributes to cell fate decisions. However, the role of Nampt in brain and stroke remains to be investigated. We used lentivirus-mediated Nampt overexpression and knockdown to manipulate Nampt expression and explore the effects of Nampt in neuronal survival on ischemic stress both in vivo and in vitro. We also used adenosine monophosphate (AMP)-activated kinase-α2 (AMPKα2) and silent mating type information regulation 2 homolog 1 (SIRT1) knockout mice to investigate the underlying mechanisms of Nampt neuroprotection. Nampt inhibition by a highly-specific Nampt inhibitor, FK866, aggravated brain infarction in experimentally cerebral ischemia rats, whereas Nampt overexpression in local brain and Nampt enzymatic product nicotinamide mononucleotide (NMN) reduced ischemia-induced cerebral injuries. Nampt overexpression and knockdown regulated neuron survival via the AMPK pathway. Neuroprotection of Nampt was abolished in AMPKα2(-/-) neurons. In neurons, Nampt positively modulated NAD(+) levels and thereby controlled SIRT1 activity. SIRT1 coprecipitated with serine/threonine kinase 11 (LKB1), an upstream kinase of AMPK, and promoted LKB1 deacetylation in neurons. Nampt-induced LKB1 deacetylation and AMPK activation disappeared in SIRT1(-/-) neurons. In contrast, Ca(2+) /calmodulin-dependent protein kinase kinase-β (CaMKK-β), another upstream kinase of AMPK, was not involved in the neuroprotection of Nampt. More important, Nampt overexpression-induced neuroprotection was abolished in SIRT1(+/-) and AMPKα2(-/-) mice. Our findings reveal that Nampt protects against ischemic stroke through rescuing neurons from death via the SIRT1-dependent AMPK pathway and indicate that Nampt is a new therapeutic target for stroke. Copyright © 2011 American Neurological Association.

  2. Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47 phox phosphorylation of NADPH oxidase in SK Hep-1 cells.

    PubMed

    de Araújo, Glaucy Rodrigues; Rabelo, Ana Carolina Silveira; Meira, Janaína Serenato; Rossoni-Júnior, Joamyr Victor; Castro-Borges, William de; Guerra-Sá, Renata; Batista, Maurício Azevedo; Silveira-Lemos, Denise da; Souza, Gustavo Henrique Bianco de; Brandão, Geraldo Célio; Chaves, Míriam Martins; Costa, Daniela Caldeira

    2017-02-01

    Baccharis trimera, popularly known as "carqueja", is a native South-American plant possessing a high concentration of polyphenolic compounds and therefore high antioxidant potential. Despite the antioxidant potential described for B. trimera, there are no reports concerning the signaling pathways involved in this process. So, the aim of the present study was to assess the influence of B. trimera on the modulation of PKC signaling pathway and to characterize the effect of the nicotinamide adenine dinucleotide phosphate oxidase enzyme (NOX) on the generation of reactive oxygen species in SK Hep-1 cells. SK-Hep 1 cells were treated with B. trimera, quercetin, or rutin and then stimulated or not with PMA/ionomycin and labeled with carboxy H 2 DCFDA for detection of reactive oxygen species by flow cytometer. The PKC expression by Western blot and enzyme activity was performed to evaluate the influence of B. trimera and quercetin on PKC signaling pathway. p47 phox and p47 phox phosphorylated expression was performed by Western blot to evaluate the influence of B. trimera on p47 phox phosphorylation. The results showed that cells stimulated with PMA/ionomycin (activators of PKC) showed significantly increased reactive oxygen species production, and this production returned to baseline levels after treatment with DPI (NOX inhibitor). Both B. trimera and quercetin modulated reactive oxygen species production through the inhibition of PKC protein expression and enzymatic activity, also with inhibition of p47 phox phosphorylation. Taken together, these results suggest that B. trimera has a potential mechanism for inhibiting reactive oxygen species production through the PKC signaling pathway and inhibition subunit p47 phox phosphorylation of nicotinamide adenine dinucleotide phosphate oxidase.

  3. UV-Vis Action Spectroscopy Reveals a Conformational Collapse in Hydrogen-Rich Dinucleotide Cation Radicals.

    PubMed

    Korn, Joseph A; Urban, Jan; Dang, Andy; Nguyen, Huong T H; Tureček, František

    2017-09-07

    We report the generation of deoxyriboadenosine dinucleotide cation radicals by gas-phase electron transfer to dinucleotide dications and their noncovalent complexes with crown ether ligands. Stable dinucleotide cation radicals of a novel hydrogen-rich type were generated and characterized by tandem mass spectrometry and UV-vis photodissociation (UVPD) action spectroscopy. Electron structure theory analysis indicated that upon electron attachment the dinucleotide dications underwent a conformational collapse followed by intramolecular proton migrations between the nucleobases to give species whose calculated UV-vis absorption spectra matched the UVPD action spectra. Hydrogen-rich cation radicals generated from chimeric riboadenosine 5'-diesters gave UVPD action spectra that pointed to novel zwitterionic structures consisting of aromatic π-electron anion radicals intercalated between stacked positively charged adenine rings. Analogies with DNA ionization are discussed.

  4. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation*♦

    PubMed Central

    Nebel, Merle; Schwoerer, Alexander P.; Warszta, Dominik; Siebrands, Cornelia C.; Limbrock, Ann-Christin; Swarbrick, Joanna M.; Fliegert, Ralf; Weber, Karin; Bruhn, Sören; Hohenegger, Martin; Geisler, Anne; Herich, Lena; Schlegel, Susan; Carrier, Lucie; Eschenhagen, Thomas; Potter, Barry V. L.; Ehmke, Heimo; Guse, Andreas H.

    2013-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy. PMID:23564460

  5. Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

    PubMed

    Kurdrid, Pavinee; Subudhi, Sanjukta; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Hongsthong, Apiradee

    2007-12-01

    When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

  6. Administration of nicotinamide riboside prevents oxidative stress and organ injury in sepsis.

    PubMed

    Hong, Guangliang; Zheng, Dong; Zhang, Lulu; Ni, Rui; Wang, Grace; Fan, Guo-Chang; Lu, Zhongqiu; Peng, Tianqing

    2018-08-01

    Sepsis-caused multiple organ failure remains the major cause of morbidity and mortality in intensive care units. Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD + ), which is important in regulating oxidative stress. This study investigated whether administration of NR prevented oxidative stress and organ injury in sepsis. Mouse sepsis models were induced by injection of lipopolysaccharides (LPS) or feces-injection-in-peritoneum. NR was given before sepsis onset. Cultured macrophages and endothelial cells were incubated with various agents. Administration of NR elevated the NAD + levels, and elicited a reduction of oxidative stress, inflammation and caspase-3 activity in lung and heart tissues, which correlated with attenuation of pulmonary microvascular permeability and myocardial dysfunction, leading to less mortality in sepsis models. These protective effects of NR were associated with decreased levels of plasma high mobility group box-1 (HMGB1) in septic mice. Consistently, pre-treatment of macrophages with NR increased NAD + content and reduced HMGB1 release upon LPS stimulation. NR also prevented reactive oxygen species (ROS) production and apoptosis in endothelial cells induced by a conditioned-medium collected from LPS-treated macrophages. Furthermore, inhibition of SIRT1 by EX527 offset the negative effects of NR on HMGB1 release in macrophages, and ROS and apoptosis in endothelial cells. Administration of NR prevents lung and heart injury, and improves the survival in sepsis, likely by inhibiting HMGB1 release and oxidative stress via the NAD + /SIRT1 signaling. Given NR has been used as a health supplement, it may be a useful agent to prevent organ injury in sepsis. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Mitochondrial respiratory complex I probed by delayed luminescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Baran, Irina; Ionescu, Diana; Privitera, Simona; Scordino, Agata; Mocanu, Maria Magdalena; Musumeci, Francesco; Grasso, Rosaria; Gulino, Marisa; Iftime, Adrian; Tofolean, Ioana Teodora; Garaiman, Alexandru; Goicea, Alexandru; Irimia, Ruxandra; Dimancea, Alexandru; Ganea, Constanta

    2013-12-01

    The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 μs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.

  8. Interactive response of photosynthetic characteristics in Haloxylon ammodendron and Hedysarum scoparium exposed to soil water and air vapor pressure deficits.

    PubMed

    Gong, Chunmei; Wang, Jiajia; Hu, Congxia; Wang, Junhui; Ning, Pengbo; Bai, Juan

    2015-08-01

    C4 plants possess better drought tolerance than C3 plants. However, Hedysarum scoparium, a C3 species, is dominant and widely distributed in the desert areas of northwestern China due to its strong drought tolerance. This study compared it with Haloxylon ammodendron, a C4 species, regarding the interactive effects of drought stress and different leaf-air vapor pressure deficits. Variables of interest included gas exchange, the activity levels of key C4 photosynthetic enzymes, and cellular anatomy. In both species, gas exchange parameters were more sensitive to high vapor pressure deficit than to strong water stress, and the net CO2 assimilation rate (An) was enhanced as vapor pressure deficits increased. A close relationship between An and stomatal conductance (gs) suggested that the species shared a similar response mechanism. In H. ammodendron, the activity levels of key C4 enzymes were higher, including those of phosphoenolpyruvate carboxylase (PEPC) and nicotinamide adenine dinucleotide phosphate-malate enzyme (NADP-ME), whereas in H. scoparium, the activity level of nicotinamide adenine dinucleotide-malate enzyme (NAD-ME) was higher. Meanwhile, H. scoparium utilized adaptive structural features, including a larger relative vessel area and a shorter distance from vein to stomata, which facilitated the movement of water. These findings implied that some C4 biochemical pathways were present in H. scoparium to respond to environmental challenges. Copyright © 2015. Published by Elsevier B.V.

  9. Enzymatic Basis for Differentiation of Rhizobium into Fast- and Slow-Growing Groups

    PubMed Central

    Drets, G. Martinez-De; Arias, A.

    1972-01-01

    Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and other enzymes related to carbohydrate metabolism were studied in rhizobia. A nicotinamide adenine dinucleotide phosphate-6-phosphogluconate dehydrogenase was detected in strains of the fast-growing group of Rhizobium but not in strains of the slow-growing group. An enzymatic differentiation of rhizobia was established. PMID:4400417

  10. Nicotinamide Phosphoribosyltransferase Upregulation by Phenylephrine Reduces Radiation Injury in Submandibular Gland

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xiang, Bin, E-mail: xiangbin72@163.com; Han, Lichi; Wang, Xinyue

    Purpose: Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. Methods and Materials: The cell viability of primary cultured submandibular gland cells was determinedmore » using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 (SIRT1) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. Results: NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1, which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. Conclusions: These findings reveal the regulatory mechanisms of NAMPT

  11. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  12. Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study.

    PubMed

    Dellinger, Ryan W; Santos, Santiago Roel; Morris, Mark; Evans, Mal; Alminana, Dan; Guarente, Leonard; Marcotulli, Eric

    2017-01-01

    NRPT is a combination of nicotinamide riboside (NR), a nicotinamide adenine dinucleotide (NAD +) precursor vitamin found in milk, and pterostilbene (PT), a polyphenol found in blueberries. Here, we report this first-in-humans clinical trial designed to assess the safety and efficacy of a repeat dose of NRPT (commercially known as Basis). NRPT was evaluated in a randomized, double-blind, and placebo-controlled study in a population of 120 healthy adults between the ages of 60 and 80 years. The study consisted of three treatment arms: placebo, recommended dose of NRPT (NRPT 1X), and double dose of NRPT (NRPT 2X). All subjects took their blinded supplement daily for eight weeks. Analysis of NAD + in whole blood demonstrated that NRPT significantly increases the concentration of NAD + in a dose-dependent manner. NAD + levels increased by approximately 40% in the NRPT 1X group and approximately 90% in the NRPT 2X group after 4 weeks as compared to placebo and baseline. Furthermore, this significant increase in NAD + levels was sustained throughout the entire 8-week trial. NAD + levels did not increase for the placebo group during the trial. No serious adverse events were reported in this study. This study shows that a repeat dose of NRPT is a safe and effective way to increase NAD + levels sustainably.

  13. Germination Requirements of Bacillus macerans Spores

    PubMed Central

    Sacks, L. E.; Thompson, P. A.

    1971-01-01

    2-Phenylacetamide is an effective germinant for spores of five strains of Bacillus macerans, particularly in the presence of fructose. Benzyl penicillin, the phenyl acetamide derivative of penicillin, and phenylacetic acid are also good germinants. l-Asparagine is an excellent germinant for four strains. α-Amino-butyric acid is moderately effective. Pyridoxine, pyridoxal, adenine, and 2,6-diaminopurine are potent germinants for NCA strain 7X1 only. d-Glucose is a powerful germinant for strain B-70 only. d-Fructose and d-ribose strongly potentiate germination induced by other germinants (except l-asparagine) but have only weak activity by themselves. Niacinamide and nicotinamide-adenine dinucleotide, inactive by themselves, are active in the presence of fructose or ribose. Effects of pH, ion concentration, and temperature are described. PMID:4251279

  14. Restoring NAD(+) Levels with NAD(+) Intermediates, the Second Law of Thermodynamics and Aging Delay.

    PubMed

    Poljsak, Borut; Milisav, Irina

    2018-04-26

    The hypothesis regarding the role of increased nicotinamide adenine dinucleotide (NAD+) levels with reference to the fundamental concepts of ageing and entropy is presented. Considering the second law of thermodynamics, NAD+ seems the appropriate candidate for reversing many aging-associated pathologies. NAD+ is presented as an essential compound that enables organisms to stay highly organized and well-maintained, with a lower entropy state.

  15. Raman Spectroscopy of the Interferon-Induced 2’,5’-Oligoadenylates

    DTIC Science & Technology

    1987-06-25

    generation of the Raman spectrum of triethyl ammonium ion ••••••••••••••••••••••••••••••• 41 12. structures of purine, adenine, purine riboside , adenosine...ribose 5 1-phosphate, AMP, and ATP........ 48 13. Raman spectra of adenine and purine •••••••.••••••••• 49 14. Raman spectra of purine riboside and... nicotinamide adenine dinucleotide; TFAB, triethyl anunonium bicarbonate; TFA, triethyl amm::mium. ion; CD circular _dichroism; NMR, nuclear magnetic

  16. Protein kinase C epsilon regulates mitochondrial pools of Nampt and NAD following resveratrol and ischemic preconditioning in the rat cortex

    PubMed Central

    Morris-Blanco, Kahlilia C; Cohan, Charles H; Neumann, Jake T; Sick, Thomas J; Perez-Pinzon, Miguel A

    2014-01-01

    Preserving mitochondrial pools of nicotinamide adenine dinucleotide (NAD) or nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in NAD production, maintains mitochondrial function and confers neuroprotection after ischemic stress. However, the mechanisms involved in regulating mitochondrial-localized Nampt or NAD have not been defined. In this study, we investigated the roles of protein kinase C epsilon (PKCɛ) and AMP-activated protein kinase (AMPK) in regulating mitochondrial pools of Nampt and NAD after resveratrol or ischemic preconditioning (IPC) in the cortex and in primary neuronal-glial cortical cultures. Using the specific PKCɛ agonist ψɛRACK, we found that PKCɛ induced robust activation of AMPK in vitro and in vivo and that AMPK was required for PKCɛ-mediated ischemic neuroprotection. In purified mitochondrial fractions, PKCɛ enhanced Nampt levels in an AMPK-dependent manner and was required for increased mitochondrial Nampt after IPC or resveratrol treatment. Analysis of intrinsic NAD autofluorescence using two-photon microscopy revealed that PKCɛ modulated NAD in the mitochondrial fraction. Further assessments of mitochondrial NAD concentrations showed that PKCɛ has a key role in regulating the mitochondrial NAD+/nicotinamide adenine dinucleotide reduced (NADH) ratio after IPC and resveratrol treatment in an AMPK- and Nampt-dependent manner. These findings indicate that PKCɛ is critical to increase or maintain mitochondrial Nampt and NAD after pathways of ischemic neuroprotection in the brain. PMID:24667915

  17. Nicotinamide N-methyltransferase: more than a vitamin B3 clearance enzyme

    PubMed Central

    Pissios, Pavlos

    2017-01-01

    Nicotinamide N-methyltransferase (NNMT) was originally identified as the enzyme responsible for the methylation of nicotinamide (NAM), one of the forms of vitamin B3. Methylated NAM (MNAM) is eventually excreted from the body. Recent evidence has expanded the role of NNMT beyond clearance of excess vitamin B3. NNMT has been implicated in the regulation of multiple metabolic pathways in tissues such as the adipose tissue and liver, as well as cancer cells, through consumption of methyl donors and generation of active metabolites. This review examines recent findings regarding the function of NNMT in physiology and disease and highlights potential new avenues for therapeutic intervention. Finally, key gaps in our knowledge for this enzymatic system and future areas of investigation are discussed. PMID:28291578

  18. Time-dependent disturbances of chloride salts on overall redox reaction and luminescence in Vibrio fischeri.

    PubMed

    Yu, Zhenyang; Zhang, Jing; Hou, Meifang

    2018-05-01

    The redox state of NADH/NADPH balance (nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate) is crucial in cellular homeostasis. Recent studies reported that sodium halide ions (NaX, X = F - , Cl - , Br - and I - ) stimulated NAD(P)H in Vibrio fischeri (VF). However, it remained unanswered whether this pattern applied in salts with other cations, e.g., K + , Mg 2+ and Ca 2+ , whose aquatic concentrations were increased by anthropogenic activities and climate change. Currently, VF were incubated with chloride salts, including KCl, MgCl 2 and CaCl 2 , and effects were measured in a time-dependent fashion. Both NADH and NADPH showed stimulation that increased over time, and the greatest maximum stimulation at 24 h was CaCl 2  > MgCl 2  > KCl. The changes of NADH/NADPH ratios over time in CaCl 2 , MgCl 2 and KCl were descendent, ascendant and stable, respectively. Simultaneously, FMN:NAD(P)H reaction catalyst (luciferase, in the form of expression levels of lux A and lux B), adenosine triphosphate and the expression levels of its regulating gene adk were also stimulated. The luminescence showed even more significant stimulations than the overall redox reaction. Together with earlier reported effects of NaCl, the chloride salts commonly disturbed the redox state and influenced the adaption of organisms to challenging environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

    PubMed Central

    Schraufstatter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1986-01-01

    To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis. PMID:2937805

  20. Deproteinization is Necessary for the Accurate Determination of Ammonia Levels by Glutamate Dehydrogenase Assay in Blood Plasma From Subjects With Liver Injury.

    PubMed

    Vodenicarovova, Melita; Skalska, Hana; Holecek, Milan

    2017-11-08

    To determine the effect of presence of high concentrations of nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes on the accuracy of glutamate dehydrogenase (GLDH) assay for ammonia. We measured ammonia concentrations using GLDH and NADH or NADPH in blood-plasma specimens and specimens deproteinized by sulfosalicylic acid from CCl4-treated or control rats. The nonspecific oxidation of NADH and NADPH was measured in mixtures without GLDH. We observed a gradual decrease (~0.5%) in absorbance in the plasma of controls after the addition of NADH but not after adding NADPH. The decrease in absorbance in plasma of CCl4-treated animals was 13.2% and 5.2% after the addition of NADH and NADPH, respectively. The decrease in absorbance was not detected in deproteinized specimens. The values of ammonia concentration were higher in the plasma specimens compared with the deproteinized ones. Deproteinization is necessary for accurate measurement of ammonia using GLDH assay in the blood plasma of subjects with liver injury. © American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  1. Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

    PubMed Central

    Cohen, G. N.; Stanier, R. Y.; Bras, Gisele Le

    1969-01-01

    The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed. PMID:4391829

  2. Primitive Photosynthetic Architectures Based on Self-Organization and Chemical Evolution of Amino Acids and Metal Ions.

    PubMed

    Liu, Kai; Ren, Xiaokang; Sun, Jianxuan; Zou, Qianli; Yan, Xuehai

    2018-06-01

    The emergence of light-energy-utilizing metabolism is likely to be a critical milestone in prebiotic chemistry and the origin of life. However, how the primitive pigment is spontaneously generated still remains unknown. Herein, a primitive pigment model based on adaptive self-organization of amino acids (Cystine, Cys) and metal ions (zinc ion, Zn 2+ ) followed by chemical evolution under hydrothermal conditions is developed. The resulting hybrid microspheres are composed of radially aligned cystine/zinc (Cys/Zn) assembly decorated with carbonate-doped zinc sulfide (C-ZnS) nanocrystals. The part of C-ZnS can work as a light-harvesting antenna to capture ultraviolet and visible light, and use it in various photochemical reactions, including hydrogen (H 2 ) evolution, carbon dioxide (CO 2 ) photoreduction, and reduction of nicotinamide adenine dinucleotide (NAD + ) to nicotinamide adenine dinucleotide hydride (NADH). Additionally, guest molecules (e.g., glutamate dehydrogenase, GDH) can be encapsulated within the hierarchical Cys/Zn framework, which facilitates sustainable photoenzymatic synthesis of glutamate. This study helps deepen insight into the emergent functionality (conversion of light energy) and complexity (hierarchical architecture) from interaction and reaction of prebiotic molecules. The primitive pigment model is also promising to work as an artificial photosynthetic microreactor.

  3. Catabolism of phloroglucinol by the rumen anaerobe coprococcus.

    PubMed

    Patel, T R; Jure, K G; Jones, G A

    1981-12-01

    A rumen isolate, Coprococcus, sp. Pe(1)5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K(m) values of 3.0 x 10 M and 29.0 x 10 M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.

  4. Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

    PubMed

    Hühner, Jens; Ingles-Prieto, Álvaro; Neusüß, Christian; Lämmerhofer, Michael; Janovjak, Harald

    2015-02-01

    Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study.

    PubMed

    Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

    2016-11-10

    Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the functions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM.

  6. Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study

    PubMed Central

    Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

    2016-01-01

    Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the fprotein expression in unctions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM. PMID:27754828

  7. Effect on oxidative stress, hepatic chemical metabolizing parameters, and genotoxic damage of mad honey intake in rats.

    PubMed

    Eraslan, G; Kanbur, M; Karabacak, M; Arslan, K; Siliğ, Y; Soyer Sarica, Z; Tekeli, M Y; Taş, A

    2017-01-01

    A total of 66 male Wistar rats were used and six groups (control: 10 animals and experimental: 12 animals) were formed. While a separate control group was established for each study period, mad honey application to the animals in the experimental group was carried out with a single dose (12.5 g kg -1 body weight (b.w.); acute stage), at a dose of 7.5 g kg -1 b.w. for 21 days (subacute stage), and at a dose of 5 g kg -1 b.w. for 60 days (chronic stage). Tissue and blood oxidative stress markers (malondialdehyde (MDA), nitric oxide (NO), 4-hydroxynonenal (HNE), superoxide dismutase, catalase, glutathione (GSH) peroxidase, and glucose-6-phosphate dehydrogenase), hepatic chemical metabolizing parameters in the liver (cytochrome P450 2E1, nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase, nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase (CYTC), GSH S-transferase (GST), and GSH), and micronucleus and comet test in some samples were examined. Findings from the study showed that single and repeated doses given over the period increased MDA, NO, and HNE levels while decreasing/increasing tissue and blood antioxidant enzyme activities. From hepatic chemical metabolizing parameters, GST activity increased in the subacute and chronic stages and CYTC activity increased in the acute period, whereas GSH level decreased in the subacute stage. Changes in tail and head intensities were found in most of the comet results. Mad honey caused oxidative stresses for each exposure period and made some significant changes on the comet test in certain periods for some samples obtained. In other words, according to the available research results obtained, careless consumption of mad honey for different medical purposes is not appropriate.

  8. Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor.

    PubMed

    Vidal, Juan-C; Espuelas, Javier; Castillo, Juan-R

    2004-10-01

    A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.

  9. [The release of flavin adenine dinucleotide upon local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase].

    PubMed

    Lomtev, A S; Bobrov, A G; Vekshin, N L

    2004-01-01

    The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

  10. STUDIES ON MAMMALIAN AND HUMAN PYRUVATE AND ALPHA-KETOGLUTARATE DEHYDROGENATION COMPLEXES.

    DTIC Science & Technology

    Enzyme systems that catalyze a coenzyme A- and nicotinamide adenine dinucleotide-linked oxidative decarboxylation of pyruvate and alpha - ketoglutarate ...The pig heart pyruvate dehydrogenase complex was strongly inhibited by EDTA at low concentration, but the pig heart alpha - ketoglutarate ...On the oxidative decarboxylation of alpha -keto acids in pig heart complexes, Ca(2+) was strongly stimulatory to the same or more extent than Mg(2

  11. 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.

    PubMed

    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

  12. Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease.

    PubMed

    Watson, Alanna; Nong, Zengxuan; Yin, Hao; O'Neil, Caroline; Fox, Stephanie; Balint, Brittany; Guo, Linrui; Leo, Oberdan; Chu, Michael W A; Gros, Robert; Pickering, J Geoffrey

    2017-06-09

    The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD + ) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown. To determine whether a Nampt-NAD + control system exists within the aortic media and is required for aortic health. Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD + control system in SMCs impacts aortic integrity, mice with Nampt -deficient SMCs were generated. SMC- Nampt knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD + in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD + with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT. NAMPT promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with NAMPT expression. The aortic media depends on an intrinsic NAD + fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy. © 2017 American Heart

  13. An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers.

    PubMed

    Airhart, Sophia E; Shireman, Laura M; Risler, Linda J; Anderson, Gail D; Nagana Gowda, G A; Raftery, Daniel; Tian, Rong; Shen, Danny D; O'Brien, Kevin D

    2017-01-01

    The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels. Though mitochondrial dysfunction plays a critical role in the development and progression of heart failure, no mitochondria-targeted therapies have been translated into clinical practice. Recent murine studies have reported associations between imbalances in the NADH/NAD+ ratio with mitochondrial dysfunction in multiple tissues, including myocardium. Moreover, an NAD+ precursor, nicotinamide mononucleotide, improved cardiac function, while another NAD+ precursor, nicotinamide riboside (NR), improved mitochondrial function in muscle, liver and brown adipose. Thus, PK studies of NR in humans is critical for future clinical trials. In this non-randomized, open-label PK study of 8 healthy volunteers, 250 mg NR was orally administered on Days 1 and 2, then uptitrated to peak dose of 1000 mg twice daily on Days 7 and 8. On the morning of Day 9, subjects completed a 24-hour PK study after receiving 1000 mg NR at t = 0. Whole-blood levels of NR, clinical blood chemistry, and NAD+ levels were analyzed. Oral NR was well tolerated with no adverse events. Significant increases comparing baseline to mean concentrations at steady state (Cave,ss) were observed for both NR (p = 0.03) and NAD+ (p = 0.001); the latter increased by 100%. Absolute changes from baseline to Day 9 in NR and NAD+ levels correlated highly (R2 = 0.72, p = 0.008). Because NR increases circulating NAD+ in humans, NR may have potential as a therapy in patients with mitochondrial dysfunction due to genetic and/or acquired diseases.

  14. An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers

    PubMed Central

    Airhart, Sophia E.; Shireman, Laura M.; Risler, Linda J.; Anderson, Gail D.; Nagana Gowda, G. A.; Raftery, Daniel; Tian, Rong; Shen, Danny D.; O’Brien, Kevin D.

    2017-01-01

    Objectives The co-primary objectives of this study were to determine the human pharmacokinetics (PK) of oral NR and the effect of NR on whole blood nicotinamide adenine dinucleotide (NAD+) levels. Background Though mitochondrial dysfunction plays a critical role in the development and progression of heart failure, no mitochondria-targeted therapies have been translated into clinical practice. Recent murine studies have reported associations between imbalances in the NADH/NAD+ ratio with mitochondrial dysfunction in multiple tissues, including myocardium. Moreover, an NAD+ precursor, nicotinamide mononucleotide, improved cardiac function, while another NAD+ precursor, nicotinamide riboside (NR), improved mitochondrial function in muscle, liver and brown adipose. Thus, PK studies of NR in humans is critical for future clinical trials. Methods In this non-randomized, open-label PK study of 8 healthy volunteers, 250 mg NR was orally administered on Days 1 and 2, then uptitrated to peak dose of 1000 mg twice daily on Days 7 and 8. On the morning of Day 9, subjects completed a 24-hour PK study after receiving 1000 mg NR at t = 0. Whole-blood levels of NR, clinical blood chemistry, and NAD+ levels were analyzed. Results Oral NR was well tolerated with no adverse events. Significant increases comparing baseline to mean concentrations at steady state (Cave,ss) were observed for both NR (p = 0.03) and NAD+ (p = 0.001); the latter increased by 100%. Absolute changes from baseline to Day 9 in NR and NAD+ levels correlated highly (R2 = 0.72, p = 0.008). Conclusions Because NR increases circulating NAD+ in humans, NR may have potential as a therapy in patients with mitochondrial dysfunction due to genetic and/or acquired diseases. PMID:29211728

  15. Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3beta.

    PubMed

    Feng, Jianhua; Lucchinetti, Eliana; Ahuja, Preeti; Pasch, Thomas; Perriard, Jean-Claude; Zaugg, Michael

    2005-11-01

    Postischemic administration of volatile anesthetics activates reperfusion injury salvage kinases and decreases myocardial damage. However, the mechanisms underlying anesthetic postconditioning are unclear. Isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane (1.5 minimum alveolar concentration) administered at the onset of reperfusion. In some experiments, atractyloside (10 microm), a mitochondrial permeability transition pore (mPTP) opener, and LY294002 (15 microm), a phosphatidylinositol 3-kinase inhibitor, were coadministered with isoflurane. Western blot analysis was used to determine phosphorylation of protein kinase B/Akt and its downstream target glycogen synthase kinase 3beta after 15 min of reperfusion. Myocardial tissue content of nicotinamide adenine dinucleotide served as a marker for mPTP opening. Accumulation of MitoTracker Red 580 (Molecular Probes, Invitrogen, Basel, Switzerland) was used to visualize mitochondrial function. Anesthetic postconditioning significantly improved functional recovery and decreased infarct size (36 +/- 1% in unprotected hearts vs. 3 +/- 2% in anesthetic postconditioning; P < 0.05). Isoflurane-mediated protection was abolished by atractyloside and LY294002. LY294002 inhibited isoflurane-induced phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3beta and opened mPTP as determined by nicotinamide adenine dinucleotide measurements. Atractyloside, a direct opener of the mPTP, did not inhibit phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3beta by isoflurane but reversed isoflurane-mediated cytoprotection. Microscopy showed accumulation of the mitochondrial tracker in isoflurane-protected functional mitochondria but no staining in mitochondria of unprotected hearts. Anesthetic postconditioning by isoflurane effectively protects against reperfusion damage by preventing

  16. Nicotinamide riboside attenuates alcohol induced liver injuries via activation of SirT1/PGC-1α/mitochondrial biosynthesis pathway.

    PubMed

    Wang, Sufan; Wan, Ting; Ye, Mingtong; Qiu, Yun; Pei, Lei; Jiang, Rui; Pang, Nengzhi; Huang, Yuanling; Liang, Baoxia; Ling, Wenhua; Lin, Xiaojun; Zhang, Zhenfeng; Yang, Lili

    2018-07-01

    Nicotinamide riboside (NR) is a nicotinamide adenine dinucleotide (NAD + ) precursor which is present in foods such as milk and beer. It was reported that NR can prevent obesity, increase longevity, and promote liver regeneration. However, whether NR can prevent ethanol-induced liver injuries is not known. This study aimed to explore the effect of NR on ethanol induced liver injuries and the underlying mechanisms. We fed C57BL/6 J mice with Lieber-DeCarli ethanol liquid diet with or without 400 mg/kg·bw NR for 16 days. Liver injuries and SirT1-PGC-1α-mitochondrial function were analyzed. In in vitro experiments, HepG2 cells (CYP2E1 over-expressing cells) were incubated with ethanol ± 0.5 mmol/L NR. Lipid accumulation and mitochondrial function were compared. SirT1 knockdown in HepG2 cells were further applied to confirm the role of SirT1 in the protection of NR on lipid accumulation. We found that ethanol significantly decreased the expression and activity of hepatic SirT1 and induced abnormal expression of enzymes of lipid metabolism in mice. Both in vivo and in vitro experiments showed that NR activated SirT1 through increasing NAD + levels, decreased oxidative stress, increased deacetylation of PGC-1α and mitochondrial function. In SirT1 knockdown HepG2 cells, NR lost its ability in enhancing mitochondrial function, and its protection against lipid accumulation induced by ethanol. NR can protect against ethanol induced liver injuries via replenishing NAD + , reducing oxidative stress, and activating SirT1-PGC-1α-mitochondrial biosynthesis. Our data indicate that SirT1 plays an important role in the protection of NR against lipid accumulation and mitochondrial dysfunctions induced by ethanol. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

    PubMed

    Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

    2012-07-01

    We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

  18. ROS open roads to roundworm infection.

    PubMed

    Feng, Baomin; Shan, Libo

    2014-04-08

    The rapid production of reactive oxygen species (ROS) upon pathogen attack is generally considered a defense mechanism for microbial killing and an initiation of host defense responses in plants and animals. In this issue, Siddique et al. show that nicotinamide adenine dinucleotide phosphate oxidase-derived ROS function as a pathogenicity factor to promote the roundworm nematode infection in Arabidopsis thaliana, revealing the complex action of ROS in host-pathogen interactions.

  19. Unprecedented head-to-head right-handed cross-links between the antitumor bis(mu-N,N'-di-p-tolylformamidinate) dirhodium(II,II) core and the dinucleotide d(ApA) with the adenine bases in the rare imino form.

    PubMed

    Chifotides, Helen T; Dunbar, Kim R

    2007-10-17

    Reactions of the anticancer active compound cis-[Rh2(DTolF)2(CH3CN)6](BF4)2 with 9-ethyladenine (9-EtAdeH) or the dinucleotide d(ApA) proceed with bridging adenine bases in the rare imino form (A*), spanning the Rh-Rh bond at equatorial positions via N7/N6. The inflection points for the pH-dependent H2 and H8 NMR resonance curves of cis-[Rh2(DTolF)2(9-EtAdeH)2](BF4)2 correspond to N1H deprotonation of the metal-stabilized rare imino tautomer, which takes place at pKa approximately 7.5 in CD3CN-d3, a considerably reduced value as compared to that of the imino form of 9-EtAdeH. Similarly, coordination of the metal atoms to the N7/N6 adenine sites in Rh2(DTolF)2{d(ApA)} induces formation of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H sites (pKa approximately 7.0 in CD3CN-d3), as compared to the imino form of the free dinucleotide. The presence of the adenine bases in the rare imino form, due to bidentate metalation of the N6/N7 sites, is further corroborated by DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of Rh2(DTolF)2{d(ApA)} in CD3CN-d3 at -38 degrees C. Due to the N7/N6 bridging mode of the adenine bases in Rh2(DTolF)2{d(ApA)}, only the anti orientation of the imino tautomer is possible. The imino form A* of adenine in DNA may result in AT-->CG transversions or AT-->GC transitions, which can eventually lead to lethal mutations. The HH arrangement of the bases in Rh2(DTolF)2{d(ApA)} is indicated by the H8/H8 NOE cross-peaks in the 2D ROESY NMR spectrum, whereas the formamidinate bridging groups dictate the presence of one right-handed conformer HH1R in solution. Complete characterization of Rh2(DTolF)2{d(ApA)} by 2D NMR spectroscopy and molecular modeling supports the presence of the HH1R conformer, anti orientation of both sugar residues about the glycosyl bonds, and N-type conformation for the 5'-A base.

  20. Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

    PubMed

    Conforti, Laura; Wilbrey, Anna; Morreale, Giacomo; Janeckova, Lucie; Beirowski, Bogdan; Adalbert, Robert; Mazzola, Francesca; Di Stefano, Michele; Hartley, Robert; Babetto, Elisabetta; Smith, Trevor; Gilley, Jonathan; Billington, Richard A; Genazzani, Armando A; Ribchester, Richard R; Magni, Giulio; Coleman, Michael

    2009-02-23

    The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

  1. Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

    PubMed

    Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

    Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

  2. Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Koenig, Karsten

    1992-07-01

    The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NADPH fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor of 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior--probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component was most pronounced for the intact strain DL1. Time- resolved fluorescence seems therefore to be an appropriate method of probe the function of the respiratory chain and--in the further step--to differentiate between various types of cells and tissues in medical diagnosis or environmental research.

  3. Catabolism of Phloroglucinol by the Rumen Anaerobe Coprococcus

    PubMed Central

    Patel, T. R.; Jure, K. G.; Jones, G. A.

    1981-01-01

    A rumen isolate, Coprococcus, sp. Pe15, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with Km values of 3.0 × 10−5 M and 29.0 × 10−5 M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique. PMID:16345897

  4. Blood modulates the kinetics of reactive oxygen release in pancreatic ischemia-reperfusion injury.

    PubMed

    Neeff, Hannes P; Sommer, Olaf; Meyer, Sebastian; Tinelli, Anja; Scholtes, Moritz; Hopt, Ulrich T; Drognitz, Oliver; von Dobschuetz, Ernst

    2012-10-01

    Reason for the unsuccessful use of antioxidants in transplantation might be the unknown kinetics of reactive oxygen species (ROS) release. In this study, we compared the kinetics of ROS release from rat pancreata in the presence and absence of blood. In vivo, ischemia-reperfusion injury (IRI) was induced in pancreata of male Wistar rats by occlusion of the arterial blood supply for 1 or 2 hours. In vitro, isolated pancreata were single-pass perfused with Krebs-Henseleit bicarbonate solution. Reactive oxygen species were quantified by electron spin resonance spectroscopy using CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) as spin label. Thiols (glutathione), nicotinamide adenine dinucleotide phosphate-oxidase activity, myeloperoxidase activity, and adenosine triphosphate content were measured. During reperfusion, an increase in IRI-induced ROS in arterial blood was noted after 2 hours of warm ischemia. In sharp contrast, ROS release was immediate and short lived in blood-free perfused organs. The degree of tissue damage correlated with nicotinamide adenine dinucleotide phosphate-oxidase activity and adenosine triphosphate content. Antioxidative capacity of tissues was reduced. Electron spin resonance spectroscopy in conjunction with spin labels allows for the detection of ROS kinetics in pancreatic IRI. Reactive oxygen species kinetics are dependent on the length of the ischemic period and the presence or absence of blood.

  5. Human sperm NADH and NADPH diaphorase cytochemistry: correlation with sperm motility.

    PubMed

    Zini, A; O'Bryan, M K; Israel, L; Schlegel, P N

    1998-03-01

    We have examined the correlation between the retention of residual sperm cytoplasm and sperm motility in semen from men presenting for infertility evaluation. Semen samples (n = 12) were obtained from nonazoospermic men presenting for infertility evaluation at our institution. Samples were fractionated into high-, intermediate-, and low-density subpopulations by Percoll gradients in order to examine the correlation between the retention of residual sperm cytoplasm and sperm motility. Residual sperm cytoplasm retention was detected by cytochemical staining of sperm for nicotinamide adenine dinucleotide (NADH)- or nicotinamide adenine dinucleotide phosphate (NADPH)-dependent diaphorase activity. The different sperm subpopulations (low, intermediate, and high density) had significantly different percentages of sperm with droplet retention (analysis of variance, P < 0.05). Using either NADH or NADPH diaphorase staining as a marker of the cytoplasmic space, a significant negative correlation was observed between the percentage of sperm with residual cytoplasmic droplets and the percentage of motile sperm (r = -0.58 and -0.61, respectively, P < 0.05). Assessment of residual sperm cytoplasm retention is a simple diagnostic test. Although this test is of unproven value in the management of infertile men, this and other studies suggest that it may provide useful data on sperm function.

  6. Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation.

    PubMed

    Yoon, Ji Hye; Lim, Tae-Gyu; Lee, Kyung Mi; Jeon, Ae Ji; Kim, Su Yeon; Lee, Ki Won

    2011-01-12

    The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.

  7. Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

    PubMed

    Brickman, Timothy J; Suhadolc, Ryan J; McKelvey, Pamela J; Armstrong, Sandra K

    2017-02-01

    Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis. © 2016 John Wiley & Sons Ltd.

  8. Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kang, Liang; Hu, Jia; Weng, Yuxiong

    Intervertebral disc degeneration (IDD) is marked by imbalanced metabolism of the extracellular matrix (ECM) in the nucleus pulposus (NP) of intervertebral discs. This study aimed to determine whether sirtuin 6 (SIRT6), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, protects the NP from ECM degradation in IDD. Our study showed that expression of SIRT6 markedly decreased during IDD progression. Overexpression of wild-type SIRT6, but not a catalytically inactive mutant, prevented IL-1β-induced NP ECM degradation. SIRT6 depletion by RNA interference in NP cells caused ECM degradation. Moreover, SIRT6 physically interacted with nuclear factor-κB (NF-κB) catalytic subunit p65, transcriptionalmore » activity of which was significantly suppressed by SIRT6 overexpression. These results suggest that SIRT6 prevented NP ECM degradation in vitro via inhibiting NF-κB-dependent transcriptional activity and that this effect depended on its deacetylase activity. - Highlights: • SIRT6 expression is decreased in degenerative nucleus pulposus (NP) tissues. • SIRT6 overexpression lowers IL-1β-induced matrix degradation of NP. • SIRT6 inhibition induces matrix degradation of NP. • SIRT6 prevents matrix degradation of NP via the NF-κB signaling pathway.« less

  9. The B Vitamins Nicotinamide (B3) and Riboflavin (B2) Stimulate Metamorphosis in Larvae of the Deposit-Feeding Polychaete Capitella teleta: Implications for a Sensory Ligand-Gated Ion Channel

    PubMed Central

    Burns, Robert T.; Pechenik, Jan A.; Biggers, William J.; Scavo, Gia; Lehman, Christopher

    2014-01-01

    Marine sediments can contain B vitamins, presumably incorporated from settled, decaying phytoplankton and microorganisms associated with decomposition. Because B vitamins may be advantageous for the energetically intensive processes of metamorphosis, post-metamorphic growth, and reproduction, we tested several B vitamins to determine if they would stimulate larvae of the deposit-feeding polychaete Capitella teleta to settle and metamorphose. Nicotinamide and riboflavin individually stimulated larvae of C. teleta to settle and metamorphose, generally within 1–2 hours at nicotinamide concentrations as low as 3 µM and riboflavin concentrations as low as 50 µM. More than 80% of the larvae metamorphosed within 30 minutes at a nicotinamide concentration of 7 µM. The pyridine channel agonist pyrazinecarboxamide also stimulated metamorphosis at very low concentrations. In contrast, neither lumichrome, thiamine HCl, pyridoxine HCl, nor vitamin B12 stimulated larvae of C. teleta to metamorphose at concentrations as high as 500 µM. Larvae also did not metamorphose in response to either nicotinamide or pyrazinecarboxamide in calcium-free seawater or with the addition of 4-acetylpyridine, a competitive inhibitor of the pyridine receptor. Together, these results suggest that larvae of C. teleta are responding to nicotinamide and riboflavin via a chemosensory pyridine receptor similar to that previously reported to be present on crayfish chela and involved with food recognition. Our data are the first to implicate B vitamins as possible natural chemical settlement cues for marine invertebrate larvae. PMID:25390040

  10. Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways

    NASA Astrophysics Data System (ADS)

    Shirshin, Evgeny; Cherkasova, Olga; Tikhonova, Tatiana; Berlovskaya, Elena; Priezzhev, Alexander; Fadeev, Victor

    2015-05-01

    We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

  11. Nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus; the H+/H- ratio and the activation state of the enzyme during reduction of acetyl pyridine adenine dinucleotide.

    PubMed

    Palmer, T; Jackson, J B

    1992-02-21

    Chromatophores from Rhodobacter capsulatus were incubated in the dark with NADPH and acetylpyridineadenine dinucleotide (AcPdAD+) in the presence of different concentrations of myxothiazol. The transhydrogenase activity was monitored until an appropriate mass action ratio, [AcPdAD+][NADPH]/[AcPdADH][NADP+], was reached. The sample was then illuminated and the initial rate of either AcPdAD+ reduction by NADPH or AcPdADH oxidation by NADP+ was recorded. The ratio of H+ translocated per H- equivalent transferred by transhydrogenase was calculated from the value of the membrane potential (delta pH = 0) at which illumination caused no net reaction in either direction. The mean value for the H+/H- ratio was 0.55. At greater values of [AcPdAD+][NADPH]/[AcPdADH][NADP+] than were employed in the above experiments and over a wider range of concentrations of myxothiazol, it was found that incremental increases in the membrane potential always gave rise to a decrease, never an increase in the rate of AcPdAD+ reduction. In contrast to the H(+)-ATP synthase, there is no evidence of any activation/deactivation of H(+)-transhydrogenase by the protonmotive force.

  12. Quantitative Analysis of NAD Synthesis-Breakdown Fluxes.

    PubMed

    Liu, Ling; Su, Xiaoyang; Quinn, William J; Hui, Sheng; Krukenberg, Kristin; Frederick, David W; Redpath, Philip; Zhan, Le; Chellappa, Karthikeyani; White, Eileen; Migaud, Marie; Mitchison, Timothy J; Baur, Joseph A; Rabinowitz, Joshua D

    2018-05-01

    The redox cofactor nicotinamide adenine dinucleotide (NAD) plays a central role in metabolism and is a substrate for signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. NAD concentration falls during aging, which has triggered intense interest in strategies to boost NAD levels. A limitation in understanding NAD metabolism has been reliance on concentration measurements. Here, we present isotope-tracer methods for NAD flux quantitation. In cell lines, NAD was made from nicotinamide and consumed largely by PARPs and sirtuins. In vivo, NAD was made from tryptophan selectively in the liver, which then excreted nicotinamide. NAD fluxes varied widely across tissues, with high flux in the small intestine and spleen and low flux in the skeletal muscle. Intravenous administration of nicotinamide riboside or mononucleotide delivered intact molecules to multiple tissues, but the same agents given orally were metabolized to nicotinamide in the liver. Thus, flux analysis can reveal tissue-specific NAD metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. A Phosphoenzyme Mimic, Overlapping Catalytic Sites and Reaction Coordinate Motion for Human NAMPT

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Burgos, E.; Ho, M; Almo, S

    2009-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD+) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF3- as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reactionmore » coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.« less

  14. The Vitamin Nicotinamide: Translating Nutrition into Clinical Care

    PubMed Central

    Maiese, Kenneth; Chong, Zhao Zhong; Hou, Jinling; Shang, Yan Chen

    2009-01-01

    Nicotinamide, the amide form of vitamin B3 (niacin), is changed to its mononucleotide compound with the enzyme nicotinic acide/nicotinamide adenylyl-transferase, and participates in the cellular energy metabolism that directly impacts normal physiology. However, nicotinamide also influences oxidative stress and modulates multiple pathways tied to both cellular survival and death. During disorders that include immune system dysfunction, diabetes, and aging-related diseases, nicotinamide is a robust cytoprotectant that blocks cellular inflammatory cell activation, early apoptotic phosphatidylserine exposure, and late nuclear DNA degradation. Nicotinamide relies upon unique cellular pathways that involve forkhead transcription factors, sirtuins, protein kinase B (Akt), Bad, caspases, and poly (ADP-ribose) polymerase that may offer a fine line with determining cellular longevity, cell survival, and unwanted cancer progression. If one is cognizant of the these considerations, it becomes evident that nicotinamide holds great potential for multiple disease entities, but the development of new therapeutic strategies rests heavily upon the elucidation of the novel cellular pathways that nicotinamide closely governs. PMID:19783937

  15. Streptozotocin Diabetes CORRELATION WITH EXTENT OF DEPRESSION OF PANCREATIC ISLET NICOTINAMIDE ADENINE DINUCLEOTIDE

    PubMed Central

    Anderson, Tom; Schein, Philip S.; McMenamin, Mary G.; Cooney, David A.

    1974-01-01

    The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 μmol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 μmol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 μmol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 μmol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-14C]streptozotocin by islets was 3.8 times that of [methyl-14C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets. PMID:4369217

  16. Nicotinamide dependence of uropathogenic Escherichia coli UTI89 and application of nadB as a neutral insertion site.

    PubMed

    Li, Zhaoli; Bouckaert, Julie; Deboeck, Francine; De Greve, Henri; Hernalsteens, Jean-Pierre

    2012-03-01

    NAD and NADP are ubiquitous in the metabolism of Escherichia coli K-12. NAD auxotrophy can be rendered by mutation in any of the three genes nadB, nadA and nadC. The nadB and nadA genes were defined as antivirulence loci in Shigella spp., as a mutation (mainly in nadB) disrupting the synthesis of quinolinate is required for virulence. Uropathogenic E. coli (UPEC) isolates from acute cystitis patients, exhibiting nicotinamide auxotrophy, were of serotype O18 : K1 : H7. E. coli UTI89, the model uropathogenic and O18 : K1 : H7 strain, requires nicotinamide or quinolinate for growth. A mutation in the nadB gene, encoding L-aspartate oxidase, was shown to be responsible for the nicotinamide requirement of UTI89. This was further confirmed by complementation of UTI89 with a recombinant plasmid harbouring the nadB gene of E. coli K-12. An Ala28Val point mutant of the recombinant plasmid failed to support the growth of UTI89 in minimal medium. This proves that the Ala28Val mutation in the NadB gene of UTI89 completely impedes de novo synthesis of nicotinamide. In spontaneous prototrophic revertants of UTI89, the nadB gene has a Val28Ala mutation. Both analyses implicate that the nicotinamide auxotrophy of UTI89 is caused by a single Ala28Val mutation in NadB. We showed that the same mutation is also present in other NAD auxotrophic E. coli O18 strains. No significant differences were observed between the virulence of isogenic NAD auxotrophic and prototrophic strains in the murine ascending urinary tract infection model. Considering these data, we applied the nadB locus as a neutral site for DNA insertions in the bacterial chromosome. We successfully restored the parental phenotype of a fimH mutant by inserting fimH, with a synthetic em7 promoter, into the nadB gene. This neutral insertion site is of significance for further research on the pathogenicity of UPEC.

  17. Enhancement of ethanol-oxygen biofuel cell output using a CNT based nano-composite as bioanode.

    PubMed

    Gouranlou, Farideh; Ghourchian, Hedayatollah

    2016-04-15

    The present research, describes preparation and application of a novel bioanode for ethanol-oxygen biofuel cells. We applied an enzyme based nanocomposite consisting of polymethylene green as electron transfer mediator, carboxylated-multiwall carbon nanotubes as electron transfer accelerator, alcohol dehydrogenase as biocatalyst and polydiallyldimethylammonium chloride as supporting agent. In the presence of β-nicotinamide adenine dinucleotide as cofactor, and ethanol as fuel, the feasibility of the bioanode for increasing the power was evaluated under the ambient conditions. In the optimum conditions the biofuel cell produced the power density of 1.713 mW cm(-2) and open circuit voltage of 0.281 V. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells*

    PubMed Central

    Arredouani, Abdelilah; Ruas, Margarida; Collins, Stephan C.; Parkesh, Raman; Clough, Frederick; Pillinger, Toby; Coltart, George; Rietdorf, Katja; Royle, Andrew; Johnson, Paul; Braun, Matthias; Zhang, Quan; Sones, William; Shimomura, Kenju; Morgan, Anthony J.; Lewis, Alexander M.; Chuang, Kai-Ting; Tunn, Ruth; Gadea, Joaquin; Teboul, Lydia; Heister, Paula M.; Tynan, Patricia W.; Bellomo, Elisa A.; Rutter, Guy A.; Rorsman, Patrik; Churchill, Grant C.; Parrington, John; Galione, Antony

    2015-01-01

    Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells. PMID:26152717

  19. Application of Negative Curvature Hollow-Core Fiber in an Optical Fiber Sensor Setup for Multiphoton Spectroscopy

    PubMed Central

    Stawska, Hanna Izabela; Mazur, Leszek Mateusz; Kosolapov, Alexey; Kolyadin, Anton; Bereś-Pawlik, Elżbieta

    2017-01-01

    In this paper, an application of negative curvature hollow core fiber (NCHCF) in an all-fiber, multiphoton fluorescence sensor setup is presented. The dispersion parameter (D) of this fiber does not exceed the value of 5 ps/nm × km across the optical spectrum of (680–750) nm, making it well suited for the purpose of multiphoton excitation of biological fluorophores. Employing 1.5 m of this fiber in a simple, all-fiber sensor setup allows us to perform multiphoton experiments without any dispersion compensation methods. Multiphoton excitation of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) with this fiber shows a 6- and 9-fold increase, respectively, in the total fluorescence signal collected when compared with the commercial solution in the form of a hollow-core photonic band gap fiber (HCPBF). To the author’s best knowledge, this is the first time an NCHCF was used in an optical-fiber sensor setup for multiphoton fluorescence experiments. PMID:28984838

  20. Application of Negative Curvature Hollow-Core Fiber in an Optical Fiber Sensor Setup for Multiphoton Spectroscopy.

    PubMed

    Popenda, Maciej Andrzej; Stawska, Hanna Izabela; Mazur, Leszek Mateusz; Jakubowski, Konrad; Kosolapov, Alexey; Kolyadin, Anton; Bereś-Pawlik, Elżbieta

    2017-10-06

    In this paper, an application of negative curvature hollow core fiber (NCHCF) in an all-fiber, multiphoton fluorescence sensor setup is presented. The dispersion parameter (D) of this fiber does not exceed the value of 5 ps/nm × km across the optical spectrum of (680-750) nm, making it well suited for the purpose of multiphoton excitation of biological fluorophores. Employing 1.5 m of this fiber in a simple, all-fiber sensor setup allows us to perform multiphoton experiments without any dispersion compensation methods. Multiphoton excitation of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) with this fiber shows a 6- and 9-fold increase, respectively, in the total fluorescence signal collected when compared with the commercial solution in the form of a hollow-core photonic band gap fiber (HCPBF). To the author's best knowledge, this is the first time an NCHCF was used in an optical-fiber sensor setup for multiphoton fluorescence experiments.

  1. Ultrafast photo-initiated molecular quantum dynamics in the DNA dinucleotide d(ApG) revealed by broadband transient absorption spectroscopy.

    PubMed

    Stuhldreier, Mayra C; Temps, Friedrich

    2013-01-01

    The ultrafast photo-initiated quantum dynamics of the adenine-guanine dinucleotide d(ApG) in aqueous solution (pH 7) has been studied by femtosecond time-resolved spectroscopy after excitation at lambda = 260 nm. The results reveal a hierarchy of processes on time scales from tau < 100 fs to tau > 100 ps. Characteristic spectro-temporal signatures are observed indicating the transformation of the molecules in the electronic relaxation from the photo-excited state to a long-lived exciplex. In particular, broadband UV/VIS excited-state absorption (ESA) measurements detected a distinctive absorption by the excited dinucleotide around lambda = 335 nm, approximately 0.5 eV to the blue compared to the maximum of the broad and unstructured ESA spectrum after excitation of an equimolar mixture of the mononucleotides dAMP and dGMP. A similar feature has been identified as signature of the excimer in the dynamics of the adenine dinucleotide d(ApA). The lifetime of the d(ApG) exciplex was found to be tau = 124 +/- 4 ps both from the ESA decay time and from the ground-state recovery time, far longer than the sub-picosecond lifetimes of excited dAMP or dGMP. Fluorescence-time profiles measured by the up-conversion technique indicate that the exciplex state is reached around approximately 6 ps after excitation. Very weak residual fluorescence at longer times red-shifted to the emission from the photo-excited state shows that the exciplex is almost optically dark, but still has enough oscillator strength to give rise to the dual fluorescence of the dinucleotide in the static fluorescence spectrum.

  2. The 2.5 Å Crystal Structure of the SIRT1 Catalytic Domain Bound to Nicotinamide Adenine Dinucleotide (NAD + ) and an Indole (EX527 Analogue) Reveals a Novel Mechanism of Histone Deacetylase Inhibition

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhao, Xun; Allison, Dagart; Condon, Bradley

    2013-02-14

    The sirtuin SIRT1 is a NAD+-dependent histone deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPARγ, NFκB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241–516) bound to NAD+ and the 27 analogue compound 35. 35 binds deep in themore » catalytic cleft, displacing the NAD+ nicotinamide and forcing the cofactor into an extended conformation. The extended NAD+ conformation sterically prevents substrate binding. The SIRT1/NAD+/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.« less

  3. Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

    PubMed

    Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

    2010-07-01

    Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

  4. Ethanol and liver: Recent insights into the mechanisms of ethanol-induced fatty liver

    PubMed Central

    Liu, Jinyao

    2014-01-01

    Alcoholic fatty liver disease (AFLD), a potentially pathologic condition, can progress to steatohepatitis, fibrosis, and cirrhosis, leading to an increased probability of hepatic failure and death. Alcohol induces fatty liver by increasing the ratio of reduced form of nicotinamide adenine dinucleotide to oxidized form of nicotinamide adenine dinucleotide in hepatocytes; increasing hepatic sterol regulatory element-binding protein (SREBP)-1, plasminogen activator inhibitor (PAI)-1, and early growth response-1 activity; and decreasing hepatic peroxisome proliferator-activated receptor-α activity. Alcohol activates the innate immune system and induces an imbalance of the immune response, which is followed by activated Kupffer cell-derived tumor necrosis factor (TNF)-α overproduction, which is in turn responsible for the changes in the hepatic SREBP-1 and PAI-1 activity. Alcohol abuse promotes the migration of bone marrow-derived cells (BMDCs) to the liver and then reprograms TNF-α expression from BMDCs. Chronic alcohol intake triggers the sympathetic hyperactivity-activated hepatic stellate cell (HSC) feedback loop that in turn activates the HSCs, resulting in HSC-derived TNF-α overproduction. Carvedilol may block this feedback loop by suppressing sympathetic activity, which attenuates the progression of AFLD. Clinical studies evaluating combination therapy of carvedilol with a TNF-α inhibitor to treat patients with AFLD are warranted to prevent the development of alcoholic liver disease. PMID:25356030

  5. Two continuous coupled assays for ornithine-δ-aminotransferase.

    PubMed

    Juncosa, Jose I; Lee, Hyunbeom; Silverman, Richard B

    2013-09-15

    We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

    PubMed

    Kemmer, G; Reilly, T J; Schmidt-Brauns, J; Zlotnik, G W; Green, B A; Fiske, M J; Herbert, M; Kraiss, A; Schlör, S; Smith, A; Reidl, J

    2001-07-01

    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

  7. NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

    PubMed Central

    Kemmer, Gabriele; Reilly, Thomas J.; Schmidt-Brauns, Joachim; Zlotnik, Gary W.; Green, Bruce A.; Fiske, Michael J.; Herbert, Mark; Kraiß, Anita; Schlör, Stefan; Smith, Arnold; Reidl, Joachim

    2001-01-01

    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5′-nucleotidase activity. The e (P4) protein is also shown to have NMN 5′-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed. PMID:11395461

  8. [Reference values of niacin for the Venezuelan population].

    PubMed

    Carias, Diamela; García-Casal, María Nieves; Soto de Sanabria, Ingrid; López Rodríguez, Ana Victoria

    2013-12-01

    Niacin is a water soluble vitamin, also known as nicotinic acid or Vitamin B3. Nicotinamide is a derivative of niacin (amide of nicotinic acid), and is used by the body to produce the coenzyme nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), In preparing this review about the niacin requirements for Venezuela, it was found that there is not enough national data to establish recommendations, therefore, as in the 2000 review of the Venezuelan Recommended Dietary Allowances (RDAs), the actual recommendations for intake of niacin, are based on those reported for the United States population. The RDAs for Venezuela are: 2-4 mg/day for infants less than 1 year old, 6-8 mg/day for children between 1 and 8 years, 12 mg/day for children between 9 and 13 years, 14 mg/day for adolescents and female adults, 16 mg/day for adolescents and adult males, 18 mg/day during pregnancy and 17 mg/day during lactation. The estimated average requirement (EAR) is: 6-9 mg/day for children, 11 mg/ day for adolescents and female adults and 12 mg/day for adolescents and adult males, increasing to 14 mg/day during pregnancy and to 13 mg/day during lactation. The niacin Tolerable Upper Intake Levels (UL) are: 10 mg/day for children between 1 and 3 years, 15 mg/day for children between 4 and 8 years, 20 mg/day for children between 9 and 13 years, 30 mg/day for adolescents and 35 mg/day for adults. It is necessary to perform studies where the nutritional status of this vitamin is evaluated for different population groups, including not only the estimation of consumption, but the use of biochemical indicators, such as measuring the levels of the coenzymes NAD and NADP in erythrocytes or whole blood and determination of the major urinary metabolites of the vitamin.

  9. Crystallization of human nicotinamide phosphoribosyltransferase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takahashi, Ryo; Nakamura, Shota; Yoshida, Takuya

    2007-05-01

    Human nicotinamide phosphoribosyltransferase has been crystallized using microseeding methods and X-ray diffraction data have been collected at 2.0 Å resolution. In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate. Because the diffraction pattern of the initally obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 Å resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 Å.more » Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.« less

  10. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    PubMed

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. © 2015 by the Association of Clinical Scientists, Inc.

  11. Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

    PubMed Central

    Tylichová, Martina; Briozzo, Pierre; Kopečný, David; Ferrero, Julien; Moréra, Solange; Joly, Nathalie; Snégaroff, Jacques; Šebela, Marek

    2008-01-01

    Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Amino­aldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 Å resolution at 100 K. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 Å, β = 98.1°. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit. PMID:18259056

  12. A key role for Pre-B cell colony-enhancing factor in experimental hepatitis.

    PubMed

    Moschen, Alexander R; Gerner, Romana; Schroll, Andrea; Fritz, Teresa; Kaser, Arthur; Tilg, Herbert

    2011-08-01

    Pre-B cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase or visfatin, plays an important role in metabolic, inflammatory, and malignant diseases. Recent evidence suggests that blocking its enzymatic activity using a specific small-molecule inhibitor (FK866) might be beneficial in acute experimental inflammation. We investigated the role of PBEF in human liver disease and experimental hepatitis. PBEF serum levels and hepatic expression were determined in patients with chronic liver diseases. These studies were followed by in vivo experiments using concanavalin A (ConA) and D-galactosamine/lipopolysaccharide (LPS) models of experimental hepatitis. PBEF was either overexpressed by hydrodynamic perfusion or inhibited by FK866. In vivo findings were corroborated studying inflammatory responses of lentivirally PBEF-silenced or control FL83B mouse hepatocytes. Here, we demonstrate that PBEF serum levels were increased in patients with chronic liver diseases irrespective of disease stage and etiology. In particular, we observed enhanced PBEF expression in hepatocytes. Liver-targeted overexpression of PBEF rendered mice more susceptible to ConA- and D-galactosamine/LPS-induced hepatitis compared with control animals. In contrast, inhibition of PBEF using FK866 protected mice from ConA-induced liver damage and apoptosis. Administration of FK866 resulted in depletion of liver nicotinamide adenine dinucleotide+ levels and reduced proinflammatory cytokine expression. Additionally, FK866 protected mice in the D-galactosamine/LPS model of acute hepatitis. In vitro, PBEF-silenced mouse hepatocytes showed decreased responses after stimulation with LPS, lipoteichoic acid, and tumor necrosis factor α. In primary murine Kupffer cells, FK866 suppressed LPS-induced interleukin (IL)-6 production, whereas incubation with recombinant PBEF resulted in increased IL-6 release. Our data suggest that PBEF is of key importance in experimental hepatitis

  13. Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration.

    PubMed

    Vaur, Pauline; Brugg, Bernard; Mericskay, Mathias; Li, Zhenlin; Schmidt, Mark S; Vivien, Denis; Orset, Cyrille; Jacotot, Etienne; Brenner, Charles; Duplus, Eric

    2017-12-01

    NAD + depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD + was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD + to NR prompted us to probe the effects of NAD + and NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NAD + reduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NAD + Moreover, the stronger effect of NR compared to NAD + depends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NAD + and NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NAD + in excitotoxicity-induced AxD and that axonal protection involves defending intracellular NAD + homeostasis.-Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B 3 , protects against excitotoxicity-induced axonal degeneration. © FASEB.

  14. Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats.

    PubMed

    Cowley, Allen W; Yang, Chun; Zheleznova, Nadezhda N; Staruschenko, Alexander; Kurth, Theresa; Rein, Lisa; Kumar, Vikash; Sadovnikov, Katherine; Dayton, Alex; Hoffman, Matthew; Ryan, Robert P; Skelton, Meredith M; Salehpour, Fahimeh; Ranji, Mahsa; Geurts, Aron

    2016-02-01

    This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4, resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SS(Nox4-/-)) rats. SS(Nox4-/-) rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SS(Nox4-/-) rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67(phox), and p22(phox) were found to be significantly lower (P<0.05) in SS(Nox4-/-) compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SS(Nox4-/-) rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SS(Nox4-/-) rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4. © 2015 American Heart Association, Inc.

  15. CD38 Dictates Age-Related NAD Decline and Mitochondrial Dysfunction through an SIRT3-Dependent Mechanism.

    PubMed

    Camacho-Pereira, Juliana; Tarragó, Mariana G; Chini, Claudia C S; Nin, Veronica; Escande, Carlos; Warner, Gina M; Puranik, Amrutesh S; Schoon, Renee A; Reid, Joel M; Galina, Antonio; Chini, Eduardo N

    2016-06-14

    Nicotinamide adenine dinucleotide (NAD) levels decrease during aging and are involved in age-related metabolic decline. To date, the mechanism responsible for the age-related reduction in NAD has not been elucidated. Here we demonstrate that expression and activity of the NADase CD38 increase with aging and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity. We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo, indicating that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Lipoxin A4 inhibits UV radiation-induced skin inflammation and oxidative stress in mice.

    PubMed

    Martinez, R M; Fattori, V; Saito, P; Melo, C B P; Borghi, S M; Pinto, I C; Bussmann, A J C; Baracat, M M; Georgetti, S R; Verri, W A; Casagrande, R

    2018-04-27

    Lipoxin A4 (LXA 4 ) is a metabolic product of arachidonic acid. Despite potent anti-inflammatory and pro-resolution activities, it remains to be determined if LXA 4 has effect on ultraviolet (UV) radiation-induced skin inflammation. To investigate the effects of systemic administration with LXA 4 on UV radiation-induced inflammation and oxidative damage in the skin of mice. Varied parameters of inflammation and oxidative stress in the skin of mice were evaluated after UV radiation (4.14 J/cm 2 ). Pretreatment with LXA 4 significantly inhibited UV radiation-induced skin edema and myeloperoxidase activity. LXA 4 efficacy was enhanced by increasing the time of pre-treatment to up to 72 h. LXA 4 reduced UV radiation-induced skin edema, neutrophil recruitment (myeloperoxidase activity and LysM-eGFP + cells), MMP-9 activity, deposition of collagen fibers, epidermal thickness, sunburn cell counts, and production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-33). Depending on the time point, LXA 4 increased the levels of anti-inflammatory cytokines (TGF-β and IL-10). LXA 4 significantly attenuated UV radiation-induced oxidative damage returning the oxidative status to baseline levels in parameters such as ferric reducing ability, scavenging of free radicals, GSH levels, catalase activity and superoxide anion production. LXA 4 also reduced UV radiation-induced gp91 phox [nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) subunit] mRNA expression and enhanced nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target enzyme nicotinamide adenine dinucleotide (phosphate) quinone oxidoreductase (Nqo1) mRNA expression. LXA 4 inhibited UV radiation-induced skin inflammation by diminishing pro-inflammatory cytokine production and oxidative stress as well as inducing anti-inflammatory cytokines and Nrf2. Copyright © 2018. Published by Elsevier B.V.

  17. P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage.

    PubMed

    Wang, Gelin; Han, Ting; Nijhawan, Deepak; Theodoropoulos, Pano; Naidoo, Jacinth; Yadavalli, Sivaramakrishnan; Mirzaei, Hamid; Pieper, Andrew A; Ready, Joseph M; McKnight, Steven L

    2014-09-11

    The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

    PubMed

    Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

    2017-07-25

    Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

  19. Fluorescence spectroscopy using excitation and emission matrix for quantification of tissue native fluorophores and cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Gayen, S. K.; Xu, M.

    2014-03-01

    Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.

  20. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    NASA Astrophysics Data System (ADS)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  1. 3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1*♦

    PubMed Central

    Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

    2016-01-01

    An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3′-hydroxyl group. Both products of AvrRxo1, 3′-NADP and 3′-nicotinic acid adenine dinucleotide phosphate (3′-NAADP), are substantially different from the ubiquitous co-enzyme 2′-NADP and the calcium mobilizer 2′-NAADP. Interestingly, 3′-NADP and 3′-NAADP have previously been used as inhibitors or signaling molecules but were regarded as “artificial” compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3′-phosphorylated NAD derivatives. PMID:27621317

  2. Optical biopsy using fluorescence spectroscopy for prostate cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Native fluorescence spectra are acquired from fresh normal and cancerous human prostate tissues. The fluorescence data are analyzed using a multivariate analysis algorithm such as non-negative matrix factorization. The nonnegative spectral components are retrieved and attributed to the native fluorophores such as collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD) in tissue. The retrieved weights of the components, e.g. NADH and FAD are used to estimate the relative concentrations of the native fluorophores and the redox ratio. A machine learning algorithm such as support vector machine (SVM) is used for classification to distinguish normal and cancerous tissue samples based on either the relative concentrations of NADH and FAD or the redox ratio alone. The classification performance is shown based on statistical measures such as sensitivity, specificity, and accuracy, along with the area under receiver operating characteristic (ROC) curve. A cross validation method such as leave-one-out is used to evaluate the predictive performance of the SVM classifier to avoid bias due to overfitting.

  3. 3'-NADP and 3'-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1.

    PubMed

    Schuebel, Felix; Rocker, Andrea; Edelmann, Daniel; Schessner, Julia; Brieke, Clara; Meinhart, Anton

    2016-10-28

    An arsenal of effector proteins is injected by bacterial pathogens into the host cell or its vicinity to increase virulence. The commonly used top-down approaches inferring the toxic mechanism of individual effector proteins from the host's phenotype are often impeded by multiple targets of different effectors as well as by their pleiotropic effects. Here we describe our bottom-up approach, showing that the bacterial type III effector AvrRxo1 of plant pathogens is an authentic phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), are substantially different from the ubiquitous co-enzyme 2'-NADP and the calcium mobilizer 2'-NAADP. Interestingly, 3'-NADP and 3'-NAADP have previously been used as inhibitors or signaling molecules but were regarded as "artificial" compounds so far. Our findings now necessitate a shift in thinking about the biological importance of 3'-phosphorylated NAD derivatives. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Calcium Signaling and Reactive Oxygen Species in Mitochondria.

    PubMed

    Bertero, Edoardo; Maack, Christoph

    2018-05-11

    In heart failure, alterations of Na + and Ca 2+ handling, energetic deficit, and oxidative stress in cardiac myocytes are important pathophysiological hallmarks. Mitochondria are central to these processes because they are the main source for ATP, but also reactive oxygen species (ROS), and their function is critically controlled by Ca 2+ During physiological variations of workload, mitochondrial Ca 2+ uptake is required to match energy supply to demand but also to keep the antioxidative capacity in a reduced state to prevent excessive emission of ROS. Mitochondria take up Ca 2+ via the mitochondrial Ca 2+ uniporter, which exists in a multiprotein complex whose molecular components were identified only recently. In heart failure, deterioration of cytosolic Ca 2+ and Na + handling hampers mitochondrial Ca 2+ uptake and the ensuing Krebs cycle-induced regeneration of the reduced forms of NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate), giving rise to energetic deficit and oxidative stress. ROS emission from mitochondria can trigger further ROS release from neighboring mitochondria termed ROS-induced ROS release, and cross talk between different ROS sources provides a spatially confined cellular network of redox signaling. Although low levels of ROS may serve physiological roles, higher levels interfere with excitation-contraction coupling, induce maladaptive cardiac remodeling through redox-sensitive kinases, and cell death through mitochondrial permeability transition. Targeting the dysregulated interplay between excitation-contraction coupling and mitochondrial energetics may ameliorate the progression of heart failure. © 2018 American Heart Association, Inc.

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

    PubMed

    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.

  6. Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells*

    PubMed Central

    Ali, Ramadan A.; Camick, Christina; Wiles, Katherine; Walseth, Timothy F.; Slama, James T.; Bhattacharya, Sumit; Giovannucci, David R.; Wall, Katherine A.

    2016-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+ mobilizing second messenger discovered to date, has been implicated in Ca2+ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca2+ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca2+ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca2+ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca2+ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. PMID:26728458

  7. Immobilisation of enzymes on poly(aniline)-poly(anion) composite films. Preparation of bioanodes for biofuel cell applications.

    PubMed

    Simon, Evelyne; Halliwell, Catherine M; Toh, Chee Seng; Cass, Anthony E G; Bartlett, Philip N

    2002-01-01

    Immobilisation of enzymes is important for applications such as biosensors or biofuel cells. A poly(histidine) tag had been introduced on the C terminus of a lactate dehydrogenase enzyme. This mutant enzyme was then immobilised onto poly(aniline) (PANi)-poly(anion) composite films, PANi-poly(vinylsulfonate) (PVS) or PANi-poly(acrylate) (PAA). The NADH produced by the immobilised enzyme in the presence of beta-nicotinamide adenine dinucleotide (NAD(+)) and lactate is oxidised at the poly(aniline)-coated electrode at 0.05 to 0.1 V vs. saturated calomel electrode (SCE) at 35 degrees C.

  8. Label-free assessment of endothelial cell metabolic state using autofluorescent microscopy

    NASA Astrophysics Data System (ADS)

    Pullen, Benjamin J.; Nguyen, Tam; Gosnell, Martin; Anwer, Ayad G.; Goldys, Ewa; Nicholls, Stephen J.; Psaltis, Peter J.

    2016-12-01

    To examine the process of endothelial cell aging we utilised hyperspectral imaging to collect broad autofluorescence emission at the individual cellular level and mathematically isolate the characteristic spectra of nicotinamide and flavin adenine dinucleotides (NADH and FAD, respectively). Quantitative analysis of this data provides the basis for a non-destructive spatial imaging method for cells and tissue. FAD and NADH are important factors in cellular metabolism and have been shown to be involved with the redox state of the cell; with the ratio between the two providing the basis for an `optical redox ratio'.

  9. The role of β-adrenergic blockers in Parkinson's disease: possible genetic and cell-signaling mechanisms.

    PubMed

    Luong, Khanh vinh quoc; Nguyen, Lan Thi Hoàng

    2013-06-01

    Genetic studies have identified numerous factors linking β-adrenergic blockade to Parkinson's disease (PD), including human leukocyte antigen genes, the renin-angiotensin system, poly(adenosine diphosphate-ribose) polymerase 1, nerve growth factor, vascular endothelial growth factor, and the reduced form of nicotinamide adenine dinucleotide phosphate. β-Adrenergic blockade has also been implicated in PD via its effects on matrix metalloproteinases, mitogen-activated protein kinase pathways, prostaglandins, cyclooxygenase 2, and nitric oxide synthase. β-Adrenergic blockade may have a significant role in PD; therefore, the characterization of β-adrenergic blockade in patients with PD is needed.

  10. Cassava root diet induces low pyruvate levels.

    PubMed

    Golay, Van K

    2010-01-01

    The high cyanogenic-glucoside carbohydrate of the cassava root (Manihot esculenta) has special properties that make it an ideal therapeutic food for lowering nicotinamide adenine dinucleotide reduced form (NADH) and inducing Sirtuin (Sirt) gene overexpression when eaten in an exclusive mono-food diet regime. The author, using himself as the sole test subject, repeatedly induced low pyruvate levels (reflective of NADH levels) after being on the diet for 1-2 weeks at a time. The possible influences of exclusive cassava dieting on redox control and Sirtuin activation will be discussed.

  11. Laser induced fluorescence of biochemical for UV LIDAR application.

    PubMed

    Gupta, L; Sharma, R C; Razdan, A K; Maini, A K

    2014-05-01

    Laser induced fluorescence spectroscopy in the ultraviolet regime has been used for the detection of biochemical through a fiber coupled CCD detector from a distance of 2 m. The effect of concentration and laser excitation energy on the fluorescence spectra of nicotinamide adenine dinucleotide (NADH) has been investigated. The signature fluorescence peak of NADH was centred about 460 nm. At lower concentration Raman peak centred at 405 nm was also observed. The origin of this peak has been discussed. Detection limit with the proposed set up is found to be 1 ppm.

  12. What is the tryptophan kynurenine pathway and why is it important to neurotherapy?

    PubMed Central

    Davis, Ian

    2015-01-01

    The kynurenine pathway has received increasing attention as its connection to inflammation, the immune system, and neurological conditions became more apparent. It is the primary route for tryptophan catabolism in the liver and the starting point for the synthesis of nicotinamide adenine dinucleotide in mammals. Dysregulation or overactivation of this pathway can lead to immune system activation and accumulation of potentially neurotoxic compounds. These aspects make the kynurenine pathway a promising target for therapeutic development to treat inflammation and some diseases with neurological aspects, especially in cancer patients undergoing chemotherapy. PMID:26004930

  13. Head to Head Comparison of Short-Term Treatment with the NAD(+) Precursor Nicotinamide Mononucleotide (NMN) and 6 Weeks of Exercise in Obese Female Mice.

    PubMed

    Uddin, Golam M; Youngson, Neil A; Sinclair, David A; Morris, Margaret J

    2016-01-01

    Obesity is well known to be a major cause of several chronic metabolic diseases, which can be partially counteracted by exercise. This is due, in part, to an upregulation of mitochondrial activity through increased nicotinamide adenine dinucleotide (NAD(+)). Recent studies have shown that NAD(+) levels can be increased by using the NAD(+) precursor, nicotinamide mononucleotide (NMN) leading to the suggestion that NMN could be a useful intervention in diet related metabolic disorders. In this study we compared the metabolic, and especially mitochondrial-associated, effects of exercise and NMN in ameliorating the consequences of high-fat diet (HFD) induced obesity in mice. Sixty female 5 week old C57BL6/J mice were allocated across five groups: Chow sedentary: CS; Chow exercise: CEX; HFD sedentary: HS; HFD NMN: HNMN; HFD exercise: HEX (12/group). After 6 weeks of diet, exercise groups underwent treadmill exercise (15 m/min for 45 min), 6 days per week for 6 weeks. NMN or vehicle (500 mg/kg body weight) was injected (i.p.) daily for the last 17 days. No significant alteration in body weight was observed in response to exercise or NMN. The HFD significantly altered adiposity, glucose tolerance, plasma insulin, NADH levels and citrate synthase activity in muscle and liver. HEX and HNMN groups both showed significantly improved glucose tolerance compared to the HS group. NAD(+) levels were increased significantly both in muscle and liver by NMN whereas exercise increased NAD(+) only in muscle. Both NMN and exercise ameliorated the HFD-induced reduction in liver citrate synthase activity. However, exercise, but not NMN, ameliorated citrate synthase activity in muscle. Overall these data suggest that while exercise and NMN-supplementation can induce similar reversal of the glucose intolerance induced by obesity, they are associated with tissue-specific effects and differential alterations to mitochondrial function in muscle and liver.

  14. Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice.

    PubMed

    Neelakantan, Harshini; Vance, Virginia; Wetzel, Michael D; Wang, Hua-Yu Leo; McHardy, Stanton F; Finnerty, Celeste C; Hommel, Jonathan D; Watowich, Stanley J

    2018-01-01

    There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD + ) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD + salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD + and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to

  15. Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

    PubMed

    Ali, Ramadan A; Camick, Christina; Wiles, Katherine; Walseth, Timothy F; Slama, James T; Bhattacharya, Sumit; Giovannucci, David R; Wall, Katherine A

    2016-02-26

    Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Association of circulating levels of nicotinamide phosphoribosyltransferase (NAMPT/Visfatin) and of a frequent polymorphism in the promoter of the NAMPT gene with coronary artery disease in diabetic and non-diabetic subjects

    PubMed Central

    2013-01-01

    Background Nicotinamide phosphoribosyltransferase (NAMPT) is the limiting enzyme in one of pathways of synthesis of Nicotinamide Adenine Dinucleotide, a redox coenzyme. NAMPT is considered as an insulin-mimetic factor and a potential regulatory factor in inflammatory and immune processes. Associations of circulating NAMPT levels with cardiovascular disease (CVD) and insulin resistance have been reported. We investigated association of circulating NAMPT levels and the rs9770242 NAMPT gene polymorphism with coronary artery disease (CAD). Methods We studied 594 Brazilian subjects undergoing a coronary angiography (49% of whom had type 2 diabetes). CAD, defined as stenosis greater than 50% in one major coronary vessel or branch, was observed in 68% of subjects. Genetic studies were also performed in 858 North-American Non-Hispanic White subjects with type 2 diabetes (49% with CAD). Results We observed an interaction between glycemic and CAD status on the comparison of NAMPT levels by CAD status. NAMPT levels were higher in type 2 diabetic patients with CAD as compared to those without CAD: 5.27 ± 2.93 ng/ml vs. 4.43 ± 2.94 ng/ml, p = 0.006 (mean ± SD). NAMPT levels were not significantly different in non-diabetic subjects with or without CAD. The T-allele of rs9770242 was associated with CAD in the Brazilian cohort (OR 1.46, 95% CI 1.06 - 2.01, p = 0.02) while no association was observed in the North-American cohort. Conclusions Our data suggest that circulating NAMPT levels are associated with CAD in type 2 diabetic patients. NAMPT rs9770242 polymorphism may be associated with CAD in some populations. PMID:23968400

  17. Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins.

    PubMed

    Martí, María C; Olmos, Enrique; Calvete, Juan J; Díaz, Isabel; Barranco-Medina, Sergio; Whelan, James; Lázaro, Juan J; Sevilla, Francisca; Jiménez, Ana

    2009-06-01

    Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

  18. Quantitative neonatal glucose-6-phosphate dehydrogenase screening: distribution, reference values, and classification by phenotype.

    PubMed

    Algur, Nurit; Avraham, Irit; Hammerman, Cathy; Kaplan, Michael

    2012-08-01

    To determine enzyme assay reference values for newborns in a Sephardic Jewish population at high risk for glucose-6-phosphate dehydrogenase (G6PD) deficiency. Quantitative G6PD testing was performed on umbilical cord blood. The reduction of nicotinamide adenine dinucleotide phosphate to nicotinamide adenine dinucleotide phosphate-oxidase, reflecting G6PD activity, was measured spectrophotometrically. Hemoglobin (Hb) was measured on the same sample. G6PD activity was recorded as U/g Hb. Males (N = 1502) were separated into 2 distinct groups: those <7 U/g Hb (n = 243 [16.2%], median 0.28 U/g Hb), designated G6PD deficient, presumably hemizygotes; and those ≥ 9 U/g Hb (n = 1256 [83.8%], 18.76 U/g Hb), designated G6PD normal, presumably hemizygotes. Female (n = 1298) values were a continuum and were categorized based on the male distribution: those <7 U/g Hb (n = 81 [6.2%], 4.84 U/g Hb), G6PD deficient, probably homozogytes; those ≥ 9.5 U/g Hb, equivalent to 50% of the male normal value, (n = 1153 (88.8%), 18.36 U/g Hb), G6PD normal, probably homozygotes; and those with intermediate values (n = 64 [4.9%], 8.61 U/g Hb), probable heterozygotes. Accurate identification of the male G6PD-deficient state was possible despite high normal neonatal G6PD values. Female values were presented as a continuum preventing accurate classification but were classified based on male phenotype for practical use. Copyright © 2012 Mosby, Inc. All rights reserved.

  19. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

    NASA Astrophysics Data System (ADS)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

    2016-03-01

    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  20. Increased rate of adenine incorporation into adenine nucleotide pool in erythrocytes of patients with chronic renal failure.

    PubMed

    Marlewski, M; Smolenski, R T; Szolkiewicz, M; Aleksandrowicz, Z; Rutkowski, B; Swierczynski, J

    2000-11-01

    Elevated purine nucleotide pool (mainly ATP) in erythrocytes of patients with chronic renal failure (CRF) is a known phenomenon, however the mechanism responsible for this abnormality is far from being clear. We hypothesize that the increased rate of adenine incorporation into adenine nucleotide pool is responsible for the elevated level of ATP in uremic erythrocytes. In chronically uremic patients we evaluated using HPLC technique: (a) plasma adenine concentration; (b) the rate of adenine incorporation into adenine nucleotide pool in uremic erythrocytes. Additionally, the effect of higher than physiological phosphate concentration (2.4 mM) and lower than physiological pH (7.1) on adenine incorporation into erythrocytes adenine nucleotide pool was investigated. Healthy volunteers with normal renal function served as control. The concentration of adenine in plasma of CRF patients was found to be significantly higher than in plasma of healthy subjects. In contrast, adenosine concentration was similar both in healthy humans and in CRF patients. In isolated erythrocytes of uremic patients (incubated in the medium pH 7.4, containing 1.2 mM inorganic phosphate) adenine was incorporated into adenine nucleotide pool at a rate approximately 2-fold higher than in erythrocytes from healthy subjects. The rate of adenosine incorporation into adenine nucleotide pool was similar in erythrocytes of both studied groups. Incubation of erythrocytes obtained from healthy subjects in the medium pH 7.4, containing 2.4 mM inorganic phosphate, caused the increase of adenine incorporation into adenine nucleotide pool by about 60%. Incubation of the cells in the pH 7.1 buffer containing 2. 4 mM inorganic phosphate increased the rate of adenine incorporation into adenylate approximately 2-fold as compared to erythrocytes incubated in the medium pH 7.4 containing 1.2 mM inorganic phosphate. Erythrocytes obtained from uremic patients and incubated in the pH 7.1 medium containing 2.4 m

  1. Evaluation of actinic cheilitis using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Cosci, Alessandro; Pratavieira, Sebastião.; Takahama, Ademar; Souza Azevedo, Rebeca; Kurachi, Cristina

    2016-03-01

    Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

  2. Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

    2017-06-01

    We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

  3. The impact of aging, hearing loss, and body weight on mouse hippocampal redox state, measured in brain slices using fluorescence imaging.

    PubMed

    Stebbings, Kevin A; Choi, Hyun W; Ravindra, Aditya; Llano, Daniel Adolfo

    2016-06-01

    The relationships between oxidative stress in the hippocampus and other aging-related changes such as hearing loss, cortical thinning, or changes in body weight are not yet known. We measured the redox ratio in a number of neural structures in brain slices taken from young and aged mice. Hearing thresholds, body weight, and cortical thickness were also measured. We found striking aging-related increases in the redox ratio that were isolated to the stratum pyramidale, while such changes were not observed in thalamus or cortex. These changes were driven primarily by changes in flavin adenine dinucleotide, not nicotinamide adenine dinucleotide hydride. Multiple regression analysis suggested that neither hearing threshold nor cortical thickness independently contributed to this change in hippocampal redox ratio. However, body weight did independently contribute to predicted changes in hippocampal redox ratio. These data suggest that aging-related changes in hippocampal redox ratio are not a general reflection of overall brain oxidative state but are highly localized, while still being related to at least one marker of late aging, weight loss at the end of life. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Electrochemical Detection of the Molecules of Life

    NASA Technical Reports Server (NTRS)

    Thomson, Seamus; Quinn, Richard; Koehne, Jessica

    2017-01-01

    All forms of life on Earth contain cellular machinery that can transform and regulate chemical energy through metabolic pathways. These processes are oxidation-reduction reactions that are performed by four key classes of molecules: flavins, nicotinamaides, porphyrins, and quinones. By detecting the electrochemical interaction of these redox-active molecules with an electrode, a method of differentiating them by their class could be established and incorporated into future life-detecting missions. This body of work investigates the electrochemistry of ubiquitous molecules found in life and how they may be detected. Molecules can oxidise or reduce the surface of an electrode - giving or receiving electrons - and these interactions are represented by changes in current with respect to an applied voltage. This relationship varies with: electrolyte type and concentration, working electrode material, the redox-active molecule itself, and scan rate. Flavin adenine dinucleotide (FAD), riboflavin, nicotinamide adenine dinucleotide (NADH), and anthraquinone are all molecules found intracellularly in almost all living organisms. An organism-synthesised extracellular redox-active molecule, Plumbagin, was also selected as part of this study. The goal of this work is to detect these molecules in seawater and assess its application in searching for life on Ocean Worlds.

  5. NAD+ : A key metabolic regulator with great therapeutic potential.

    PubMed

    Sultani, G; Samsudeen, A F; Osborne, B; Turner, N

    2017-10-01

    Nicotinamide adenine dinucleotide (NAD + ) is a ubiquitous metabolite that serves an essential role in the catabolism of nutrients. Recently, there has been a surge of interest in NAD + biology, with the recognition that NAD + influences many biological processes beyond metabolism, including transcription, signalling and cell survival. There are a multitude of pathways involved in the synthesis and breakdown of NAD + , and alterations in NAD + homeostasis have emerged as a common feature of a range of disease states. Here, we provide an overview of NAD + metabolism and summarise progress on the development of NAD + -related therapeutics. © 2017 British Society for Neuroendocrinology.

  6. Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD.

    PubMed

    Thomas, Leonard M; Harper, Angelica R; Miner, Whitney A; Ajufo, Helen O; Branscum, Katie M; Kao, Lydia; Sims, Paul A

    2013-07-01

    The crystal structure of AdhP, a recombinantly expressed alcohol dehydrogenase from Escherichia coli K-12 (substrain MG1655), was determined to 2.01 Å resolution. The structure, which was solved using molecular replacement, also included the structural and catalytic zinc ions and the cofactor nicotinamide adenine dinucleotide (NAD). The crystals belonged to space group P21, with unit-cell parameters a = 68.18, b = 118.92, c = 97.87 Å, β = 106.41°. The final R factor and Rfree were 0.138 and 0.184, respectively. The structure of the active site of AdhP suggested a number of residues that may participate in a proton relay, and the overall structure of AdhP, including the coordination to structural and active-site zinc ions, is similar to those of other tetrameric alcohol dehydrogenase enzymes.

  7. Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T-Cell Hyperactivation

    PubMed Central

    Kwon, Hye-Sook; Brent, Michael M.; Getachew, Ruth; Jayakumar, Prerana; Chen, Lin-Feng; Schnolzer, Martina; McBurney, Michael W.; Marmorstein, Ronen; Greene, Warner C.; Ott, Melanie

    2009-01-01

    Summary Symptoms of T-cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-κB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-κB-responsive genes, a function lost in SIRT1−/− cells. These results support a model where the normal function of SIRT1 as a negative regulator of T-cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals. PMID:18329615

  8. Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

    PubMed Central

    Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

    2012-01-01

    CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

  9. Co-immobilization of glucose oxidase and xylose dehydrogenase displayed whole cell on multiwalled carbon nanotube nanocomposite films modified electrode for simultaneous voltammetric detection of D-glucose and D-xylose.

    PubMed

    Li, Liang; Liang, Bo; Li, Feng; Shi, Jianguo; Mascini, Marco; Lang, Qiaolin; Liu, Aihua

    2013-04-15

    In this paper, we first report the construction of Nafion/glucose oxidase (GOD)/xylose dehydrogenase displayed bacteria (XDH-bacteria)/multiwalled carbon nanotubes (MWNTs) modified electrode for simultaneous voltammetric determination of D-glucose and D-xylose. The optimal conditions for the immobilized enzymes were established. Both enzymes retained their good stability and activities. In the mixture solution of D-glucose and D-xylose containing coenzyme NAD⁺ (the oxidized form of nicotinamide adenine dinucleotide), the Nafion/GOD/XDH-bacteria/MWNTs modified electrode exhibited quasi-reversible oxidation-reduction peak at -0.5 V (vs. saturated calomel electrode, SCE) originating from the catalytic oxidation of D-glucose, and oxidation peak at +0.55 V(vs. SCE) responding to the oxidation of NADH (the reduced form of nicotinamide adenine dinucleotide) by the carbon nanotubes, where NADH is the resultant product of coenzyme NAD⁺ involved in the catalysis of D-xylose by XDH-displayed bacteria. For the proposed biosensor, cathodic peak current at -0.5 V was linear with the concentration of D-glucose within the range of 0.25-6 mM with a low detection limit of 0.1 mM D-glucose (S/N=3), and the anodic peak current at +0.55 V was linear with the concentration of d-xylose in the range of 0.25∼4 mM with a low detection limit of 0.1 mM D-xylose (S/N=3). Further, D-xylose and D-glucose did not interfere with each other. 300-fold excess saccharides including D-maltose, D-galactose, D-mannose, D-sucrose, D-fructose, D-cellobiose, and 60-fold excess L-arabinose, and common interfering substances (100-fold excess ascorbic acid, dopamine, uric acid) as well as 300-fold excess D-xylitol did not affect the detection of D-glucose and D-xylose (both 1 mM). Therefore, the proposed biosensor is stable, specific, reproducible, simple, rapid and cost-effective, which holds great potential in real applications. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in brain mitochondria from rats and dogs.

    PubMed

    Sugiyama, Y; Fujita, T; Matsumoto, M; Okamoto, K; Imada, I

    1985-12-01

    The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe3+. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10(-5)M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10(-5)-10(-6) M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2,6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2,4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears

  11. Retinoic acid-related orphan receptor alpha reprograms glucose metabolism in glutamine-deficient hepatoma cells.

    PubMed

    Byun, Jun-Kyu; Choi, Yeon-Kyung; Kang, Yu Na; Jang, Byoung Kuk; Kang, Koo Jeong; Jeon, Yong Hyun; Lee, Ho-Won; Jeon, Jae-Han; Koo, Seung-Hoi; Jeong, Won-Il; Harris, Robert A; Lee, In-Kyu; Park, Keun-Gyu

    2015-03-01

    The metabolism of glutamine and glucose is recognized as a promising therapeutic target for the treatment of cancer; however, targeted molecules that mediate glutamine and glucose metabolism in cancer cells have not been addressed. Here, we show that restricting the supply of glutamine in hepatoma cells, including HepG2 and Hep3B cells, markedly increased the expression of retinoic acid-related orphan receptor alpha (RORα). Up-regulation of RORα in glutamine-deficient hepatoma cells resulted from an increase in the level of cellular reactive oxygen species and in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate reduced (NADP+ /NADPH) ratio, which was consistent with a reduction in the glutathione/glutathione disulfide (GSH/GSSG) ratio. Adenovirus (Ad)-mediated overexpression of RORα (Ad-RORα) or treatment with the RORα activator, SR1078, reduced aerobic glycolysis and down-regulated biosynthetic pathways in hepatoma cells. Ad-RORα and SR1078 reduced the expression of pyruvate dehydrogenase kinase 2 (PDK2) and inhibited the phosphorylation of pyruvate dehydrogenase and subsequently shifted pyruvate to complete oxidation. The RORα-mediated decrease in PDK2 levels was caused by up-regulation of p21, rather than p53. Furthermore, RORα inhibited hepatoma growth both in vitro and in a xenograft model in vivo. We also found that suppression of PDK2 inhibited hepatoma growth in a xenograft model. These findings mimic the altered glucose utilization and hepatoma growth caused by glutamine deprivation. Finally, tumor tissue from 187 hepatocellular carcinoma patients expressed lower levels of RORα than adjacent nontumor tissue, supporting a potential beneficial effect of RORα activation in the treatment of liver cancer. RORα mediates reprogramming of glucose metabolism in hepatoma cells in response to glutamine deficiency. The relationships established here between glutamine metabolism, RORα expression and signaling, and

  12. A selective and sensitive D-xylose electrochemical biosensor based on xylose dehydrogenase displayed on the surface of bacteria and multi-walled carbon nanotubes modified electrode.

    PubMed

    Li, Liang; Liang, Bo; Shi, Jianguo; Li, Feng; Mascini, Marco; Liu, Aihua

    2012-03-15

    A novel Nafion/bacteria-displaying xylose dehydrogenase (XDH)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode was fabricated and applied for the sensitive and selective determination of d-xylose (INS 967), where the XDH-displayed bacteria (XDH-bacteria) was prepared using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. The XDH-displayed bacteria can be used directly, eliminating further enzyme-extraction and purification, thus greatly improved the stability of the enzyme. The optimal conditions for the construction of biosensor were established: homogeneous Nafion-MWNTs composite dispersion (10 μL) was cast onto the inverted glassy carbon electrode, followed by casting 10-μL of XDH-bacteria aqueous solution to stand overnight to dry, then a 5-μL of Nafion solution (0.05 wt%) is syringed to the electrode surface. The bacteria-displaying XDH could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD(+) in 0.1M PBS buffer (pH7.4), where NAD(+) (nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around 0.50 V (vs. Ag/AgCl). In contrast, the bacteria-XDH-only modified electrode showed oxidation peak at higher potential of 0.7 V and less sensitivity. Therefore, the electrode/MWNTs/bacteria-XDH/Nafion exhibited good analytical performance such as long-term stability, a wide dynamic range of 0.6-100 μM and a low detection limit of 0.5 μM D-xylose (S/N=3). No interference was observed in the presence of 300-fold excess of other saccharides including D-glucose, D-fructose, D-maltose, D-galactose, D-mannose, D-sucrose, and D-cellbiose as well as 60-fold excess of L-arabinose. The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds

  13. Monocyte-derived extracellular Nampt-dependent biosynthesis of NAD+ protects the heart against pressure overload

    PubMed Central

    Yano, Masamichi; Akazawa, Hiroshi; Oka, Toru; Yabumoto, Chizuru; Kudo-Sakamoto, Yoko; Kamo, Takehiro; Shimizu, Yu; Yagi, Hiroki; Naito, Atsuhiko T.; Lee, Jong-Kook; Suzuki, Jun-ichi; Sakata, Yasushi; Komuro, Issei

    2015-01-01

    Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD+ by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD+ concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD+, were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD+ levels and NAD+-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD+ against heart failure. PMID:26522369

  14. Assimilation of NAD(+) precursors in Candida glabrata.

    PubMed

    Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

    2007-10-01

    The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.

  15. Novel Directions for Diabetes Mellitus Drug Discovery

    PubMed Central

    Maiese, Kenneth; Chong, Zhao Zhong; Shang, Yan Chen; Wang, Shaohui

    2012-01-01

    Introduction Diabetes mellitus impacts almost 200 million individuals worldwide and leads to debilitating complications. New avenues of drug discovery must target the underlying cellular processes of oxidative stress, apoptosis, autophagy, and inflammation that can mediate multi-system pathology during diabetes mellitus. Areas Covered We examine novel directions for drug discovery that involve the β-nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide, the cytokine erythropoietin, the NAD+-dependent protein histone deacetylase SIRT1, the serine/threonine-protein kinase mammalian target of rapamycin (mTOR), and the wingless pathway. Implications for the targeting of these pathways that oversee gluconeogenic genes, insulin signaling and resistance, fatty acid beta-oxidation, inflammation, and cellular survival are presented. Expert Opinion Nicotinamide, erythropoietin, and the downstram pathways of SIRT1, mTOR, forkhead transcription factors, and wingless signaling offer exciting prospects for novel directions of drug discovery for the treatment of metabolic disorders. Future investigations must dissect the complex relationship and fine modulation of these pathways for the successful translation of robust reparative and regenerative strategies against diabetes mellitus and the complications of this disorder. PMID:23092114

  16. Smoking-Induced Upregulation of AKR1B10 Expression in the Airway Epithelium of Healthy Individuals

    PubMed Central

    Wang, Rui; Wang, Guoqing; Ricard, Megan J.; Ferris, Barbara; Strulovici-Barel, Yael; Salit, Jacqueline; Hackett, Neil R.; Gudas, Lorraine J.

    2010-01-01

    Background: The aldo-keto reductase (AKR) gene superfamily codes for monomeric, soluble reduced nicotinamide adenine dinucleotide phosphate-dependent oxidoreductases that mediate elimination reactions. AKR1B10, an AKR that eliminates retinals, has been observed as upregulated in squamous metaplasia and non-small cell lung cancer and has been suggested as a diagnostic marker specific to tobacco-related carcinogenesis. We hypothesized that upregulation of AKR1B10 expression may be initiated in healthy smokers prior to the development of evidence of lung cancer. Methods: Expression of AKR1B10 was assessed at the mRNA level using microarrays with TaqMan confirmation in the large airway epithelium (21 healthy nonsmokers, 31 healthy smokers) and small airway epithelium (51 healthy nonsmokers, 58 healthy smokers) obtained by fiberoptic bronchoscopy and brushing. Results: Compared with healthy nonsmokers, AKR1B10 mRNA levels were significantly upregulated in both large and small airway epithelia of healthy smokers. Consistent with the mRNA data, AKR1B10 protein was significantly upregulated in the airway epithelium of healthy smokers as assessed by Western blot analysis and immunohistochemistry, with AKR1B10 expressed in both differentiated and basal cells. Finally, cigarette smoke extract mediated upregulation of AKR1B10 in airway epithelial cells in vitro, and transfection of AKR1B10 into airway epithelial cells enhanced the conversion of retinal to retinol. Conclusions: Smoking per se mediates upregulation of AKR1B10 expression in the airway epithelia of healthy smokers with no evidence of lung cancer. In the context of these observations and the link of AKR1B10 to the metabolism of retinals and to lung cancer, the smoking-induced upregulation of AKR1B10 may be an early process in the multiple events leading to lung cancer. PMID:20705797

  17. Studying Catabolism of Protein ADP-Ribosylation.

    PubMed

    Palazzo, Luca; James, Dominic I; Waddell, Ian D; Ahel, Ivan

    2017-01-01

    Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD + ) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

  18. Adipose tissue NAD+ biology in obesity and insulin resistance: From mechanism to therapy.

    PubMed

    Yamaguchi, Shintaro; Yoshino, Jun

    2017-05-01

    Nicotinamide adenine dinucleotide (NAD + ) biosynthetic pathway, mediated by nicotinamide phosphoribosyltransferase (NAMPT), a key NAD + biosynthetic enzyme, plays a pivotal role in controlling many biological processes, such as metabolism, circadian rhythm, inflammation, and aging. Over the past decade, NAMPT-mediated NAD + biosynthesis, together with its key downstream mediator, namely the NAD + -dependent protein deacetylase SIRT1, has been demonstrated to regulate glucose and lipid metabolism in a tissue-dependent manner. These discoveries have provided novel mechanistic and therapeutic insights into obesity and its metabolic complications, such as insulin resistance, an important risk factor for developing type 2 diabetes and cardiovascular disease. This review will focus on the importance of adipose tissue NAMPT-mediated NAD + biosynthesis and SIRT1 in the pathophysiology of obesity and insulin resistance. We will also critically explore translational and clinical aspects of adipose tissue NAD + biology. © 2017 WILEY Periodicals, Inc.

  19. NAD⁺ repletion improves mitochondrial and stem cell function and enhances life span in mice.

    PubMed

    Zhang, Hongbo; Ryu, Dongryeol; Wu, Yibo; Gariani, Karim; Wang, Xu; Luan, Peiling; D'Amico, Davide; Ropelle, Eduardo R; Lutolf, Matthias P; Aebersold, Ruedi; Schoonjans, Kristina; Menzies, Keir J; Auwerx, Johan

    2016-06-17

    Adult stem cells (SCs) are essential for tissue maintenance and regeneration yet are susceptible to senescence during aging. We demonstrate the importance of the amount of the oxidized form of cellular nicotinamide adenine dinucleotide (NAD(+)) and its effect on mitochondrial activity as a pivotal switch to modulate muscle SC (MuSC) senescence. Treatment with the NAD(+) precursor nicotinamide riboside (NR) induced the mitochondrial unfolded protein response and synthesis of prohibitin proteins, and this rejuvenated MuSCs in aged mice. NR also prevented MuSC senescence in the mdx (C57BL/10ScSn-Dmd(mdx)/J) mouse model of muscular dystrophy. We furthermore demonstrate that NR delays senescence of neural SCs and melanocyte SCs and increases mouse life span. Strategies that conserve cellular NAD(+) may reprogram dysfunctional SCs and improve life span in mammals. Copyright © 2016, American Association for the Advancement of Science.

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

    PubMed

    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.

  1. Sirtuins, Cell Senescence, and Vascular Aging.

    PubMed

    Kida, Yujiro; Goligorsky, Michael S

    2016-05-01

    The sirtuins (SIRTs) constitute a class of proteins with nicotinamide adenine dinucleotide-dependent deacetylase or adenosine diphosphate-ribosyltransferase activity. Seven SIRT family members have been identified in mammals, from SIRT1, the best studied for its role in vascular aging, to SIRT7. SIRT1 and SIRT2 are localized in the nucleus and cytoplasm. SIRT3, SIRT4, and SIRT5 are mitochondrial, and SIRT6 and SIRT7 are nuclear. Extensive studies have clearly revealed that SIRT proteins regulate diverse cell functions and responses to stressors. Vascular aging involves the aging process (senescence) of endothelial and vascular smooth muscle cells. Two types of cell senescence have been identified: (1) replicative senescence with telomere attrition; and (2) stress-induced premature senescence without telomere involvement. Both types of senescence induce vascular cell growth arrest and loss of vascular homeostasis, and contribute to the initiation and progression of cardiovascular diseases. Previous mechanistic studies have revealed in detail that SIRT1, SIRT3, and SIRT6 show protective functions against vascular aging, and definite vascular function of other SIRTs is under investigation. Thus, direct SIRT modulation and nicotinamide adenine dinucleotide stimulation of SIRT are promising candidates for cardiovascular disease therapy. A small number of pilot studies have been conducted to assess SIRT modulation in humans. These clinical studies have not yet provided convincing evidence that SIRT proteins alleviate morbidity and mortality in patients with cardiovascular diseases. The outcomes of multiple ongoing clinical trials are awaited to define the efficacy of SIRT modulators and SIRT activators in cardiovascular diseases, along with the potential adverse effects of chronic SIRT modulation. Copyright © 2016 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

  2. Raman spectroscopic study of acute oxidative stress induced changes in mice skeletal muscles

    NASA Astrophysics Data System (ADS)

    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.

  3. Effects of argan oil on the mitochondrial function, antioxidant system and the activity of NADPH- generating enzymes in acrylamide treated rat brain.

    PubMed

    Aydın, Birsen

    2017-03-01

    Argan oil (AO) is rich in minor compounds such as polyphenols and tocopherols which are powerful antioxidants. Acrylamide (ACR) has been classified as a neurotoxic agent in animals and humans. Mitochondrial oxidative stress and dysfunction is one of the most probable molecular mechanisms of neurodegenerative diseases. Female Sprague Dawley rats were exposed to ACR (50mg/kg i.p. three times a week), AO (6ml/kg,o.p, per day) or together for 30days. The activities of cytosolic enzymes such as xanthine oxidase (XO), glucose 6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), mitochondrial oxidative stress, oxidative phosphorylation (OXPHOS) and tricarboxylic acid cycle (TCA) enzymes, mitochondrial metabolic function, adenosine triphosphate (ATP) level and acetylcholinesterase (AChE) activity were assessed in rat brain. Cytosolic and mitochondrial antioxidant enzymes were significantly diminished in the brains of rats treated with ACR compared to those in control. Besides, ACR treatment resulted in a significant reduction in brain ATP level, mitochondrial metabolic function, OXPHOS and TCA enzymes. Administration of AO restored both the cytosolic and mitochondrial oxidative stress by normalizing nicotinamide adenine dinucleotide phosphate (NADPH) generating enzymes. In addition, improved mitochondrial function primarily enhancing nicotinamide adenine dinucleotide (NADH) generated enzymes activities and ATP level in the mitochondria. The reason for AO's obvious beneficial effects in this study may be due to synergistic effects of its different bioactive compounds which is especially effective on mitochondria. Modulation of the brain mitochondrial functions and antioxidant systems by AO may lead to the development of new mitochondria-targeted antioxidants in the future. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  4. Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

    PubMed Central

    Diekert, Gabriele B.; Thauer, Rudolf K.

    1978-01-01

    Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO2. Rates up to 0.4 μmol min−1 mg of wet cells−1 were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO2 reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min−1 mg of protein−1 (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO2 to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO2 do not possess a CO-oxidizing system. PMID:711675

  5. Resolution of mitochondrial oxidant stress improves aged-cardiovascular performance

    PubMed Central

    Owada, Takashi; Yamauchi, Hiroyuki; Miura, Shunsuke; Machii, Hirofumi; Takeishi, Yasuchika

    2017-01-01

    Background Senescence is a major factor that increases oxidative stress in mitochondria, which contributes toward the pathogenesis of heart disease. However, the effect of antioxidant therapy on cardiac mitochondria in aged-cardiac performance remains elusive. Objectives We postulated that the mitochondrial targeting of superoxide scavenging would have benefits in the aged heart. Methods and results Generation of superoxide in the mitochondria and nicotinamide adenine dinucleotide phosphate oxidase activity increased in the heart of old mice compared with that in young mice. In old mice treated with a mitochondria-targeted antioxidant MitoTEMPO (180 µg/kg/day, 28 days) co-infusion using a subcutaneously implanted minipump, levels of superoxide in the mitochondria and nicotinamide adenine dinucleotide phosphate oxidase activity as well as hydrogen peroxide decreased markedly in cardiomyocytes. Treatment with MitoTEMPO in old mice improved the systolic and diastolic function assessed by echocardiography. Endothelium-dependent vasodilation in isolated coronary arteries and endothelial nitric-oxide synthase phosphorylation were impaired in old mice compared with that in young mice and were improved by MitoTEMPO treatment. Mitochondria from the old mice myocardium showed lower rates of complex I-dependent and II-dependent respiration compared with that from young mice. Supplementation of MitoTEMPO in old mice improved the respiration rates and efficiency of ATP generation in mitochondria to a level similar to that of young mice. Conclusion Resolution of oxidative stress in mitochondria by MitoTEMPO in old mice restored cardiac function and the capacity of coronary vasodilation to the same magnitude observed in young mice. An antioxidant strategy targeting mitochondria could have a therapeutic benefit in heart disease with senescence. PMID:27740971

  6. Salvianolic acid B protects against paraquat-induced pulmonary injury by mediating Nrf2/Nox4 redox balance and TGF-β1/Smad3 signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Bin, E-mail: iamicehe@163.com

    The present study was aimed at exploring the protective effects of Salvianolic acid B (SalB) against paraquat (PQ)-induced lung injury in mice. Lung fibrotic injuries were induced in mice by a single intragastrical administration of 300 mg/kg PQ, then the mice were administrated with 200 mg/kg, 400 mg/kg SalB, 100 mg/kg vitamin C (Vit C) and dexamethasone (DXM) for 14 days. PQ-triggered structure distortion, collagen overproduction, excessive inflammatory infiltration, pro-inflammatory cytokine release, and oxidative stress damages in lung tissues and mortality of mice were attenuated by SalB in a dose-dependent manner. Furthermore, SalB was noted to enhance the expression andmore » nuclear translocation of nuclear factor erythroid 2–related factor 2 (Nrf2) and reduce expression of the reactive oxygen species-generating enzyme Nox4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-4]. SalB also inhibited the increasing expression of transforming growth factor (TGF)-β1 and the phosphorylation of its downstream target Smad3 which were enhanced by PQ. These results suggest that SalB may exert protective effects against PQ-induced lung injury and pulmonary fibrosis. Its mechanisms involve the mediation of Nrf2/Nox4 redox balance and TGF-β1/Smad3 signaling. - Highlights: • Salvianolic acid B (SalB) reduced Paraquat-induced mortality and pulmonary injury in mice. • SalB has anti-oxidation, anti-inflammatory and anti-fibrogenic effects simultaneously. • Its mechanisms were targeting Nrf2-Nox4 redox balance and TGF-β1/Smad3 signaling.« less

  7. Nicotinamide improves sevoflurane-induced cognitive impairment through suppression of inflammation and anti-apoptosis in rat

    PubMed Central

    Wang, Ying; Zuo, Min

    2015-01-01

    Nicotinamide is amide form of vitamin B3, participate in oxidation-reduction reaction, and it plays an important role in the maintenance of normal life activities in cells; it has broad application prospects in the treatment of heart blood-vessel disease, respiratory disease, type 1 diabetes and inflammatory autoimmune diseases. Thus the present study aimed to identify whether the nicotinamide improves sevoflurane-induced cognitive impairment and its potential mechanisms in rat. Firstly, Male Sprague-Dawley rats were induced by 2.1% sevoflurane for 6 h. Protective function of nicotinamide on cognitive impairment was evaluated using Morris water maze test in the rats. Next, NF-κB and caspase-3 activities, and p53, Bax and Bcl-2 protein expression was executed using commercial kits and Western blot analysis, respectively. Preconditioning with nicotinamide could improve cognitive impairment in the rats. Administrate of nicotinamide suppressed the activation of NF-κB and caspase-3, reduced the protein expression of Bax, and promoted Bcl-2 protein expression in rats. The present results suggested nicotinamide improves sevoflurane-induced cognitive impairment and has an anti-inflammatory and anti-apoptotic effect against sevoflrane-induced damages. PMID:26884920

  8. Biofuel cell anode: NAD +/glucose dehydrogenase-coimmobilized ketjenblack electrode

    NASA Astrophysics Data System (ADS)

    Miyake, T.; Oike, M.; Yoshino, S.; Yatagawa, Y.; Haneda, K.; Kaji, H.; Nishizawa, M.

    2009-09-01

    We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD +), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD + and GDH. The improved GDH/NAD +/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 μW/cm 2 at 0.3 V.

  9. Sirtuins in neurodegenerative diseases: an update on potential mechanisms

    PubMed Central

    Min, Sang-Won; Sohn, Peter D.; Cho, Seo-Hyun; Swanson, Raymond A.; Gan, Li

    2013-01-01

    Silent information regulator 2 proteins (sirtuins or SIRTs) are a group of deacetylases (or deacylases) whose activities are dependent on and regulated by nicotinamide adenine dinucleotide (NAD+). Compelling evidence supports that sirtuins play major roles in many aspects of physiology, especially in pathways related to aging – the predominant and unifying risk factor for neurodegenerative diseases. In this review, we highlight the molecular mechanisms underlying the protective effects of sirtuins in neurodegenerative diseases, focusing on protein homeostasis, neural plasticity, mitochondrial function, and sustained chronic inflammation. We will also examine the potential and challenges of targeting sirtuin pathways to block these pathogenic pathways. PMID:24093018

  10. Comparison of neutrophil functions between two strains of inbred mice.

    PubMed

    Zhang, Xiaohuan; Zhao, Sainan; Sun, Luping; Li, Wenqing; Glogauer, Michael; Hu, Yan

    2016-12-01

    In this study, differences between two strains of inbred mice in aspects of neutrophil function, namely Rac1 expression, chemotaxis, nicotinamide adenine dinucleotide phosphate oxidase activity and formation of neutrophil extracellular traps (NETs), were determined. Neutrophils from CBA/CaH mice exhibited weaker Rac1 expression and a slower chemotactic gradient than BALB/c mice. Furthermore, PMA- or fMLP-stimulated neutrophils from CBA/CaH mice generated much less superoxide and NETs than similarly stimulated neutrophils from BALB/c mice. These findings suggest that neutrophils from BALB/c mice are functionally more efficient than those from CBA/CaH mice. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  11. Assessing the photoaging process at sun exposed and non-exposed skin using fluorescence lifetime spectroscopy

    NASA Astrophysics Data System (ADS)

    Saito Nogueira, Marcelo; Kurachi, Cristina

    2016-03-01

    Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

  12. Rapid measurement of meat spoilage using fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Dahlberg, Kevin; Gao, Xin; Smith, Jason; Bailin, Jacob

    2017-02-01

    Food spoilage is mainly caused by microorganisms, such as bacteria. In this study, we measure the autofluorescence in meat samples longitudinally over a week in an attempt to develop a method to rapidly detect meat spoilage using fluorescence spectroscopy. Meat food is a biological tissue, which contains intrinsic fluorophores, such as tryptophan, collagen, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) etc. As meat spoils, it undergoes various morphological and chemical changes. The concentrations of the native fluorophores present in a sample may change. In particular, the changes in NADH and FAD are associated with microbial metabolism, which is the most important process of the bacteria in food spoilage. Such changes may be revealed by fluorescence spectroscopy and used to indicate the status of meat spoilage. Therefore, such native fluorophores may be unique, reliable and nonsubjective indicators for detection of spoiled meat. The results of the study show that the relative concentrations of all above fluorophores change as the meat samples kept in room temperature ( 19° C) spoil. The changes become more rapidly after about two days. For the meat samples kept in a freezer ( -12° C), the changes are much less or even unnoticeable over a-week-long storage.

  13. Imaging Redox State in Mouse Muscles of Different Ages.

    PubMed

    Moon, Lily; Frederick, David W; Baur, Joseph A; Li, Lin Z

    2017-01-01

    Aging is the greatest risk factor for many diseases. Intracellular concentrations of nicotinamide adenine dinucleotide (NAD + ) and the NAD + -coupled redox state have been proposed to moderate many aging-related processes, yet the specific mechanisms remain unclear. The concentration of NAD + falls with age in skeletal muscle, yet there is no consensus on whether aging will increase or decrease the redox potential of NAD + /NADH. Oxidized flavin groups (Fp) (e.g. FAD, i.e., flavin adenine dinucleotide, contained in flavoproteins) and NADH are intrinsic fluorescent indicators of oxidation and reduction status of tissue, respectively. The redox ratio, i.e., the ratio of Fp to NADH, may be a surrogate indicator of the NAD + /NADH redox potential. In this study we used the Chance redox scanner (NADH/Fp fluorescence imaging at low temperature) to investigate the effect of aging on the redox state of mitochondria in skeletal muscles. The results showed that there are borderline significant differences in nominal concentrations of Fp and NADH, but not in the redox ratio s when comparing 3.5-month and 13-month old muscles of mice (n = 6). It may be necessary to increase the number of muscle samples and study mice of more advanced age.

  14. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Osseiran, Sam; Roider, Elisabeth M.; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E.; Evans, Conor L.

    2017-12-01

    Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

  15. Electrocatalytic reaction of hydrogen peroxide and NADH based on poly(neutral red) and FAD hybrid film.

    PubMed

    Lin, Kuo Chiang; Lin, Yu Ching; Chen, Shen Ming

    2012-01-07

    A simple method to immobilize poly(neutral red) (PNR) and flavin adenine dinucleotide (FAD) hybrid film (PNR/FAD) by cyclic voltammetry is proposed. The PNR/FAD hybrid film can be easily prepared on an electrode surface involving electropolymerization of neutral red (NR) monomers and the electrostatic interaction between the positively charged PNR and the negatively charged FAD. It exhibits electroactive, stable, surface-confined, pH-dependent, nano-sized, and compatible properties. It provides good electrocatalytic properties to various species. It shows a sensitivity of 5.4 μA mM(-1) cm(-2) and 21.5 μA mM(-1) cm(-2) for hydrogen peroxide (H(2)O(2)) and nicotinamide adenine dinucleotide (NADH) with the linear range of 0.1 μM-39 mM and 5 × 10(-5) to 2.5 × 10(-4) M, respectively. It shows another linear range of 48.8-355.5 mM with the sensitivity of 12.3 μA mM(-1) cm(-2) for H(2)O(2). In particular, the PNR/FAD hybrid film has potential to replace some hemoproteins to be a cathode of biofuel cells and provide the biosensing system for glucose and ethanol. This journal is © The Royal Society of Chemistry 2012

  16. Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy.

    PubMed

    Osseiran, Sam; Roider, Elisabeth M; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E; Evans, Conor L

    2017-12-01

    Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  17. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baig, M.; Brown, A.; Eswaramoorthy, S.

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) atmore » Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.« less

  18. Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

    PubMed

    Condino-Neto, A; Whitney, C; Newburger, P E

    1998-11-01

    We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

  19. Poly(ADP-Ribose) Polymerase-1: A Novel Therapeutic Target in Necrotizing Enterocolitis

    PubMed Central

    Giannone, Peter J.; Alcamo, Alicia A.; Schanbacher, Brandon L.; Nankervis, Craig A.; Besner, Gail E.; Bauer, John A.

    2011-01-01

    Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of infancy, afflicting 11% of infants born 22–28 weeks gestational age. Both inflammation and oxidation may be involved in NEC pathogenesis through reactive nitrogen species production, protein oxidation and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) is a critical enzyme activated to facilitate DNA repair using nicotinamide adenine dinucleotide (NAD+) as a substrate. However, in the presence of severe oxidative stress and DNA damage, PARP-1 over-activation may ensue, depleting cells of NAD+ and ATP, killing them by metabolic catastrophe. Here we tested the hypothesis that NO dysregulation in intestinal epithelial cells during NEC leads to marked PARP-1 expression and that administration of a PARP-1 inhibitor (nicotinamide) attenuates intestinal injury in a newborn rat model of NEC. In this model, 56% of control pups developed NEC (any stage), versus 14% of pups receiving nicotinamide. Forty-four percent of control pups developed high-grade NEC (grades 3–4), whereas only 7% of pups receiving nicotinamide developed high-grade NEC. Nicotinamide treatment protects pups against intestinal injury incurred in the newborn rat NEC model. We speculate that PARP-1 over-activation in NEC may drive mucosal cell death in this disease and that PARP-1 may be a novel therapeutic target in NEC. PMID:21399558

  20. NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells.

    PubMed

    Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A J; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

    2016-10-11

    NAD + is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD + precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD + synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD + synthesis from other NAD + precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD + . Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD + synthesis, explaining the overlapping metabolic effects observed with the two compounds.

  1. NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells

    PubMed Central

    Ratajczak, Joanna; Joffraud, Magali; Trammell, Samuel A. J.; Ras, Rosa; Canela, Núria; Boutant, Marie; Kulkarni, Sameer S.; Rodrigues, Marcelo; Redpath, Philip; Migaud, Marie E.; Auwerx, Johan; Yanes, Oscar; Brenner, Charles; Cantó, Carles

    2016-01-01

    NAD+ is a vital redox cofactor and a substrate required for activity of various enzyme families, including sirtuins and poly(ADP-ribose) polymerases. Supplementation with NAD+ precursors, such as nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), protects against metabolic disease, neurodegenerative disorders and age-related physiological decline in mammals. Here we show that nicotinamide riboside kinase 1 (NRK1) is necessary and rate-limiting for the use of exogenous NR and NMN for NAD+ synthesis. Using genetic gain- and loss-of-function models, we further demonstrate that the role of NRK1 in driving NAD+ synthesis from other NAD+ precursors, such as nicotinamide or nicotinic acid, is dispensable. Using stable isotope-labelled compounds, we confirm NMN is metabolized extracellularly to NR that is then taken up by the cell and converted into NAD+. Our results indicate that mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD+ synthesis, explaining the overlapping metabolic effects observed with the two compounds. PMID:27725675

  2. Role of Microsomal Retinol/Sterol Dehydrogenase-Like Short-Chain Dehydrogenases/Reductases in the Oxidation and Epimerization of 3α-Hydroxysteroids in Human Tissues

    PubMed Central

    Belyaeva, Olga V.; Chetyrkin, Sergei V.; Clark, Amy L.; Kostereva, Natalia V.; SantaCruz, Karen S.; Chronwall, Bibie M.; Kedishvili, Natalia Y.

    2008-01-01

    Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3α-hydroxysteroids that act as positive allosteric regulators of γ-aminobutyric acid type A receptors. In addition, ADT activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to γ-aminobutyric acid type A receptors, the biological activity of ALLO and ADT depends on the 3α-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3β-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3α-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3α-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3α-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3α-hydroxysteroids in vitro. Here, we present the first evidence that microsomal nicotinamide adenine dinucleotide-dependent 3α-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney, and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3α-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3α-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation. PMID:17289849

  3. Role of microsomal retinol/sterol dehydrogenase-like short-chain dehydrogenases/reductases in the oxidation and epimerization of 3alpha-hydroxysteroids in human tissues.

    PubMed

    Belyaeva, Olga V; Chetyrkin, Sergei V; Clark, Amy L; Kostereva, Natalia V; SantaCruz, Karen S; Chronwall, Bibie M; Kedishvili, Natalia Y

    2007-05-01

    Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3alpha-hydroxysteroids that act as positive allosteric regulators of gamma-aminobutyric acid type A receptors. In addition, ADT activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to gamma-aminobutyric acid type A receptors, the biological activity of ALLO and ADT depends on the 3alpha-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3beta-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3alpha-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3alpha-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3alpha-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal nicotinamide adenine dinucleotide-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3alpha-hydroxysteroids in vitro. Here, we present the first evidence that microsomal nicotinamide adenine dinucleotide-dependent 3alpha-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney, and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3alpha-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3alpha-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation.

  4. Differential effects of exposure to parasites and bacteria on stress response in turbot Scophthalmus maximus simultaneously stressed by low water depth.

    PubMed

    Rodríguez-Quiroga, J J; Otero-Rodiño, C; Suárez, P; Nieto, T P; García Estévez, J M; San Juan, F; Soengas, J L

    2017-07-01

    The stress response of turbot Scophthalmus maximus was evaluated in fish maintained 8 days under different water depths, normal (NWD, 30 cm depth, total water volume 40 l) or low (LWD, 5 cm depth, total water volume 10 l), in the additional presence of infection-infestation of two pathogens of this species. This was caused by intraperitoneal injection of sublethal doses of the bacterium Aeromonas salmonicida subsp. salmonicida or the parasite Philasterides dicentrarchi (Ciliophora:Scuticociliatida). The LWD conditions were stressful for fish, causing increased levels of cortisol in plasma, decreased levels of glycogen in liver and nicotinamide adenine dinucleotide phosphate (NADP) and increased activities of G6Pase and GSase. The presence of bacteria or parasites in fish under NWD resulted in increased cortisol levels in plasma whereas in liver, changes were of minor importance including decreased levels of lactate and GSase activity. The simultaneous presence of bacteria and parasites in fish under NWD resulted a sharp increase in the levels of cortisol in plasma and decreased levels of glucose. Decreased levels of glycogen and lactate and activities of GSase and glutathione reductase (GR), as well as increased activities of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and levels of nicotinamide adenine dinucleotide phosphate (NADPH) occurred in the same fish in liver. Finally, the presence of pathogens in S. maximus under stressful conditions elicited by LWD resulted in synergistic actions of both type of stressors in cortisol levels. In liver, the presence of bacteria or parasites induced a synergistic action on several variables such as decreased activities of G6Pase and GSase as well as increased levels of NADP and NADPH and increased activities of GPase, G6PDH and 6PGDH. © 2017 The Fisheries Society of the British Isles.

  5. Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis.

    PubMed

    Siler, Ulrich; Romao, Susana; Tejera, Emilio; Pastukhov, Oleksandr; Kuzmenko, Elena; Valencia, Rocio G; Meda Spaccamela, Virginia; Belohradsky, Bernd H; Speer, Oliver; Schmugge, Markus; Kohne, Elisabeth; Hoenig, Manfred; Freihorst, Joachim; Schulz, Ansgar S; Reichenbach, Janine

    2017-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells in human subjects, causing hemolytic anemia linked to impaired nicotinamide adenine dinucleotide phosphate (NADPH) production and imbalanced redox homeostasis in erythrocytes. Because G6PD is expressed by a variety of hematologic and nonhematologic cells, a broader clinical phenotype could be postulated in G6PD-deficient patients. We describe 3 brothers with severe G6PD deficiency and susceptibility to bacterial infection. We sought to study the molecular pathophysiology leading to susceptibility to infection in 3 siblings with severe G6PD deficiency. Blood samples of 3 patients with severe G6PD deficiency were analyzed for G6PD enzyme activity, cellular oxidized nicotinamide adenine dinucleotide phosphate/NADPH levels, phagocytic reactive oxygen species production, neutrophil extracellular trap (NET) formation, and neutrophil elastase translocation. In these 3 brothers strongly reduced NADPH oxidase function was found in granulocytes, leading to impaired NET formation. Defective NET formation has thus far been only observed in patients with the NADPH oxidase deficiency chronic granulomatous disease, who require antibiotic and antimycotic prophylaxis to prevent life-threatening bacterial and fungal infections. Because severe G6PD deficiency can be a phenocopy of chronic granulomatous disease with regard to the cellular and clinical phenotype, careful evaluation of neutrophil function seems mandatory in these patients to decide on appropriate anti-infective preventive measures. Determining the level of G6PD enzyme activity should be followed by analysis of reactive oxygen species production and NET formation to decide on required antibiotic and antimycotic prophylaxis. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Treatment with NAD(+) inhibited experimental autoimmune encephalomyelitis by activating AMPK/SIRT1 signaling pathway and modulating Th1/Th17 immune responses in mice.

    PubMed

    Wang, Jueqiong; Zhao, Congying; Kong, Peng; Sun, Huanhuan; Sun, Zhe; Bian, Guanyun; Sun, Yafei; Guo, Li

    2016-10-01

    Nicotinamide adenine dinucleotide (NAD(+)) plays vital roles in mitochondrial functions, cellular energy metabolism and calcium homeostasis. In this study, we investigated the effect of NAD(+) administration for the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. EAE, a classical animal model of multiple sclerosis (MS), was induced by subcutaneous injection of myelin oligodendrocyteglycoprotein (MOG). The mice were treated with 250mg/kg (body weight) NAD(+) in PBS administered intraperitoneally once daily. We observed that NAD(+) treatment could lessen the severity of EAE. Additionally, NAD(+) treatment attenuated pathological injuries of EAE mice. We also found that the AMP-activated protein kinase (AMPK)/silent mating-type information regulation 2 homolog 1(SIRT1) pathway was activated in the NAD(+)-treated mice and NAD(+) treatment suppressed pro-inflammatory T cell responses. Our findings demonstrated that NAD(+) could be an effective and promising agent to treat multiple sclerosis and its effects on other autoimmune diseases should be explored. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. NNMT activation can contribute to the development of fatty liver disease by modulating the NAD + metabolism.

    PubMed

    Komatsu, Motoaki; Kanda, Takeshi; Urai, Hidenori; Kurokochi, Arata; Kitahama, Rina; Shigaki, Shuhei; Ono, Takashi; Yukioka, Hideo; Hasegawa, Kazuhiro; Tokuyama, Hirobumi; Kawabe, Hiroshi; Wakino, Shu; Itoh, Hiroshi

    2018-06-05

    Nicotinamide N-methyltransferase (NNMT) catalyses the reaction between nicotinamide (NAM) and S-adenosylmethionine to produce 1-methylnicotinamide and S-adenosylhomocysteine. Recently, this enzyme has also been reported to modulate hepatic nutrient metabolism, but its role in the liver has not been fully elucidated. We developed transgenic mice overexpressing NNMT to elucidate its role in hepatic nutrient metabolism. When fed a high fat diet containing NAM, a precursor for nicotinamide adenine dinucleotide (NAD) + , these NNMT-overexpressing mice exhibit fatty liver deterioration following increased expression of the genes mediating fatty acid uptake and decreased very low-density lipoprotein secretion. NNMT overactivation decreased the NAD + content in the liver and also decreased gene activity related to fatty acid oxidation by inhibiting NAD + -dependent deacetylase Sirt3 function. Moreover, the transgenic mice showed liver fibrosis, with the induction of inflammatory and fibrosis genes. Induced NNMT expression decreased the tissue methylation capacity, thereby reducing methylation of the connective tissue growth factor (CTGF) gene promoter, resulting in increased CTGF expression. These data indicate that NNMT links the NAD + and methionine metabolic pathways and promotes liver steatosis and fibrosis. Therefore, targeting NNMT may serve as a therapeutic strategy for treating fatty liver and fibrosis.

  8. Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer’s mouse models

    PubMed Central

    Gong, Bing; Pan, Yong; Vempati, Prashant; Zhao, Wei; Knable, Lindsay; Ho, Lap; Wang, Jun; Sastre, Magdalena; Ono, Kenjiro; Sauve, Anthony A.; Pasinetti, Giulio M.

    2013-01-01

    Nicotinamide adenine dinucleotide (NAD)+, a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD+ expression has been linked with a decrease in beta-amyloid (Aβ) toxicity in Alzheimer’s disease (AD). Nicotinamide riboside (NR) is a NAD+ precursor, it promotes peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α expression in the brain. Evidence has shown that PGC-1α is a crucial regulator of Aβ generation because it affects β-secretase (BACE1) degradation. In this study we tested the hypothesis that NR treatment in an AD mouse model could attenuate Aβ toxicity through the activation of PGC-1α-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysiological recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD+ in the cerebral cortex; (2) application of NR to hippocampal slices (10 µM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1 degradation was prevented by PGC-1α-shRNA gene silencing; and (4) NR treatment and PGC-1α overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, inpart by promoting PGC-1α-mediated BACE1

  9. [Perspectives on pharmacological and clinical benefits from sirtuin 1 activators in oxidative damage].

    PubMed

    Kutinová, Canová Nikolina; Gaier, Norbert; Farghali, Hassan

    2012-01-01

    Sirtuins, named after their homology to the Saccharomyces cerevisiae Silent Information Regulator Two, constitute a family of highly conserved nicotinamide adenine dinucleotide-dependent enzymes that deacetylate histones and residues of acetylated lysine. The main aim of this article is to put forward the pharmacological importance of major sirtuin 1 activators of natural or synthetic origin tested in last years in cases of oxidative tissue damage. The related bioactivity of these activators as "leading" compounds in the search for new drugs and remedies is also described. With the recent development of our knowledge on the cross talks between sirtuin 1 and its modulators (e.g. resveratrol), pharmacological and clinical research on this topic is getting a new horizon.

  10. Slowing ageing by design: the rise of NAD+ and sirtuin-activating compounds

    PubMed Central

    Bonkowski, Michael S.; Sinclair, David A.

    2016-01-01

    The sirtuins (SIRT1–7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases with remarkable abilities to prevent diseases and even reverse aspects of ageing. Mice engineered to express additional copies of SIRT1 or SIRT6, or treated with sirtuin-activating compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved organ function, physical endurance, disease resistance and longevity. Trials in non-human primates and in humans have indicated that STACs may be safe and effective in treating inflammatory and metabolic disorders, among others. These advances have demonstrated that it is possible to rationally design molecules that can alleviate multiple diseases and possibly extend lifespan in humans. PMID:27552971

  11. Regenerative capability of skeletal muscle in chicken muscular dystrophy.

    PubMed

    Nonaka, I; Fujita, T; Sugita, H

    1984-06-01

    To examine the morphological sequence of regenerating fibers after myonecrosis in dystrophic muscles, 0.5 ml of 0.5% bupivacaine hydrochloride (BPVC) (Marcaine) solution, a local anesthetic with a cytotoxic effect on the muscle fibers, was injected directly into the dystrophic (line 413) and nondystrophic (line 412) posterior latissimus dorsi (PLD) muscles of young and adult chickens. Although the dystrophic muscles after BPVC injection showed a rapid recovery with a similar tempo to that of nondystrophic ones, they showed different morphological behavior in the early phase of regeneration, including marked variability in the size of fibers and in the intracytoplasmic enzyme activities of nicotinamide adenine dinucleotide, reduced-tetrazolium reductase (NADH-TR), acetylcholinesterase (AChE), and nonspecific esterase (NSE).

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

    PubMed

    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.

  13. Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

    PubMed

    Mitchelson, K R

    1996-01-01

    The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

  14. STUDIES ON THE INFLUENCE OF X-RAY IRRADIATION IN VIT. B$sub 2$ METABOLISM. I. INFLUENCE OF VIT. B$sub 2$ DISTRIBUTION IN ORGANS OF RATS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yokoyama, K.; Kusumoto, T.; Nakamura, J.

    X rays were directed to the liver region of albino rats. The distribution of vitamin B/sub 2/ in the liver, kidney, intestine, heart, spleen, and blood was investigated 6, 12, 18, and 24 hr after the irradiation. Total vitamin B/sub 2/ increased in these organs except in the blood; flavin mononucleotide (FMN) and free riboflavin (FR) increased without exception, and flavin adenine dinucleotide (FAD) decreased except in the spleen, that is, abnormal distribution of vitamin B/sub 2/ fractions was significant except in the spleen. Successive estimations of the fractions suggest that the metabolic disturbances by irradiation occur in the reactionmore » FMN - FAD initially, then in FR - FMN and FR - FAD. (Absts. Japan. Med., 1: No. 7, 1960.)« less

  15. Protection by mTOR Inhibition on Zymosan-Induced Systemic Inflammatory Response and Oxidative/Nitrosative Stress: Contribution of mTOR/MEK1/ERK1/2/IKKβ/IκB-α/NF-κB Signalling Pathway.

    PubMed

    Sahan-Firat, Seyhan; Temiz-Resitoglu, Meryem; Guden, Demet Sinem; Kucukkavruk, Sefika Pinar; Tunctan, Bahar; Sari, Ayse Nihal; Kocak, Zumrut; Malik, Kafait U

    2018-02-01

    Mammalian target of rapamycin (mTOR), a serine/threonine kinase regulate variety of cellular functions including cell growth, differentiation, cell survival, metabolism, and stress response, is now appreciated to be a central regulator of immune responses. Because mTOR inhibitors enhanced the anti-inflammatory activities of regulatory T cells and decreased the production of proinflammatory cytokines by macrophages, mTOR has been a pharmacological target for inflammatory diseases. In this study, we examined the role of mTOR in the production of proinflammatory and vasodilator mediators in zymosan-induced non-septic shock model in rats. To elucidate the mechanism by which mTOR contributes to non-septic shock, we have examined the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system caused by mTOR/mitogen-activated protein kinase kinase (MEK1)/extracellular signal-regulated kinase (ERK1/2)/inhibitor κB kinase (IKKβ)/inhibitor of κB (IκB-α)/nuclear factor-κB (NF-κB) signalling pathway activation. After 1 h of zymosan (500 mg/kg, i.p.) administration to rats, mean arterial blood pressure (MAP) was decreased and heart rate (HR) was increased. These changes were associated with increased expression and/or activities of ribosomal protein S6, MEK1, ERK1/2, IKKβ, IκB-α and NF-κB p65, and NADPH oxidase system activity in cardiovascular and renal tissues. Rapamycin (1 mg/kg, i.p.), a selective mTOR inhibitor, reversed these zymosan-induced changes in these tissues. These observations suggest that activation of mTOR/MEK1/ERK1/2/IKKβ/IκB-α/NF-κB signalling pathway with proinflammatory and vasodilator mediator formation and NADPH oxidase system activity contributes to systemic inflammation in zymosan-induced non-septic shock. Thus, mTOR may be an optimal target for the treatment of the diseases characterized by the severe systemic inflammatory response.

  16. Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa

    NASA Astrophysics Data System (ADS)

    Maleki, Sepideh; Gopalakrishnan, Sandeep; Ghanian, Zahra; Sepehr, Reyhaneh; Schmitt, Heather; Eells, Janis; Ranji, Mahsa

    2013-01-01

    Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11±0.03 in the SD normal and 0.841±0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.

  17. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress

    PubMed Central

    Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R.; Audi, Said

    2012-01-01

    Abstract. Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs. PMID:22559688

  18. An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors

    PubMed Central

    Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G.; Tawfik, Dan S.

    2016-01-01

    Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose’s ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint—geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif. PMID:26938925

  19. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

  20. Nicotinamide inhibits the growth of P. falciparum and enhances the antimalarial effect of artemisinin, chloroquine and pyrimethamine.

    PubMed

    Tcherniuk, Sergey O; Chesnokova, Olga; Oleinikov, Irina V; Oleinikov, Andrew V

    2017-09-01

    Nicotinamide (vitamin B3) - is a water soluble amide derivative of nicotinic acid, which has been used at high doses for a variety of therapeutic applications. However, its antimalarial effect has not been intensively explored. In this work we analysed the effect of nicotinamide alone and in combination with artemisinin, chloroquine andpyrimethamine on the growth of blood stages of P. falciparum. Our results demonstrate that nicotinamide effectively inhibits the growth of blood stage parasites with IC 50 of 6.9±0.1mM and 2.2±0.3mM for CS2 and 3G8 strains, respectively. The combination of nicotinamide with artemisinin, chloroquine and pyrimethamine demonstrated synergistic effects at IC 10-90%. Treatment of uninfected red blood cells with high dose of nicotinamide (60mM) did not provoke the significant LDH release, demonstrating its non-toxicity for erythrocytes. Nicotinamide acts below the level of tolerance and reduces the effective concentration of anti-malarial drugs due to synergism. These in vitro results suggest that nicotinamide might be useful not only as a vitamin supplement but also as an enhancer of the anti-parasitic effect of common antimalarial drugs including artemisinin, chloroquine and pyrimethamine. Published by Elsevier B.V.

  1. Computational vibrational study on coordinated nicotinamide

    NASA Astrophysics Data System (ADS)

    Bolukbasi, Olcay; Akyuz, Sevim

    2005-06-01

    The molecular structure and vibrational spectra of zinc (II) halide complexes of nicotinamide (ZnX 2(NIA) 2; X=Cl or Br; NIA=Nicotinamide) were investigated by computational vibrational study and scaled quantum mechanical (SQM) analysis. The geometry optimisation and vibrational wavenumber calculations of zinc halide complexes of nicotinamide were carried out by using the DFT/RB3LYP level of theory with 6-31G(d,p) basis set. The calculated wavenumbers were scaled by using scaled quantum mechanical (SQM) force field method. The fundamental vibrational modes were characterised by their total energy distribution. The coordination effects on nicotinamide through the ring nitrogen were discussed.

  2. NAD+ maintenance attenuates light induced photoreceptor degeneration Δ

    PubMed Central

    Bai, Shi; Sheline, Christian T.

    2013-01-01

    Light-induced retinal damage (LD) occurs after surgery or sun exposure. We previously showed that zinc (Zn2+) accumulated in photoreceptors and RPE cells after LD but prior to cell death, and pyruvate or nicotinamide attenuated the resultant death perhaps by restoring nicotinamide adenine dinucleotide (NAD+) levels. We first examined the levels of NAD+ and the efficacy of pyruvate or nicotinamide in oxidative toxicities using primary retinal cultures. We next manipulated NAD+ levels in vivo and tested the affect on LD to photoreceptors and RPE. NAD+ levels cycle with a 24-h rhythm in mammals, which is affected by the feeding schedule. Therefore, we tested the affect of increasing NAD+ levels on LD by giving nicotinamide, inverting the feeding schedule, or using transgenic mice which overexpress cytoplasmic nicotinamide mononucleotide adenyl-transferase-1 (cytNMNAT1), an NAD+ synthetic enzyme. Zn2+ accumulation was also assessed in culture and in retinal sections. Retinas of light damaged animals were examined by OCT and plastic sectioning, and retinal NAD levels were measured. Day fed, or nicotinamide treated rats showed less NAD+ loss, and LD compared to night fed rats or untreated rats without changing the Zn2+ staining pattern. CytNMNAT1 showed less Zn2+ staining, NAD+ loss, and cell death after LD. In conclusion, intense light, Zn2+ and oxidative toxicities caused an increase in Zn2+, NAD+ loss, and cell death which were attenuated by NAD+ restoration. Therefore, NAD+ levels play a protective role in LD-induced death of photoreceptors and RPE cells. PMID:23274583

  3. Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.).

    PubMed

    Katahira, Riko; Ashihara, Hiroshi

    2009-12-01

    As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --> nicotinamide mononucleotide --> nicotinamide riboside --> nicotinic acid riboside --> nicotinic acid mononucleotide --> nicotinic acid adenine dinucleotide --> NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.

  4. Theophylline-nicotinamide cocrystal formation in physical mixture during storage.

    PubMed

    Ervasti, Tuomas; Aaltonen, Jaakko; Ketolainen, Jarkko

    2015-01-01

    Pharmaceutically relevant properties, such as solubility and dissolution rate, of active pharmaceutical ingredients can be enhanced by cocrystal formation. Theophylline and nicotinamide are known to form cocrystals, for example if subjected to solid-state grinding. However, under appropriate conditions, cocrystals can also form in physical mixtures without any mechanical activation. The purpose of this work was to study whether theophylline and nicotinamide could form cocrystals spontaneously, without mechanical activation. Crystalline theophylline and nicotinamide powders were gently mixed manually in a 1:1 molar ratio and stored at different relative humidity and temperature conditions. The solid state of the samples was analyzed by differential scanning calorimetry, Raman spectroscopy and X-ray powder diffractometry. Three different variations of theophylline were used as starting materials, e.g., two size fractions of theophylline anhydrate (large 710 μm-1 mm and small 180-355 μm), and monohydrate (recrystallized from water). As a reference, anhydrous theophylline-nicotinamide cocrystals were prepared by solid-state grinding. The results of this study indicate that theophylline-nicotinamide cocrystals can form without any mechanical activation from physical mixtures of theophylline and nicotinamide during storage. For anhydrous samples, storage humidity was found to be a critical parameter for cocrystal formation. Increasing temperature was also found to have an accelerating effect on the transformation. The effect of particle size of anhydrous theophylline on the transformation rate could not be completely resolved; DSC and Raman indicated slightly faster transformation with a physical mixture prepared from large size fraction of anhydrous theophylline, but the differences were only minor. Cocrystal formation was also observed in the physical mixture prepared from theophylline monohydrate, but the rate was not as high as with samples prepared from anhydrous

  5. Stimulation of the α7 nicotinic acetylcholine receptor protects against neuroinflammation after tibia fracture and endotoxemia in mice.

    PubMed

    Terrando, Niccolò; Yang, Ting; Ryu, Jae Kyu; Newton, Phillip T; Monaco, Claudia; Feldmann, Marc; Ma, Daqing; Akassoglou, Katerina; Maze, Mervyn

    2015-03-17

    Surgery and critical illness often associate with cognitive decline. Surgical trauma or infection can lead independently to learning and memory impairments via similar, but not identical, cellular signaling of the innate immune system that promotes neuroinflammation. In this study we explored the putative synergism between aseptic orthopedic surgery and infection, the latter reproduced by postoperative lipopolysaccharide (LPS) administration. We observed that surgery and LPS augmented systemic inflammation up to postoperative d 3 and this was associated with further neuroinflammation (CD11b and CD68 immunoreactivity) in the hippocampus in mice compared with those receiving surgery or LPS alone. Administration of a selective α7 subtype nicotinic acetylcholine receptor (α7 nAChR) agonist 2 h after LPS significantly improved neuroinflammation and hippocampal-dependent memory dysfunction. Modulation of nuclear factor-kappa B (NF-κB) activation in monocytes and regulation of the oxidative stress response through nicotinamide adenine dinucleotide phosphate (NADPH) signaling appear to be key targets in modulating this response. Overall, these results suggest that it may be conceivable to limit and possibly prevent postoperative complications, including cognitive decline and/or infections, through stimulation of the cholinergic antiinflammatory pathway.

  6. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1)*

    PubMed Central

    Harlan, Benjamin A.; Pehar, Mariana; Sharma, Deep R.; Beeson, Gyda; Beeson, Craig C.; Vargas, Marcelo R.

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD+) participates in redox reactions and NAD+-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD+-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD+ as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1–7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD+ salvage pathway capable of resynthesizing NAD+ from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD+ levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD+ salvage pathway in astrocytes. Supplementation with the NAD+ precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD+ levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1–2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD+ content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD+ salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

  7. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Activates Global and Heterogeneous Local Ca2+ Signals from NAADP- and Ryanodine Receptor-gated Ca2+ Stores in Pulmonary Arterial Myocytes*

    PubMed Central

    Jiang, Yong-Liang; Lin, Amanda H. Y.; Xia, Yang; Lee, Suengwon; Paudel, Omkar; Sun, Hui; Yang, Xiao-Ru; Ran, Pixin; Sham, James S. K.

    2013-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-mobilizing messenger that releases Ca2+ from endolysosomal organelles. Recent studies showed that NAADP-induced Ca2+ release is mediated by the two-pore channels (TPCs) TPC1 and TPC2. However, the expression of TPCs and the NAADP-induced local Ca2+ signals have not been examined in vascular smooth muscle. Here, we found that both TPC1 and TPC2 are expressed in rat pulmonary arterial smooth muscle cells (PASMCs), with TPC1 being the major subtype. Application of membrane-permeant NAADP acetoxymethyl ester to PASMCs elicited a biphasic increase in global [Ca2+]i, which was independent of extracellular Ca2+ and blocked by the NAADP antagonist Ned-19 or the vacuolar H+-ATPase inhibitor bafilomycin A1, indicating Ca2+ release from acidic endolysosomal Ca2+ stores. The Ca2+ response was unaffected by xestospongin C but was partially blocked by ryanodine or thapsigargin. NAADP triggered heterogeneous local Ca2+ signals, including a diffuse increase in cytosolic [Ca2+], Ca2+ sparks, Ca2+ bursts, and regenerative Ca2+ release. The diffuse Ca2+ increase and Ca2+ bursts were ryanodine-insensitive, presumably arising from different endolysosomal sources. Ca2+ sparks and regenerative Ca2+ release were inhibited by ryanodine, consistent with cross-activation of loosely coupled ryanodine receptors. Moreover, Ca2+ release stimulated by endothelin-1 was inhibited by Ned-19, ryanodine, or xestospongin C, suggesting that NAADP-mediated Ca2+ signals interact with both ryanodine and inositol 1,4,5-trisphosphate receptors during agonist stimulation. Our results show that NAADP mediates complex global and local Ca2+ signals. Depending on the physiological stimuli, these diverse Ca2+ signals may serve to regulate different cellular functions in PASMCs. PMID:23443655

  8. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells.

    PubMed

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells.

  9. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  10. Nicotinamide extends replicative lifespan of human cells.

    PubMed

    Kang, Hyun Tae; Lee, Hyung Il; Hwang, Eun Seong

    2006-10-01

    We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6-fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho-Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide-treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro-aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production.

  11. Amperometric biosensor based on glassy carbon electrode modified with long-length carbon nanotube and enzyme

    NASA Astrophysics Data System (ADS)

    Furutaka, Hajime; Nemoto, Kentaro; Inoue, Yuki; Hidaka, Hiroki; Muguruma, Hitoshi; Inoue, Hitoshi; Ohsawa, Tatsuya

    2016-05-01

    An amperometric biosensor based on a glassy carbon electrode modified with long-length multiwalled carbon nanotubes (MWCNTs) and enzyme nicotinamide-adenine-dinucleotide-dependent glucose dehydrogenase (GDH) is presented. We demonstrate the effect of the MWCNT length on the amperometric response of the enzyme biosensor. The long length of MWCNT is 200 µm (average), whereas the normal length of MWCNT is 1 µm (average). The response of the long MWCNT-GDH electrode is 2 times more sensitive than that of the normal-length MWCNT-GDH electrode in the concentration range from 0.25-35 mM. The result of electrochemical impedance spectroscopy measurements suggest that the long-length MWCNT-GDH electrode formed a better electron transfer network than the normal-length one.

  12. Formate Dehydrogenase of Clostridium thermoaceticum: Incorporation of Selenium-75, and the Effects of Selenite, Molybdate, and Tungstate on the Enzyme

    PubMed Central

    Andreesen, Jan R.; Ljungdahl, Lars G.

    1973-01-01

    The formation of the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase in Clostridium thermoaceticum is stimulated by the presence of molybdate and selenite in the growth medium. The highest formate dehydrogenase activity was obtained with 2.5 × 10−4 M Na2MoO4 and 5 × 10−5 Na2SeO3. Tungstate but not vanadate could replace molybdate and stimulate the formation of formate dehydrogenase. Tungstate stimulated activity more than molybdate, and in combination with molybdate the stimulation of formation of formate dehydrogenase was additive. Formate dehydrogenase was isolated from cells grown in the presence of Na275SeO2, and a correlation was observed between bound 75Se and enzyme activity. PMID:4147651

  13. Frex and FrexH: Indicators of metabolic states in living cells.

    PubMed

    Zhao, Yuzheng; Yang, Yi

    2012-01-01

    Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form play central roles in energy and redox metabolisms. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level in living cells. We recently reported a series of genetically encoded fluorescent sensors highly specific for NADH. These sensors allow real-time, quantitative measurement of this significant molecule in different subcellular compartments. In this study, we provide a more detailed discussion of the benefits and limitations of these genetically encoded fluorescent sensors. These sensors are utilized in most laboratories without the need for sophisticated instruments because of their superior sensitivity and specificity. They are also viable alternatives to existing techniques for measuring the endogenous fluorescence of intracellular NAD(P)H.

  14. Enzymatic formation of the bisfuran structure in aflatoxin biosynthesis.

    PubMed Central

    Wan, N C; Hsieh, D P

    1980-01-01

    A relatively stable enzyme system that converts versiconal hemiacetal acetate to versicolorin A was isolated from the soluble fraction of the homogenized cells of Aspergillus parasiticus ATCC 15517. The cell-free preparation did not require oxygen or oxidized nicotinamide adenine dinucleotide phosphate for activity, nor did it require dithiothreitol, polyclar (polyvinyl pyrrolidone), or glycerol for stabilization of activity. It was susceptible to inhibition by dichlorvos and cysteine. Isotope tracer studies revealed involvement of several intermediates in the conversion of versiconal hemiacetal acetate to versicolorin A. These findings confirm the biogenetic relationship of versiconal hemiacetal acetate and versicolorin A, and they confirm that the bisfuran ring structure in aflatoxins and related fungal metabolites is derived from the hemiacetal structure of versiconal hemiacetal acetate. Images PMID:7356313

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

    PubMed

    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.

  16. Modulating NAD+ metabolism, from bench to bedside.

    PubMed

    Katsyuba, Elena; Auwerx, Johan

    2017-09-15

    Discovered in the beginning of the 20 th century, nicotinamide adenine dinucleotide (NAD + ) has evolved from a simple oxidoreductase cofactor to being an essential cosubstrate for a wide range of regulatory proteins that include the sirtuin family of NAD + -dependent protein deacylases, widely recognized regulators of metabolic function and longevity. Altered NAD + metabolism is associated with aging and many pathological conditions, such as metabolic diseases and disorders of the muscular and neuronal systems. Conversely, increased NAD + levels have shown to be beneficial in a broad spectrum of diseases. Here, we review the fundamental aspects of NAD + biochemistry and metabolism and discuss how boosting NAD + content can help ameliorate mitochondrial homeostasis and as such improve healthspan and lifespan. © 2017 The Authors.

  17. Carbon Nanotubes (CNTs) for the Development of Electrochemical Biosensors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lin, Yuehe; Yantasee, Wassana; Wang, Joseph

    2005-01-01

    Carbon nanotube (CNT) is a very attractive material for the development of biosensors because of its capability to provide strong electrocatalytic activity and minimize surface fouling of the sensors. This article reviews our recent developments of oxidase- and dehydrogenase-amperometric biosensors based on the immobilization of CNTs, the co-immobilization of enzymes on the CNTs/Nafion or the CNT/Teflon composite materials, or the attachment of enzymes on the controlled-density aligned CNT-nanoelectrode arrays. The excellent electrocatalytic activities of the CNTs on the redox reactions of hydrogen peroxide, nicotinamide adenine dinucleotide (NADH), and homocysteine have been demonstrated. Successful applications of the CNT-based biosensors reviewed hereinmore » include the low-potential detections of glucose, organophosphorus compounds, and alcohol.« less

  18. P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury.

    PubMed

    Yin, Terry C; Britt, Jeremiah K; De Jesús-Cortés, Héctor; Lu, Yuan; Genova, Rachel M; Khan, Michael Z; Voorhees, Jaymie R; Shao, Jianqiang; Katzman, Aaron C; Huntington, Paula J; Wassink, Cassie; McDaniel, Latisha; Newell, Elizabeth A; Dutca, Laura M; Naidoo, Jacinth; Cui, Huxing; Bassuk, Alexander G; Harper, Matthew M; McKnight, Steven L; Ready, Joseph M; Pieper, Andrew A

    2014-09-25

    The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. NAMPT and NAMPT-controlled NAD Metabolism in Vascular Repair.

    PubMed

    Wang, Pei; Li, Wen-Lin; Liu, Jian-Min; Miao, Chao-Yu

    2016-06-01

    Vascular repair plays important roles in postischemic remodeling and rehabilitation in cardiovascular and cerebrovascular disease, such as stroke and myocardial infarction. Nicotinamide adenine dinucleotide (NAD), a well-known coenzyme involved in electron transport chain for generation of adenosine triphosphate, has emerged as an important controller regulating various biological signaling pathways. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for NAD biosynthesis in mammals. NAMPT may also act in a nonenzymatic manner, presumably mediated by unknown receptor(s). Rapidly accumulating data in the past decade show that NAMPT and NAMPT-controlled NAD metabolism regulate fundamental biological functions in endothelial cells, vascular smooth muscle cells, and endothelial progenitor cells. The NAD-consuming proteins, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38, may contribute to the regulatory effects of NAMPT-NAD axis in these cells and vascular repair. This review discusses the current data regarding NAMPT and NAMPT-controlled NAD metabolism in vascular repair and the clinical potential translational application of NAMPT-related products in treatment of cardiovascular and cerebrovascular disease.

  20. Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McLaughlin, K.J.; Soares, A.; Strain-Damerell, C. M.

    2010-05-28

    Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD{sup +} is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD{sup +} ratio. Here we investigate the molecular mechanism for NADH/NAD{sup +} sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD{sup +}, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rexmore » from the DNA site following a 40{sup o} closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.« less

  1. Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations

    PubMed Central

    Griffin, Síle M.; Pickard, Mark R.; Orme, Rowan P.; Hawkins, Clive P.; Williams, Adrian C.

    2017-01-01

    Introduction Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington’s disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons. Methods Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods. Results Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an

  2. RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-κB pathway in human endothelial cells.

    PubMed

    Ning, R B; Zhu, J; Chai, D J; Xu, C S; Xie, H; Lin, X Y; Zeng, J Z; Lin, J X

    2013-12-13

    An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- κB). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-κB activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-κB. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-κB signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose.

  3. Time-domain laser-induced fluorescence spectroscopy apparatus for clinical diagnostics

    NASA Astrophysics Data System (ADS)

    Fang, Qiyin; Papaioannou, Thanassis; Jo, Javier A.; Vaitha, Russel; Shastry, Kumar; Marcu, Laura

    2004-01-01

    We report the design and development of a compact optical fiber-based apparatus for in situ time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) of biological systems. The apparatus is modular, optically robust, and compatible with the clinical environment. It incorporates a dual output imaging spectrograph, a gated multichannel plate photomultiplier (MCP-PMT), an intensified charge-coupled-device (ICCD) camera, and a fast digitizer. It can accommodate various types of light sources and optical fiber probes for selective excitation and remote light delivery/collection as required by different applications. The apparatus allows direct recording of the entire fluorescence decay with high sensitivity (nM range fluorescein dye concentration with signal-to-noise ratio of 46) and with four decades dynamic range. It is capable of resolving a broad range of fluorescence lifetimes from hundreds of picoseconds (as low as 300 ps) using the MCP-PMT coupled to the digitizer to milliseconds using the ICCD. The data acquisition and analysis process is fully automated, enabling fast recording of fluorescence intensity decay across the entire emission spectrum (0.8 s per wavelength or ˜40 s for a 200 nm wavelength range at 5 nm increments). The spectral and temporal responses of the apparatus were calibrated and its performance was validated using fluorescence lifetime standard dyes (Rhodamin B, 9-cyanoanthracene, and rose Bengal) and tissue endogenous fluorophores (elastin, collagen, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide). Fluorescence decay lifetimes and emission spectra of all tested compounds measured with the current tr-LIFS apparatus were found in good agreement with the values reported in the literature. The design and performance of tr-LIFS apparatus have enabled in vivo studies of atherosclerotic plaques and brain tumors.

  4. Effects of exogenous nicotinamide adenine dinucleotide (NAD+) in the rat heart are mediated by P2 purine receptors.

    PubMed

    Kuzmin, Vladislav S; Pustovit, Ksenia B; Abramochkin, Denis V

    2016-06-27

    Recently, NAD+ has been considered as an essential factor, participating in nerve control of physiological functions and intercellular communication. NAD+ also has been supposed as endogenous activator of P1 and P2 purinoreceptors. Effects of extracellular NAD+ remain poorly investigated in cardiac tissue. This study aims to investigate the effects of extracellular NAD+ in different types of supraventricular and ventricular working myocardium from rat and their potential mechanisms. The standard technique of sharp microelectrode action potential recording in cardiac multicellular preparations was used to study the effects of NAD+. Extracellular NAD+ induced significant changes in bioelectrical activity of left auricle (LA), right auricle (RA), pulmonary veins (PV) and right ventricular wall (RV) myocardial preparations. 10-100 μM NAD+ produced two opposite effects in LA and RA - quickly developing and transient prolongation of action potentials (AP) and delayed sustained AP shortening, which follows the initial positive effect. In PV and RV only AP shortening was observed in response to NAD+ application. In PV preparations AP shortening induced by NAD+ may be considered as a potential proarrhythmic effect. Revealed cardiotropic effects of NAD+ are likely to be mediated by P2 purine receptors, since P1 blocker DPCPX failed to affect them and P2 antagonist suramin abolished NAD + -induced alterations of electrical activity. P2X receptors may be responsible for NAD + -induced short-lasting AP prolongation, while P2Y receptors mediate persistent AP shortening. The latter effect is partially removed by PLC inhibitor U73122 showing the potential involvement of phosphoinositide signaling pathway in mediation of NAD+ cardiotropic effects. Extracellular NAD+ is supposed to be a novel regulator of cardiac electrical activity. P2 receptors represent the main target of NAD+ at least in the rat heart.

  5. Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models.

    PubMed

    Gong, Bing; Pan, Yong; Vempati, Prashant; Zhao, Wei; Knable, Lindsay; Ho, Lap; Wang, Jun; Sastre, Magdalena; Ono, Kenjiro; Sauve, Anthony A; Pasinetti, Giulio M

    2013-06-01

    Nicotinamide adenine dinucleotide (NAD)(+), a coenzyme involved in redox activities in the mitochondrial electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the activation of NAD(+) expression has been linked with a decrease in beta-amyloid (Aβ) toxicity in Alzheimer's disease (AD). Nicotinamide riboside (NR) is a NAD(+) precursor, it promotes peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α expression in the brain. Evidence has shown that PGC-1α is a crucial regulator of Aβ generation because it affects β-secretase (BACE1) degradation. In this study we tested the hypothesis that NR treatment in an AD mouse model could attenuate Aβ toxicity through the activation of PGC-1α-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysiological recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD(+) in the cerebral cortex; (2) application of NR to hippocampal slices (10 μM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1 degradation was prevented by PGC-1α-shRNA gene silencing; and (4) NR treatment and PGC-1α overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, in part by promoting PGC-1α-mediated BACE1

  6. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Nicotinamide-ascorbic acid complex. 172.315... HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed conditions...

  7. Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I.

    PubMed

    Lorenz, Nicole; Olsovská, Jana; Sulc, Miroslav; Tudzynski, Paul

    2010-03-01

    Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

  8. Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3

    PubMed Central

    Khan, Nahid A; Auranen, Mari; Paetau, Ilse; Pirinen, Eija; Euro, Liliya; Forsström, Saara; Pasila, Lotta; Velagapudi, Vidya; Carroll, Christopher J; Auwerx, Johan; Suomalainen, Anu

    2014-01-01

    Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD+/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD+ precursor, was previously shown to boost NAD+ levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD+ levels are a promising treatment strategy for mitochondrial myopathy. PMID:24711540

  9. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistancemore » in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.« less

  10. First genetic characterization of Fasciola hepatica in Argentina by nuclear and mitochondrial gene markers.

    PubMed

    Carnevale, Silvana; Malandrini, Jorge Bruno; Pantano, María Laura; Soria, Claudia Cecilia; Rodrigues-Silva, Rosângela; Machado-Silva, José Roberto; Velásquez, Jorge Néstor; Kamenetzky, Laura

    2017-10-15

    Fasciola hepatica is a trematode showing genetic variation among isolates from different regions of the world. The objective of this work was to characterize for the first time F. hepatica isolates circulating in different regions of Argentina. Twenty-two adult flukes were collected from naturally infected bovine livers in different areas from Argentina and used for DNA extraction. We carried out PCR amplification and sequence analysis of the ribosomal internal transcribed spacer 1 (ITS1), mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunits 4 and 5 (nad4 and nad5) and mitochondrial cytochrome c oxidase subunit I (cox1) genes as genetic markers. Phylogenies were reconstructed using maximum parsimony algorithm. A total of 6 haplotypes were found for cox1, 4 haplotypes for nad4 and 3 haplotypes for nad5. The sequenced ITS1 fragment was identical in all samples. The analyzed cox1 gene fragment is the most variable marker and is recommended for future analyses. No geographic association was found in the Argentinean samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

    PubMed

    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. © 2016 by The American Society for Biochemistry and Molecular

  12. Time-gated FLIM microscope for corneal metabolic imaging

    NASA Astrophysics Data System (ADS)

    Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel

    2016-03-01

    Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

  13. Novel fiber optic-based needle redox imager for cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Kanniyappan, Udayakumar; Xu, He N.; Tang, Qinggong; Gaitan, Brandon; Liu, Yi; Li, Lin Z.; Chen, Yu

    2018-02-01

    Despite various technological advancements in cancer diagnosis, the mortality rates were not decreased significantly. We aim to develop a novel optical imaging tool to assist cancer diagnosis effectively. Fluorescence spectroscopy/imaging is a fast, rapid, and minimally invasive technique which has been successfully applied to diagnosing cancerous cells/tissues. Recently, the ratiometric imaging of intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), as pioneered by Britton Chance and the co-workers in 1950-70's, has gained much attention to quantify the physiological parameters of living cells/tissues. The redox ratio, i.e., FAD/(FAD+NADH) or FAD/NADH, has been shown to be sensitive to various metabolic changes in in vivo and in vitro cells/tissues. Optical redox imaging has also been investigated for providing potential imaging biomarkers for cancer transformation, aggressiveness, and treatment response. Towards this goal, we have designed and developed a novel fiberoptic-based needle redox imager (NRI) that can fit into an 11G clinical coaxial biopsy needle for real time imaging during clinical cancer surgery. In the present study, the device is calibrated with tissue mimicking phantoms of FAD and NADH along with various technical parameters such as sensitivity, dynamic range, linearity, and spatial resolution of the system. We also conducted preliminary imaging of tissues ex vivo for validation. We plan to test the NRI on clinical breast cancer patients. Once validated this device may provide an effective tool for clinical cancer diagnosis.

  14. RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides.

    PubMed

    Liu, Su; Liang, Qing-Mei; Zhou, Wen-Wu; Jiang, Yan-Dong; Zhu, Qing-Zi; Yu, Hang; Zhang, Chuan-Xi; Gurr, Geoff M; Zhu, Zeng-Rong

    2015-01-01

    NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens. © 2014 Society of Chemical Industry.

  15. Oxygen sensing PLIM together with FLIM of intrinsic cellular fluorophores for metabolic mapping

    NASA Astrophysics Data System (ADS)

    Kalinina, Sviatlana; Schaefer, Patrick; Breymayer, Jasmin; Bisinger, Dominik; Chakrabortty, Sabyasachi; Rueck, Angelika

    2018-02-01

    Otical imaging techniques based on time correlated single photon counting (TCSPC) has found wide applications in medicine and biology. Non-invasive and information-rich fluorescence lifetime imaging microscopy (FLIM) is successfully used for monitoring fluorescent intrinsic metabolic coenzymes as NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) and FAD+ (flavin adenine dinucleotide) in living cells and tissues. The ratio between proteinbound and free coenzymes gives an information about the balance between oxidative phosphorylation and glycolysis in the cells. The changes of the ratio reflects major cellular disorders. A correlation exists between metabolic activity, redox ratio and fluorescence lifetime during stem cell differentiation, neurodegenerative diseases, and carcinogenesis. A multichannel FLIM detection system was designed for monitoring the redox state of NAD(P)H and FAD+ and other intrinsic fluorophores as protoporphyrin IX. In addition, the unique upgrade is useful to perform FLIM and PLIM (phosphorescence lifetime imaging microscopy) simultaneously. PLIM is a promising method to investigate oxygen sensing in biomedical samples. In detail, the oxygen-dependent quenching of phosphorescence of some compounds as transition metal complexes enables measuring of oxygen partial pressure (pO2). Using a two-channel FLIM/PLIM system we monitored intrinsic pO2 by PLIM simultaneously with NAD(P)H by FLIM providing complex metabolic and redox imaging of living cells. Physico-chemical properties of oxygen sensitive probes define certain parameters including their localisation. We present results of some ruthenium based complexes including those specifically bound to mitochondria.

  16. Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy

    NASA Astrophysics Data System (ADS)

    Schweitzer, Dietrich; Deutsch, Lydia; Klemm, Matthias; Jentsch, Susanne; Hammer, Martin; Peters, Sven; Haueisen, Jens; Müller, Ulrich A.; Dawczynski, Jens

    2015-06-01

    The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included in the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τi, amplitudes αi, and relative contributions Qi were statistically compared between corresponding groups in two spectral channels (490450 ps, and the shift of τ3 from ˜3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine dinucleotide at the fundus. AGE also accumulated in the crystalline lens.

  17. Cu2+ -Modified Metal-Organic Framework Nanoparticles: A Peroxidase-Mimicking Nanoenzyme.

    PubMed

    Chen, Wei-Hai; Vázquez-González, Margarita; Kozell, Anna; Cecconello, Alessandro; Willner, Itamar

    2018-02-01

    The synthesis and characterization of UiO-type metal-organic framework nanoparticles (NMOFs) composed of Zr 4+ ions bridged by 2,2'-bipyridine-5,5'-dicarboxylic acid ligands and the postmodification of the NMOFs with Cu 2+ ions are described. The resulting Cu 2+ -modified NMOFs, Cu 2+ -NMOFs, exhibit peroxidase-like catalytic activities reflected by the catalyzed oxidation of Amplex-Red to the fluorescent Resorufin by H 2 O 2 , the catalyzed oxidation of dopamine to aminochrome by H 2 O 2 , and the catalyzed generation of chemiluminescence in the presence of luminol/H 2 O 2 . Also, the Cu 2+ -NMOFs mimic NADH peroxidase functions and catalyze the oxidation of dihydronicotinamide adenine dinucleotide, NADH, to nicotinamide adenine dinucleotide, NAD + , in the presence of H 2 O 2 . The Cu 2+ -NMOFs-catalyzed generation of chemiluminescence in the presence of luminol/H 2 O 2 is used to develop a glucose sensor by monitoring the H 2 O 2 formed by the aerobic oxidation of glucose to gluconic acid in the presence of glucose oxidase. Furthermore, loading the Cu 2+ -NMOFs with fluorescein and activating the catalyzed generation of chemiluminescence in the presence of luminol/H 2 O 2 yield an efficient chemiluminescence resonance energy transfer (CRET) process to the fluorescein reflected by the activation of the fluorescence of the dye (λ = 520 nm, CRET efficiency 35%). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  19. Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase.

    PubMed

    Tretter, Laszlo; Adam-Vizi, Vera

    2004-09-08

    Alpha-ketoglutarate dehydrogenase (alpha-KGDH), a key enzyme in the Krebs' cycle, is a crucial early target of oxidative stress (Tretter and Adam-Vizi, 2000). The present study demonstrates that alpha-KGDH is able to generate H(2)O(2) and, thus, could also be a source of reactive oxygen species (ROS) in mitochondria. Isolated alpha-KGDH with coenzyme A (HS-CoA) and thiamine pyrophosphate started to produce H(2)O(2) after addition of alpha-ketoglutarate in the absence of nicotinamide adenine dinucleotide-oxidized (NAD(+)). NAD(+), which proved to be a powerful inhibitor of alpha-KGDH-mediated H(2)O(2) formation, switched the H(2)O(2) forming mode of the enzyme to the catalytic [nicotinamide adenine dinucleotide-reduced (NADH) forming] mode. In contrast, NADH stimulated H(2)O(2) formation by alpha-KGDH, and for this, neither alpha-ketoglutarate nor HS-CoA were required. When all of the substrates and cofactors of the enzyme were present, the NADH/NAD(+) ratio determined the rate of H(2)O(2) production. The higher the NADH/NAD(+) ratio the higher the rate of H(2)O(2) production. H(2)O(2) production as well as the catalytic function of the enzyme was activated by Ca(2+). In synaptosomes, using alpha-ketoglutarate as respiratory substrate, the rate of H(2)O(2) production increased by 2.5-fold, and aconitase activity decreased, indicating that alpha-KGDH can generate H(2)O(2) in in situ mitochondria. Given the NADH/NAD(+) ratio as a key regulator of H(2)O(2) production by alpha-KGDH, it is suggested that production of ROS could be significant not only in the respiratory chain but also in the Krebs' cycle when oxidation of NADH is impaired. Thus alpha-KGDH is not only a target of ROS but could significantly contribute to generation of oxidative stress in the mitochondria.

  20. Progressive thermopreconditioning attenuates rat cardiac ischemia/reperfusion injury by mitochondria-mediated antioxidant and antiapoptotic mechanisms.

    PubMed

    Chien, Chen-Yen; Chien, Chiang-Ting; Wang, Shoei-Shen

    2014-08-01

    Progressive thermal preconditioning (PTP) provides vascular protection with less hemodynamic fluctuations, endoplasmic reticulum (ER), and oxidative stress compared with whole body hyperthermia. We suggest PTP might efficiently diminish cardiac ischemia/reperfusion-induced apoptosis and autophagy injury. A total of 67 male Wistar rats were divided into a non-PTP control group, 24 or 72 hours after a single cycle or 3 consecutive cycles of PTP in a 42°C water bath (1-24, 1-72, 3-24, and 3-72 groups). We measured the cardiac O2(-) amount in vivo in response to left anterior descending coronary artery ligation for 2 hours and reperfusion for 3 hours. Cardiac function and injury were determined by microcirculation, electrocardiography, and infarct size. The PTP-induced protective effects on nicotinamide adenine dinucleotide phosphate oxidase gp91-mediated oxidative stress, ER stress, and apoptosis- and autophagy-related mechanisms were examined using Western blot and immunohistochemistry. Coronary arterial ischemia/reperfusion depressed cardiac microcirculation, induced ST-segment elevation and increased infarct size in non-PTP and PTP rats. Ischemia/reperfusion enhanced the cardiac O2(-) levels by enhanced nicotinamide adenine dinucleotide phosphate oxidase gp91 expression, cytosolic cytochrome C release, and decreased mitochondrial Bcl-2 expression. Cardiac injury activated ER stress-78-kDa glucose-regulated protein expression, increased the Bax/Bcl-2 ratio, cleaved caspase 3 expression and poly-(ADP-ribose)-polymerase fragments, leading to apoptosis formation, and promoted LC3-II expression, resulting in autophagy formation. PTP treatment elevated heat shock protein 70, heat shock protein 32, Bcl-2, Bcl-xL, and manganese superoxide dismutase in the rat heart, especially in the 3-72 group. PTP treatment significantly restored cardiac microcirculation, decreased oxidative stress, ER stress, apoptosis, autophagy, and infarct size. PTP significantly reduced cardiac

  1. Intraoperative microwave ablation of pulmonary malignancies with tumor permittivity feedback control: ablation and resection study in 10 consecutive patients.

    PubMed

    Wolf, Farrah J; Aswad, Bassam; Ng, Thomas; Dupuy, Damian E

    2012-01-01

    To determine histologic changes induced by microwave ablation (MWA) in patients with pulmonary malignancy by using an ablation system with tumor permittivity feedback control, enabling real-time modulation of energy power and frequency. Institutional review board approval and patient informed consent were obtained for this prospective HIPAA-complaint ablation and resection study. Between March 2009 and January 2010, 10 patients (four women, six men; mean age, 71 years; age range, 52-82 years) underwent intraoperative MWA of pulmonary malignancies. Power (10-32 W) and frequency (908-928 MHz) were continuously adjusted by the generator to maintain a temperature of 110°-120°C at the 14-gauge antenna tip for one 10-minute application. After testing for an air leak, tumors were resected surgically. Gross inspection, slicing, and hematoxylin-eosin (10 specimens) and nicotinamide adenine dinucleotide (six specimens) staining were performed. Tumors included adenocarcinomas (n = 5), squamous cell carcinomas (n = 3), and metastases from endometrial (n = 1) and colorectal (n = 1) primary carcinomas. Mean maximum tumor diameter was 2.4 cm (range, 0.9-5.0 cm), and mean maximum volume was 8.6 cm(3) (range, 0.5-52.7 cm(3)). One air leak was detected. Five of 10 specimens were grossly measurable, revealing a mean maximum ablation zone diameter of 4.8 cm (range, 3.0-6.5 cm) and a mean maximum ablation zone volume of 15.1 cm(3) (range, 7.3-25.1 cm(3)). At hematoxylin-eosin staining, coagulation necrosis was observed in all ablation zones, extended into the normal lung in nine of 10 specimens, and up to blood vessel walls without evidence of vessel (>4 mm) thrombosis. Nicotinamide adenine dinucleotide staining enabled confirmation of no viability within ablation zones extending into normal lung in five of six specimens. MWA with tumor permittivity feedback control results in cytotoxic intratumoral temperatures and extension of ablation zones into aerated peritumoral pulmonary

  2. Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I ▿

    PubMed Central

    Lorenz, Nicole; Olšovská, Jana; Šulc, Miroslav; Tudzynski, Paul

    2010-01-01

    Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I. PMID:20118373

  3. Adenine specific DNA chemical sequencing reaction.

    PubMed Central

    Iverson, B L; Dervan, P B

    1987-01-01

    Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions. Images PMID:3671067

  4. Supramolecular polymer formation by cyclic dinucleotides and intercalators affects dinucleotide enzymatic processing

    PubMed Central

    Nakayama, Shizuka; Zhou, Jie; Zheng, Yue; Szmacinski, Henryk; Sintim, Herman O

    2016-01-01

    Background: Cyclic dinucleotides form supramolecular aggregates with intercalators, and this property could be utilized in nanotechnology and medicine. Methods & results: Atomic force microscopy and electrophoretic mobility shift assays were used to show that cyclic diguanylic acid (c-di-GMP) forms G-wires in the presence of intercalators. The average fluorescence lifetime of thiazole orange, when bound to c-di-GMP was greater than when bound to DNA G-quadruplexes or dsDNA. The stability of c-di-GMP supramolecular polymers is dependent on both the nature of the cation present and the intercalator. C-di-GMP or cyclic diadenylic acid/intercalator complexes are more resistant to cleavage by YybT, a phosphodiesterase, than the uncomplexed nucleotides. Conclusion: Cleavage of bacterial cyclic dinucleotides could be slowed down via complexation with small molecules and that this could be utilized for diverse applications in nanotechnology and medicine. PMID:28031943

  5. Adenine alleviates iron overload by cAMP/PKA mediated hepatic hepcidin in mice.

    PubMed

    Zhang, Yingqi; Wang, Xudong; Wu, Qian; Wang, Hao; Zhao, Lu; Wang, Xinhui; Mu, Mingdao; Xie, Enjun; He, Xuyan; Shao, Dandan; Shang, Yanna; Lai, Yongrong; Ginzburg, Yelena; Min, Junxia; Wang, Fudi

    2018-03-30

    Hemochromatosis is prevalent and often associated with high rates of morbidity and mortality worldwide. The safe alternative iron-reducing approaches are urgently needed in order to better control iron overload. Our unbiased vitamin screen for modulators of hepcidin, a master iron regulatory hormone, identifies adenine (vitamin B4) as a potent hepcidin agonist. Adenine significantly induced hepcidin mRNA level and promoter activity activation in human cell lines, possibly through BMP/SMAD pathway. Further studies in mice validated the effect of adenine on hepcidin upregulation. Consistently, adenine dietary supplement in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD pathway. Notably, adenine-rich diet significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe -/- mice, a murine model of hereditary hemochromatosis. To further validate this finding, we selected pharmacological inhibitors against BMP (LDN193189). We found LDN193189 strongly blocked the hepcidin induction by adenine. Moreover, we uncovered an essential role of cAMP/PKA-dependent axis in triggering adenine-induced hepcidin expression in primary hepatocytes by using 8 br cAMP, a cAMP analog, and H89, a potent inhibitor for PKA signaling. These findings suggest a potential therapeutic role of adenine for hereditary hemochromatosis. © 2018 Wiley Periodicals, Inc.

  6. Histochemical characteristics of a tonic smooth muscle.

    PubMed

    Gilloteaux, J; Stalmans-Falys, M

    1979-09-01

    It is suggested that ABRM, smooth muscle of Mytilus edulis L. and Mytilus galloprovincialis Lmk. (Mollusca Pelecypoda), is composed of one histochemical fibre type. The fibres are characterized by a low myofibrillar ATPase activity. Succinic and nicotinamide adenine dinucleotide oxidoreductase activities are distributed in a reverse pattern than that of the ATPase activity. Glycogen phosphorylase is richly represented in ABRM fibres and this detection is in opposition with the negative detection of alkaline phosphatase activity. These preliminary histochemical observations are similar to those found in some vertebrate smooth muscles. Mitochondrial glycerol-3-phosphate, 6-phosphogluconate, lactate and octopine dehydrogenases are not detected in muscle fibres whereas glio-interstitial tissues show weak but distinct reactivity. These last results especially characterize Mytilus catch fibres and are briefly discussed in relationship with previous physiological, biochemical and morphological observations.

  7. Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

    PubMed

    Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

    2017-03-02

    The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H 2 O 2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H 2 O 2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H 2 O 2 -free conditions). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Formation of (4R)- and (4S)-4-hydroxyochratoxin A from ochratoxin A by liver microsomes from various species.

    PubMed Central

    Størmer, F C; Hansen, C E; Pedersen, J I; Hvistendahl, G; Aasen, A J

    1981-01-01

    Two metabolic products were formed from ochratoxin A by human, pig, and rat liver microsomal fractions in the presence of reduced nicotinamide adenine dinucleotide phosphate. They were isolated from the incubation mixture in the presence of pig liver microsomes by extraction, thin-layer chromatography, and high-pressure liquid chromatography Their structures are suggested to be (4R)- and (4S)-4-hydroxyochratoxin A on the basis of mass and nuclear magnetic resonance spectroscopy. Km and the maximum velocity for the formation of the two metabolites by human, pig, and rat microsomes were determined. Their formation was inhibited by carbon monoxide and metyrapone. The results indicate that the microsomal hydroxylation system is a cytochrome P-450 and that different species are involved in the formation of the two epimeric forms of 4-hydroxyochratoxin A. PMID:7316512

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

    NASA Astrophysics Data System (ADS)

    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.

  10. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging

    PubMed Central

    Li, Jun; Bonkowski, Michael S.; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P.; Ling, Alvin J. Y.; Rajman, Luis A.; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L.; Steegborn, Clemens; Sinclair, David A.

    2017-01-01

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD+ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD+ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate–ribose) polymerase], a critical DNA repair protein. As mice age and NAD+ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD+. Thus, NAD+ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. PMID:28336669

  11. Reactive Oxygen Species and Inhibitors of Inflammatory Enzymes, NADPH Oxidase, and iNOS in Experimental Models of Parkinson's Disease

    PubMed Central

    Koppula, Sushruta; Kumar, Hemant; Kim, In Su; Choi, Dong-Kug

    2012-01-01

    Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD. PMID:22577256

  12. Nitrite Formation from Hydroxylamine and Oximes by Pseudomonas aeruginosa

    PubMed Central

    Amarger, Noelle; Alexander, M.

    1968-01-01

    Nitrite was formed from hydroxylamine and several oximes by intact cells and extracts of Pseudomonas aeruginosa. The activity was induced by the presence of oximes in the culture medium. Nitroalkanes were not intermediates in the conversion of acetaldoxime, acetone oxime, or butanone oxime to nitrite, since nitromethane inhibited the formation of nitrite from the nitro compounds but not from the corresponding oximes. The oxime apparently functions as a constant source of hydroxylamine during growth of the bacterium. Hydroxylamine at low concentration was converted stoichiometrically to nitrite by extracts of the bacterium; high concentrations were inhibitory. Nicotinamide adenine dinucleotide phosphate, oxygen, and other unidentified cofactors were necessary for the reaction. Actively nitrifying extracts possessed no hydroxylamine-cytochrome c reductase activity. Hyponitrite, nitrous oxide, and nitric oxide were not metabolized. PMID:4384968

  13. Partial transformation from fast to slow muscle fibers induced by deafferentation of capsaicin-sensitive muscle afferents.

    PubMed

    Brunetti, O; Barazzoni, A M; Della Torre, G; Clavenzani, P; Pettorossi, V E; Bortolami, R

    1997-11-01

    Mechanical and histochemical characteristics of the lateral gastrocnemius (LG) muscle of the rat were examined 21 days after capsaicin injection into the LG muscle. The capsaicin caused a decrease in generation rate of twitch and tetanic tension and an increase in fatigue resistance of LG muscle. The histochemical muscle fiber profile evaluated by myosin adenosine triphosphatase and reduced nicotinamide adenine dinucleotide tetrazolium reductase methods showed an increase of type I and IIC fibers and a decrease of the type IIB in whole muscle, and a decrease of the IIA, IIX fibers in the red part accompanied by their increase in the white part. Therefore the capsaicin treatment, which selectively eliminated fibers belonging to the III and IV groups of muscle afferents, induced muscle fiber transformation from fast contracting fatiguing fibers to slowly contracting nonfatiguing ones.

  14. Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

    PubMed

    Kishore, G; Sugumaran, M; Vaidyanathan, C S

    1976-10-01

    A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate.

  15. Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

    PubMed Central

    Kishore, G; Sugumaran, M; Vaidyanathan, C S

    1976-01-01

    A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate. PMID:10273

  16. First Report of Echinococcus equinus in a Donkey in Turkey

    PubMed Central

    Simsek, Sami; Roinioti, Erifylli; Eroksuz, Hatice

    2015-01-01

    A 2-year-old female donkey (Equus asinus) was euthanized in the Pathology Department of Firat University, Elazig, Turkey. Necropsy disclosed the presence of 7 hydatid cysts distributed throughout the lung parenchyma. One of those cysts represented the parasite material of the present study and was molecularly identified through sequencing of a fragment of cytochrome c oxidase subunit 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (NADH1) gene, as Echinococcus equinus. The generated CO1 sequence supports the presence of the dominant haplotype as has been described in Europe and Africa. The NADH1 sequence was found similar to sequences reported in equids in Egypt and the United Kingdom. The molecular identification of E. equinus in a donkey is being reported for the first time in Turkey. PMID:26797441

  17. First Report of Echinococcus equinus in a Donkey in Turkey.

    PubMed

    Simsek, Sami; Roinioti, Erifylli; Eroksuz, Hatice

    2015-12-01

    A 2-year-old female donkey (Equus asinus) was euthanized in the Pathology Department of Firat University, Elazig, Turkey. Necropsy disclosed the presence of 7 hydatid cysts distributed throughout the lung parenchyma. One of those cysts represented the parasite material of the present study and was molecularly identified through sequencing of a fragment of cytochrome c oxidase subunit 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (NADH1) gene, as Echinococcus equinus. The generated CO1 sequence supports the presence of the dominant haplotype as has been described in Europe and Africa. The NADH1 sequence was found similar to sequences reported in equids in Egypt and the United Kingdom. The molecular identification of E. equinus in a donkey is being reported for the first time in Turkey.

  18. Uric acid and transforming growth factor in fructose-induced production of reactive oxygen species in skeletal muscle

    PubMed Central

    Maarman, Gerald J.; Ojuka, Edward

    2016-01-01

    The consumption of fructose, a major constituent of the modern diet, has raised increasing concern about the effects of fructose on health. Research suggests that excessive intake of fructose (>50 g/d) causes hyperuricemia, insulin resistance, mitochondrial dysfunction, de novo lipogenesis by the liver, and increased production of reactive oxygen species (ROS) in muscle. In a number of tissues, uric acid has been shown to stimulate the production of ROS via activation of transforming growth factor β1 and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4. The role of uric acid in fructose-induced production of ROS in skeletal muscle, however, has not been investigated. This review examines the evidence for fructose-induced production of ROS in skeletal muscle, highlights proposed mechanisms, and identifies gaps in current knowledge. PMID:26946251

  19. Abnormal Septation and Inhibition of Sporulation by Accumulation of l-α-Glycerophosphate in Bacillus subtilis Mutants

    PubMed Central

    Oh, Yong K.; Freese, Elisabeth B.; Freese, Ernst

    1973-01-01

    Accumulation of l-α-glycerophosphate, in cells of Bacillus subtilis mutants lacking the nicotinamide adenine dinucleotide-independent glycerophosphate dehydrogenase activity, suppresses both growth and sporulation. After growth has stopped, the cells slowly develop one and later more asymmetric septa that are thicker than normal prespore septa and apparently contain too much cell wall material to allow further membrane development into forespores or spores. l-Malate prevents accumulation of glycerophosphate and restores sporulation of the mutant. Glucose or gluconate cannot resotre sporulation, because they still effect glycerophosphate accumulation via de novo synthesis. If that accumulation is blocked in a double mutant, which is unable to make glycerophosphate from or to metabolize it into Embden-Meyerhof compounds, then nonsuppressing amounts of glucose or gluconate can restore sporulation. Images PMID:4632310

  20. Cerebral ischemia and neuroregeneration

    PubMed Central

    Lee, Reggie H. C.; Lee, Michelle H. H.; Wu, Celeste Y. C.; Couto e Silva, Alexandre; Possoit, Harlee E.; Hsieh, Tsung-Han; Minagar, Alireza; Lin, Hung Wen

    2018-01-01

    Cerebral ischemia is one of the leading causes of morbidity and mortality worldwide. Although stroke (a form of cerebral ischemia)-related costs are expected to reach 240.67 billion dollars by 2030, options for treatment against cerebral ischemia/stroke are limited. All therapies except anti-thrombolytics (i.e., tissue plasminogen activator) and hypothermia have failed to reduce neuronal injury, neurological deficits, and mortality rates following cerebral ischemia, which suggests that development of novel therapies against stroke/cerebral ischemia are urgently needed. Here, we discuss the possible mechanism(s) underlying cerebral ischemia-induced brain injury, as well as current and future novel therapies (i.e., growth factors, nicotinamide adenine dinucleotide, melatonin, resveratrol, protein kinase C isozymes, pifithrin, hypothermia, fatty acids, sympathoplegic drugs, and stem cells) as it relates to cerebral ischemia. PMID:29623912

  1. Cyclic Dinucleotides in Oral Bacteria and in Oral Biofilms.

    PubMed

    Gürsoy, Ulvi K; Gürsoy, Mervi; Könönen, Eija; Sintim, Herman O

    2017-01-01

    Oral cavity acts as a reservoir of bacterial pathogens for systemic infections and several oral microorganisms have been linked to systemic diseases. Quorum sensing and cyclic dinucleotides, two "decision-making" signaling systems, communicate to regulate physiological process in bacteria. Discovery of cyclic dinucleotides has a long history, but the progress in our understanding of how cyclic dinucleotides regulate bacterial lifestyle is relatively new. Oral microorganisms form some of the most intricate biofilms, yet c-di-GMP, and c-di-AMP signaling have been rarely studied in oral biofilms. Recent studies demonstrated that, with the aid of bacterial messenger molecules and their analogs, it is possible to activate host innate and adaptive immune responses and epithelial integrity with a dose that is relevant to inhibit bacterial virulence mechanisms, such as fimbriae and exopolysaccharide production, biofilm formation, and host cell invasion. The aim of this perspective article is to present available information on cyclic dinucleotides in oral bacteria and in oral biofilms. Moreover, technologies that can be used to detect cyclic dinucleotides in oral biofilms are described. Finally, directions for future research are highlighted.

  2. Enzymatic synthesis of polymers containing nicotinamide mononucleotide

    NASA Technical Reports Server (NTRS)

    Liu, Rihe

    1995-01-01

    Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

  3. Enzymatic Synthesis of Polymers Containing Nicotinamide Mononucleotide

    NASA Technical Reports Server (NTRS)

    Liu, Rihe; Orgel, Leslie E.

    1995-01-01

    Nicotinamide mononucleoside 5'-diphosphate in its reduced form is an excellent substrate for polynucleotide phosphorylase from Micrococcus luteus both in de novo polymerization reactions and in primer extension reactions. The oxidized form of the diphosphate is a much less efficient substrate; it can be used to extend primers but does not oligomerize in the absence of a primer. The cyanide adduct of the oxidized substrate, like the reduced substrate, polymerizes efficiently. Loss of cyanide yields high molecular weight polymers of the oxidized form. Terminal transferase from calf thymus accepts nicotinamide mononucleoside 5'-triphosphate as a substrate and efficiently adds one residue to the 3'-end of an oligodeoxynucleotide. T4 polynucleotide kinase accepts oligomers of nicotinamide mononucleotide as substrates. However, RNA polymerases do not incorporate nicotinamide mononucleoside 5'-triphosphate into products on any of the templates that we used.

  4. Influence of large intrahepatic blood vessels on the gross and histological characteristics of lesions produced by radiofrequency ablation in a pig liver model.

    PubMed

    Tamaki, Katsuyoshi; Shimizu, Ichiro; Oshio, Atsuo; Fukuno, Hiroshi; Inoue, Hiroshi; Tsutsui, Akemi; Shibata, Hiroshi; Sano, Nobuya; Ito, Susumu

    2004-12-01

    To determine whether the presence of large intrahepatic blood vessels (>/=3 mm) affect radiofrequency (RF)-induced coagulation necrosis, the gross and histological characteristics of RF-ablated areas proximal to or around vessels were examined in normal pig livers. An RF ablation treatment using a two-stepwise extension technique produced 12 lesions: six contained vessels (Group A), and the other six were localized around vessels (Group B). Gross examination revealed that the longest and shortest diameters of the ablated lesions were significantly larger in Group B than in Group A. In Group A, patent vessels contiguous to the lesion were present in a tongue-shaped area, whereas the lesions in Group B were spherical. Staining with nicotinamide adenine dinucleotide diaphorase was negative within the ablated area; but, if vessels were present in the ablated area, the cells around the vessels in an opposite direction to the ablation were stained blue. Roll-off can be achieved with 100% cellular destruction within a lesion that does not contain large vessels. The ablated area was decreased in lesions that contained large vessels, suggesting that the presence of large vessels in the ablated area further increases the cooling effect and may require repeated RF ablation treatment to achieve complete coagulation necrosis.

  5. The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage.

    PubMed

    Douiev, Liza; Saada, Ann

    2018-06-07

    Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50 μM KCN for 24 h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD + ) deficit combined with a replicative state, rather than oxidative stress and energy depletion. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3.

    PubMed

    Khan, Nahid A; Auranen, Mari; Paetau, Ilse; Pirinen, Eija; Euro, Liliya; Forsström, Saara; Pasila, Lotta; Velagapudi, Vidya; Carroll, Christopher J; Auwerx, Johan; Suomalainen, Anu

    2014-06-01

    Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD(+)/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD(+) precursor, was previously shown to boost NAD(+) levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD(+) levels are a promising treatment strategy for mitochondrial myopathy. © 2014 The Authors. Published under the terms of the CC BY license.

  7. Nicotinamide Riboside and Nicotinic Acid Riboside Salvage in Fungi and Mammals

    PubMed Central

    Belenky, Peter; Christensen, Kathryn C.; Gazzaniga, Francesca; Pletnev, Alexandre A.; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

  8. Radioresistance of Adenine to Cosmic Rays.

    PubMed

    Vignoli Muniz, Gabriel S; Mejía, Christian F; Martinez, Rafael; Auge, Basile; Rothard, Hermann; Domaracka, Alicja; Boduch, Philippe

    2017-04-01

    The presence of nucleobases in carbonaceous meteorites on Earth is an indication of the existence of this class of molecules in outer space. However, space is permeated by ionizing radiation, which can have damaging effects on these molecules. Adenine is a purine nucleobase that amalgamates important biomolecules such as DNA, RNA, and ATP. Adenine has a unique importance in biochemistry and therefore life. The aim of this work was to study the effects of cosmic ray analogues on solid adenine and estimate its survival when exposed to corpuscular radiation. Adenine films were irradiated at GANIL (Caen, France) and GSI (Darmstadt, Germany) by 820 MeV Kr 33+ , 190 MeV Ca 10+ , 92 MeV Xe 23+ , and 12 MeV C 4+ ion beams at low temperature. The evolution of adenine molecules under heavy ion irradiation was studied by IR absorption spectroscopy as a function of projectile fluence. It was found that the adenine destruction cross section (σ d ) follows an electronic stopping power (S e ) power law under the form: CS e n ; C is a constant, and the exponential n is a dimensionless quantity. Using the equation above to fit our results, we determined σ d  = 4 × 10 -17 S e 1.17 , with S e in kiloelectronvolts per micrometer (keV μm -1 ). New IR absorption bands arise under irradiation of adenine and can be attributed to HCN, CN - , C 2 H 4 N 4 , CH 3 CN, and (CH 3 ) 3 CNC. These findings may help to understand the stability and chemistry related to complex organic molecules in space. The half-life of solid adenine exposed to the simulated interstellar medium cosmic ray flux was estimated as (10 ± 8) × 10 6 years. Key Words: Heavy ions-Infrared spectroscopy-Astrochemistry-Cosmic rays-Nucleobases-Adenine. Astrobiology 17, 298-308.

  9. Responses of Adenine Nucleotides in Germinating Soybean Embryonic Axes to Exogenously Applied Adenine and Adenosine

    PubMed Central

    Anderson, James D.

    1977-01-01

    The ATP content of soybean (Glycine max [L.] Merr. cv. Kent) axes incubated for 3 hours in 1 mm solutions of adenine and adenosine increased over 100% and 75%, respectively, over axes incubated in water. The increase in ATP was primarily due to the conversion of these purines to nucleotides via the nucleotide salvage pathway. The ATP formed was in a metabolically active pool because label from adenine was incorporated into acid-insoluble material. Adenine also increased the levels of GTP, UTP, and CTP, but not to the extent of the ATP level. PMID:16660165

  10. Identification of novel resistance mechanisms to NAMPT inhibition via the de novo NAD+ biosynthesis pathway and NAMPT mutation.

    PubMed

    Guo, Jun; Lam, Lloyd T; Longenecker, Kenton L; Bui, Mai H; Idler, Kenneth B; Glaser, Keith B; Wilsbacher, Julie L; Tse, Chris; Pappano, William N; Huang, Tzu-Hsuan

    2017-09-23

    Cancer cells have an unusually high requirement for the central and intermediary metabolite nicotinamide adenine dinucleotide (NAD + ), and NAD + depletion ultimately results in cell death. The rate limiting step within the NAD + salvage pathway required for converting nicotinamide to NAD + is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Targeting NAMPT has been investigated as an anti-cancer strategy, and several highly selective small molecule inhibitors have been found to potently inhibit NAMPT in cancer cells, resulting in NAD + depletion and cytotoxicity. To identify mechanisms that could cause resistance to NAMPT inhibitor treatment, we generated a human fibrosarcoma cell line refractory to the highly potent and selective NAMPT small molecule inhibitor, GMX1778. We uncovered novel and unexpected mechanisms of resistance including significantly increased expression of quinolinate phosphoribosyl transferase (QPRT), a key enzyme in the de novo NAD + synthesis pathway. Additionally, exome sequencing of the NAMPT gene in the resistant cells identified a single heterozygous point mutation that was not present in the parental cell line. The combination of upregulation of the NAD + de novo synthesis pathway through QPRT over-expression and NAMPT mutation confers resistance to GMX1778, but the cells are only partially resistant to next-generation NAMPT inhibitors. The resistance mechanisms uncovered herein provide a potential avenue to continue exploration of next generation NAMPT inhibitors to treat neoplasms in the clinic. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem-mass spectrometry.

    PubMed

    Yaku, Keisuke; Okabe, Keisuke; Nakagawa, Takashi

    2018-06-01

    Nicotinamide adenine dinucleotide (NAD) is a major co-factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging-related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS-based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism. Copyright © 2018 John Wiley & Sons, Ltd.

  12. Radioresistance of Adenine to Cosmic Rays

    NASA Astrophysics Data System (ADS)

    Vignoli Muniz, Gabriel S.; Mejía, Christian F.; Martinez, Rafael; Auge, Basile; Rothard, Hermann; Domaracka, Alicja; Boduch, Philippe

    2017-04-01

    The presence of nucleobases in carbonaceous meteorites on Earth is an indication of the existence of this class of molecules in outer space. However, space is permeated by ionizing radiation, which can have damaging effects on these molecules. Adenine is a purine nucleobase that amalgamates important biomolecules such as DNA, RNA, and ATP. Adenine has a unique importance in biochemistry and therefore life. The aim of this work was to study the effects of cosmic ray analogues on solid adenine and estimate its survival when exposed to corpuscular radiation. Adenine films were irradiated at GANIL (Caen, France) and GSI (Darmstadt, Germany) by 820 MeV Kr33+, 190 MeV Ca10+, 92 MeV Xe23+, and 12 MeV C4+ ion beams at low temperature. The evolution of adenine molecules under heavy ion irradiation was studied by IR absorption spectroscopy as a function of projectile fluence. It was found that the adenine destruction cross section (σd) follows an electronic stopping power (Se) power law under the form: CSen; C is a constant, and the exponential n is a dimensionless quantity. Using the equation above to fit our results, we determined σd = 4 × 10-17 Se1.17, with Se in kiloelectronvolts per micrometer (keV μm-1). New IR absorption bands arise under irradiation of adenine and can be attributed to HCN, CN-, C2H4N4, CH3CN, and (CH3)3CNC. These findings may help to understand the stability and chemistry related to complex organic molecules in space. The half-life of solid adenine exposed to the simulated interstellar medium cosmic ray flux was estimated as (10 ± 8) × 106 years.

  13. Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes through NADPH oxidase -dependent activation of NF-κB.

    PubMed

    Zheng, Shuai; Zhong, Zhao-Ming; Qin, Shuai; Chen, Guo-Xian; Wu, Qian; Zeng, Ji-Huan; Ye, Wen-Bin; Li, Wei; Yuan, Kai; Yao, Ling; Chen, Jian-Ting

    2013-01-01

    Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro. FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin(IL)-1β, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB). Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47(phox), triggered an interaction between p47(phox), p22(phox) and gp91(phox), and significantly upregulated expression of NADPH oxidase subunits p47(phox), p22(phox) and gp91(phox). IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin. These data indicate that

  14. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Seongmin; Verdine, Gregory L.; Harvard)

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases havemore » been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.« less

  15. NMR-based metabonomic analysis of MnO-embedded iron oxide nanoparticles as potential dual-modal contrast agents

    NASA Astrophysics Data System (ADS)

    Li, Jinquan; Zhou, Zijian; Feng, Jianghua; Cai, Shuhui; Gao, Jinhao; Chen, Zhong

    2014-05-01

    MnO-embedded iron oxide nanoparticles (MnIO-NPs) can be treated as potential dual-modal contrast agents. However, their overall bio-effects and potential toxicity remain unknown. In this study, the metabolic effects of MnIO-NPs (dosed at 1 and 5 mg Fe/kg) on Sprague-Dawley rats were investigated using metabonomic analysis, histopathological examination, and conventional biochemical analysis. The histological changes included a focal inflammation in the liver at high-dose and a slightly enlarged area of splenic white pulp after 48 h post-dose. Blood biochemical analysis showed that albumin, globulins, aspartate aminotransferase, lactate dehydrogenase, blood urea nitrogen, and glucose changed distinctly compared to the control. The metabonomic analysis of body fluids (serum and urine) and tissues (liver, kidney, and spleen) indicated that MnIO-NPs induced metabolic perturbation in rats including energy, nucleotides, amino acids and phospholipid metabolisms. Besides, the variations of supportive nutrients: valine, leucine, isoleucine, nicotinamide adenine dinucleotide (phosphate), and nicotinamide, and the conjugation substrates: glycine, taurine, glutamine, glutathione, and methyl donors (formate, sarcosine, dimethylglycine, choline, and betaine) were involved in detoxification reaction of MnIO-NPs. The obtained information would provide identifiable ground for the candidate selection and optimization.

  16. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

    PubMed Central

    Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F.

    2015-01-01

    The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases. PMID:25678560

  17. Evaluation of nicotinamide microemulsion on the skin penetration enhancement.

    PubMed

    Boonme, Prapaporn; Boonthongchuay, Chalida; Wongpoowarak, Wibul; Amnuaikit, Thanaporn

    2016-01-01

    This study purposed to evaluate a microemulsion containing nicotinamide for its characteristics, stability, and skin penetration and retention comparing with a solution of nicotinamide in 2:1 mixture of water and isopropyl alcohol (IPA). The microemulsion system was composed of 1:1 mixture of Span80 and Tween80 as a surfactant mixture, isopropyl palmitate (IPP) as an oil phase, and 2:1 mixture of water and IPA as an aqueous phase. Nicotinamide microemulsion was prepared by dissolving the active in the aqueous phase before simply mixing with the other components. It was determined for its characteristics and stability under various conditions. The skin penetration and retention studies of nicotinamide microemulsion and solution were performed by modified Franz diffusion cells, using newborn pig skin as the membrane. The results showed that nicotinamide microemulsion could be obtained as clear yellowish liquid, was water-in-oil (w/o) type, possessed Newtonian flow, and exhibited physicochemical stability when kept at 4 °C and room temperature (≈30 ± 2 °C) during 3 months. From the skin penetration data, the microemulsion could enhance the skin penetration of nicotinamide comparing with the solution. Additionally, nicotinamide microemulsion could provide much higher amount of skin retention than that of skin penetration, resulting in suitability for a cosmeceutical product.

  18. Nicotinamide Inhibits Vasculogenic Mimicry, an Alternative Vascularization Pathway Observed in Highly Aggressive Melanoma

    PubMed Central

    Shalmon, Bruria; Kubi, Adva; Treves, Avraham J.; Shapira-Frommer, Ronnie; Avivi, Camilla; Ortenberg, Rona; Ben-Ami, Eytan; Schachter, Jacob; Besser, Michal J.; Markel, Gal

    2013-01-01

    Vasculogenic mimicry (VM) describes functional vascular channels composed only of tumor cells and its presence predicts poor prognosis in melanoma patients. Inhibition of this alternative vascularization pathway might be of clinical importance, especially as several anti-angiogenic therapies targeting endothelial cells are largely ineffective in melanoma. We show the presence of VM structures histologically in a series of human melanoma lesions and demonstrate that cell cultures derived from these lesions form tubes in 3D cultures ex vivo. We tested the ability of nicotinamide, the amide form of vitamin B3 (niacin), which acts as an epigenetic gene regulator through unique cellular pathways, to modify VM. Nicotinamide effectively inhibited the formation of VM structures and destroyed already formed ones, in a dose-dependent manner. Remarkably, VM formation capacity remained suppressed even one month after the complete withdrawal of Nicotimamid. The inhibitory effect of nicotinamide on VM formation could be at least partially explained by a nicotinamide-driven downregulation of vascular endothelial cadherin (VE-Cadherin), which is known to have a central role in VM. Further major changes in the expression profile of hundreds of genes, most of them clustered in biologically-relevant clusters, were observed. In addition, nicotinamide significantly inhibited melanoma cell proliferation, but had an opposite effect on their invasion capacity. Cell cycle analysis indicated moderate changes in apoptotic indices. Therefore, nicotinamide could be further used to unravel new biological mechanisms that drive VM and tumor progression. Targeting VM, especially in combination with anti-angiogenic strategies, is expected to be synergistic and might yield substantial anti neoplastic effects in a variety of malignancies. PMID:23451174

  19. CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

    PubMed

    Wrona, Dominik; Siler, Ulrich; Reichenbach, Janine

    2017-03-13

    Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47 phox -deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47 phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47 phox -subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47 phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47 phox -deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

  20. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  1. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  2. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  3. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  4. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  5. Excess nicotinamide inhibits methylation-mediated degradation of catecholamines in normotensives and hypertensives.

    PubMed

    Sun, Wu-Ping; Li, Da; Lun, Yong-Zhi; Gong, Xiao-Jie; Sun, Shen-Xia; Guo, Ming; Jing, Li-Xin; Zhang, Li-Bin; Xiao, Fu-Cheng; Zhou, Shi-Sheng

    2012-02-01

    Nicotinamide and catecholamines are both degraded by S-adenosylmethionine-dependent methylation. Whether excess nicotinamide affects the degradation of catecholamines is unknown. The aim of this study was to investigate the effect of nicotinamide on the methylation status of the body and methylation-mediated catecholamine degradation in both normotensives and hypertensives. The study was conducted in 19 normotensives and 27 hypertensives, using a nicotinamide-loading test (100 mg orally). Plasma nicotinamide, N(1)-methylnicotinamide, homocysteine (Hcy), betaine, norepinephrine, epinephrine, normetanephrine and metanephrine levels before and 5 h after nicotinamide loading were measured. Compared with normotensives, hypertensives had higher baseline (fasting) levels of plasma nicotinamide, Hcy and norepinephrine, but lower levels of plasma normetanephrine, a methylated norepinephrine derivative. Nicotinamide loading induced a significant increase in the levels of plasma N(1)-methylnicotinamide and norepinephrine, and a significant decrease in the levels of O-methylated epinephrine (metanephrine) and betaine, a major methyl donor, in both hypertensives and normotensives. Moreover, nicotinamide-loading significantly increased plasma Hcy levels, but decreased plasma normetanephrine levels in normotensives. The baseline levels of plasma epinephrine in hypertensives were similar to those of normotensives, but the post-nicotinamide-loading levels of plasma epinephrine in hypertensives were higher than those of normotensives. This study demonstrated that excess nicotinamide might deplete the labile methyl pool, increase Hcy generation and inhibit catecholamine degradation. It also revealed that hypertensives had an abnormal methylation pattern, characterized by elevated fasting plasma levels of unmethylated substrates, nicotinamide, Hcy and norepinephrine. Therefore, it seems likely that high nicotinamide intake may be involved in the pathogenesis of Hcy

  6. Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fraser, Marie E., E-mail: frasm@ucalgary.ca; Cherney, Maia M.; Marcato, Paola

    2006-07-01

    Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active asmore » an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.« less

  7. NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation

    PubMed Central

    Ryu, Dongryeol; Zhang, Hongbo; Ropelle, Eduardo R.; Sorrentino, Vincenzo; Mázala, Davi A. G.; Mouchiroud, Laurent; Marshall, Philip L.; Campbell, Matthew D.; Ali, Amir Safi; Knowels, Gary M.; Bellemin, Stéphanie; Iyer, Shama R.; Wang, Xu; Gariani, Karim; Sauve, Anthony A.; Cantó, Carles; Conley, Kevin E.; Walter, Ludivine; Lovering, Richard M.; Chin, Eva R.; Jasmin, Bernard J.; Marcinek, David J.; Menzies, Keir J.; Auwerx, Johan

    2017-01-01

    Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene’s muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5’-diphosphate (ADP)–ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr−/− mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. PMID:27798264

  8. NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

    PubMed

    Ryu, Dongryeol; Zhang, Hongbo; Ropelle, Eduardo R; Sorrentino, Vincenzo; Mázala, Davi A G; Mouchiroud, Laurent; Marshall, Philip L; Campbell, Matthew D; Ali, Amir Safi; Knowels, Gary M; Bellemin, Stéphanie; Iyer, Shama R; Wang, Xu; Gariani, Karim; Sauve, Anthony A; Cantó, Carles; Conley, Kevin E; Walter, Ludivine; Lovering, Richard M; Chin, Eva R; Jasmin, Bernard J; Marcinek, David J; Menzies, Keir J; Auwerx, Johan

    2016-10-19

    Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD + ) synthesis, consistent with a potential role for the essential cofactor NAD + in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD + and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD + levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD + biosynthesis. Replenishing NAD + stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr -/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD + repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD + may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures. Copyright © 2016, American Association for the Advancement of Science.

  9. Optical imaging of metabolic adaptability in metastatic and non-metastatic breast cancer

    NASA Astrophysics Data System (ADS)

    Rebello, Lisa; Rajaram, Narasimhan

    2018-02-01

    Accurate methods for determining metastatic risk from the primary tumor are crucial for patient survival. Cell metabolism could potentially be used as a marker of metastatic risk. Optical imaging of the endogenous fluorescent molecules nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) provides a non-destructive and label-free method for determining cell metabolism. The optical redox ratio (FAD/FAD+NADH) is sensitive to the balance between glycolysis and oxidative phosphorylation (OXPHOS). We have previously established that hypoxia-reoxygenation stress leads to metastatic potential-dependent changes in optical redox ratio. The objective of this study was to monitor the changes in optical redox ratio in breast cancer cells in response to different periods of hypoxic stress as well various levels of hypoxia to establish an optimal protocol. We measured the optical redox ratio of highly metastatic 4T1 murine breast cancer cells under normoxic conditions and after exposure to 30, 60, and 120 minutes of 0.5% O2. This was followed by an hour of reoxygenation. We found an increase in the optical redox ratio following reoxygenation from hypoxia for all durations. Statistically significant differences were observed at 60 and 120 minutes (p˂0.01) compared with normoxia, implying an ability to adapt to OXPHOS after reoxygenation. The switch to OXPHOS has been shown to be a key promoter of cell invasion. We will present our results from these investigations in human breast cancer cells as well as non-metastatic breast cancer cells exposed to various levels of hypoxia.

  10. Intrinsic fluorescence biomarkers in cells treated with chemopreventive drugs

    NASA Astrophysics Data System (ADS)

    Kirkpatrick, Nathaniel D.; Brands, William R.; Zou, Changping; Brewer, Molly A.; Utzinger, Urs

    2005-03-01

    Non-invasive monitoring of cellular metabolism offers promising insights into areas ranging from biomarkers for drug activity to cancer diagnosis. Fluorescence spectroscopy can be utilized in order to exploit endogenous fluorophores, typically metabolic co-factors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), and estimate the redox status of the sample. Fluorescence spectroscopy was applied to follow metabolic changes in epithelial ovarian cells as well as bladder epithelial cancer cells during treatment with a chemopreventive drug that initiates cellular quiescence. Fluorescence signals consistent with NADH, FAD, and tryptophan were measured to monitor cellular activity, redox status, and protein content. Cells were treated with varying concentrations of N-4-(hydroxyphenyl) retinamide (4-HPR) and measured in a stable environment with a sensitive fluorescence spectrometer. A subset of measurements was completed on a low concentration of cells to demonstrate feasibility for medical application such as in bladder or ovary washes. Results suggest that all of the cells responded with similar dose dependence but started at different estimated redox ratio baseline levels correlating with cell cycle, growth inhibition, and apoptosis assays. NADH and tryptophan related fluorescence changed significantly while FAD related fluorescence remained unaltered. Fluorescence data collected from approximately 1000 - 2000 cells, comparable to a bladder or ovary wash, was measurable and useful for future experiments. This study suggests that future intrinsic biomarker measurements may need to be most sensitive to changes in NADH and tryptophan related fluorescence while using FAD related fluorescence to help estimate the baseline redox ratio and predict response to chemopreventive agents.

  11. Optical diagnosis of the progression and reversal of CCl4-induced liver injury in rodent model using minimally invasive autofluorescence spectroscopy.

    PubMed

    Nazeer, Shaiju S; Sandhyamani, S; Jayasree, Ramapurath S

    2015-06-07

    Worldwide, liver cancer is the fifth most common cancer in men and seventh most common cancer in women. Intoxicant-induced liver injury is one of the major causes for severe structural damage with fibrosis and functional derangement of the liver leading to cancer in its later stages. This report focuses on the minimally invasive autofluorescence spectroscopic (AFS) studies on intoxicant, carbon tetrachloride (CCl4)-induced liver damage in a rodent model. Different stages of liver damage, including the reversed stage, on stoppage of the intoxicant are examined. Emission from prominent fluorophores, such as collagen, nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD), and variations in redox ratio have been studied. A direct correlation between the severity of the disease and the levels of collagen and redox ratio was observed. On withdrawal of the intoxicant, a gradual reversal of the disease to normal conditions was observed as indicated by the decrease in collagen levels and redox ratio. Multivariate statistical techniques and principal component analysis followed by linear discriminant analysis (PC-LDA) were used to develop diagnostic algorithms for distinguishing different stages of the liver disease based on spectral features. The PC-LDA modeling on a minimally invasive AFS dataset yielded diagnostic sensitivities of 93%, 87% and 87% and specificities of 90%, 98% and 98% for pairwise classification among normal, fibrosis, cirrhosis and reversal conditions. We conclude that AFS along with PC-LDA algorithm has the potential for rapid and accurate minimally invasive diagnosis and detection of structural changes due to liver injury resulting from various intoxicants.

  12. Physical limits to autofluorescence signals in vivo recordings in the rat olfactory bulb: a Monte Carlo study

    NASA Astrophysics Data System (ADS)

    L'Heureux, B.; Gurden, H.; Pinot, L.; Mastrippolito, R.; Lefebvre, F.; Lanièce, P.; Pain, F.

    2007-07-01

    Understanding the cellular mechanisms of energy supply to neurons following physiological activation is still challenging and has strong implications to the interpretation of clinical functional images based on metabolic signals such as Blood Oxygen Level Dependent Magnetic Resonance Imaging or 18F-Fluorodexoy-Glucose Positron Emission Tomography. Intrinsic Optical Signal Imaging provides with high spatio temporal resolution in vivo imaging in the anaesthetized rat. In that context, intrinsic signals are mainly related to changes in the optical absorption of haemoglobin depending on its oxygenation state. This technique has been validated for imaging of the rat olfactory bulb, providing with maps of the actived olfactory glomeruli, the functional modules involved in the first step of olfactory coding. A complementary approach would be autofluorescence imaging relying on the fluorescence properties of endogenous Flavin Adenine Dinucleotide (FAD) or Nicotinamide Adenine Dinucleotide (NADH) both involved in intracellular metabolic pathways. The purpose of the present study was to investigate the feasibility of in vivo autofluorescence imaging in the rat olfactory bulb. We performed standard Monte Carlo simulations of photons scattering and absorption at the excitation and emission wavelengths of FAD and NADH fluorescence. Characterization of the fluorescence distribution in the glomerulus, effect of hemoglobin absorption at the excitation and absorption wavelengths as well as the effect of the blurring due to photon scattering and the depth of focus of the optical apparatus have been studied. Finally, optimal experimental parameters are proposed to achieve in vivo validation of the technique in the rat olfactory bulb.

  13. Compact point-detection fluorescence spectroscopy system for quantifying intrinsic fluorescence redox ratio in brain cancer diagnostics

    NASA Astrophysics Data System (ADS)

    Liu, Quan; Grant, Gerald; Li, Jianjun; Zhang, Yan; Hu, Fangyao; Li, Shuqin; Wilson, Christy; Chen, Kui; Bigner, Darell; Vo-Dinh, Tuan

    2011-03-01

    We report the development of a compact point-detection fluorescence spectroscopy system and two data analysis methods to quantify the intrinsic fluorescence redox ratio and diagnose brain cancer in an orthotopic brain tumor rat model. Our system employs one compact cw diode laser (407 nm) to excite two primary endogenous fluorophores, reduced nicotinamide adenine dinucleotide, and flavin adenine dinucleotide. The spectra were first analyzed using a spectral filtering modulation method developed previously to derive the intrinsic fluorescence redox ratio, which has the advantages of insensitivty to optical coupling and rapid data acquisition and analysis. This method represents a convenient and rapid alternative for achieving intrinsic fluorescence-based redox measurements as compared to those complicated model-based methods. It is worth noting that the method can also extract total hemoglobin concentration at the same time but only if the emission path length of fluorescence light, which depends on the illumination and collection geometry of the optical probe, is long enough so that the effect of absorption on fluorescence intensity due to hemoglobin is significant. Then a multivariate method was used to statistically classify normal tissues and tumors. Although the first method offers quantitative tissue metabolism information, the second method provides high overall classification accuracy. The two methods provide complementary capabilities for understanding cancer development and noninvasively diagnosing brain cancer. The results of our study suggest that this portable system can be potentially used to demarcate the elusive boundary between a brain tumor and the surrounding normal tissue during surgical resection.

  14. Interrogation of metabolic and oxygen states of tumors with fiber-based luminescence lifetime spectroscopy.

    PubMed

    Lukina, Maria; Orlova, Anna; Shirmanova, Marina; Shirokov, Daniil; Pavlikov, Anton; Neubauer, Antje; Studier, Hauke; Becker, Wolfgang; Zagaynova, Elena; Yoshihara, Toshitada; Tobita, Seiji; Shcheslavskiy, Vladislav

    2017-02-15

    The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for tumor development and provides background for the relevant tumor's treatment. Here, we show that a specially designed implantable fiber-optic probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of a nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on an iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions and in living mice.

  15. A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity.

    PubMed

    Liao, Ya-Fan; Hsieh, Hui-Chieh; Liu, Guang-Yaw; Hung, Hui-Chih

    2005-12-15

    A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.

  16. In situ chemichromic studies of interactions between a lutetium bis-octaalkyl-substituted phthalocyanine and selected biological cofactors

    PubMed Central

    Pal, C.; Cammidge, A. N.; Cook, M. J.; Sosa-Sanchez, J. L.; Sharma, A. K.; Ray, A. K.

    2012-01-01

    Spin-coated films, approximately 100 nm thick, of a newly synthesized bis[octakis(octyl)phthalocyaninato] lutetium(III) complex on ultrasonically cleaned glass substrates exhibit pronounced chemichromic behaviour with potential application in healthcare. In situ kinetic optical absorption spectroscopic measurements show that the phthalocyanine Q-band is red shifted by 60 nm upon oxidation arising from exposure to bromine vapour. Recovery to the original state is achieved by the treatment of the oxidized films with nicotinamide adenine dinucleotide and l-ascorbic acid (vitamin C) in an aqueous solution containing 1.5 M lithium perchlorate. The neutralization process is found to be governed by first-order kinetics. The linear increase of the reduction rate with increasing concentration of cofactors provides a basis for calibration of analyte concentrations ranging from 3.5 mM down to 0.03 mM. PMID:21676969

  17. Occurrence and Distribution of Proteinase of Streptococcus faecalis var. liquefaciens1

    PubMed Central

    Shugart, Lee R.; Beck, Raymond W.

    1966-01-01

    Shugart, Lee R. (University of Tennessee, Knoxville), and Raymond W. Beck. Occurrence and distribution of proteinase of Streptococcus faecalis var. liquefaciens. J. Bacteriol. 92:338–341. 1966.—The proteolytic enzyme produced by Streptococcus faecalis var. liquefaciens (ATCC 13398) was shown to be an exoenzyme. The production of the proteinase was followed in growing cultures, and its distribution was compared with that of the intracellular enzymes reduced nicotinamide adenine dinucleotide (NADH2) peroxidase and lactate dehydrogenase. The proteinase appeared in the culture medium prior to the stationary phase of growth, whereas the other enzymes could be found only in whole cells. Fractionation of whole cells by sonic treatment and by treatment with lysozyme showed the proteinase to be associated primarily with the cell wall and cell membrane, and NADH2 peroxidase to be associated only with the cytoplasmic fractions. PMID:16562116

  18. Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity.

    PubMed

    Liu, George Y; Essex, Anthony; Buchanan, John T; Datta, Vivekanand; Hoffman, Hal M; Bastian, John F; Fierer, Joshua; Nizet, Victor

    2005-07-18

    Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. Here we demonstrate a role of this hallmark phenotype in virulence. Compared with the wild-type (WT) bacterium, a S. aureus mutant with disrupted carotenoid biosynthesis is more susceptible to oxidant killing, has impaired neutrophil survival, and is less pathogenic in a mouse subcutaneous abscess model. The survival advantage of WT S. aureus over the carotenoid-deficient mutant is lost upon inhibition of neutrophil oxidative burst or in human or murine nicotinamide adenine dinucleotide phosphate oxidase-deficient hosts. Conversely, heterologous expression of the S. aureus carotenoid in the nonpigmented Streptococcus pyogenes confers enhanced oxidant and neutrophil resistance and increased animal virulence. Blocking S. aureus carotenogenesis increases oxidant sensitivity and decreases whole-blood survival, suggesting a novel target for antibiotic therapy.

  19. Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD⁺ on NiO Film Modified by GO and MBs.

    PubMed

    Chou, Jung-Chuan; Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

    2017-07-12

    We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide ( NAD + ) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH- NAD + -MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD⁺/GPTS/GO/NiO film and LDH- NAD + /GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated.

  20. Fabrication of Flexible Arrayed Lactate Biosensor Based on Immobilizing LDH-NAD+ on NiO Film Modified by GO and MBs

    PubMed Central

    Yan, Siao-Jie; Liao, Yi-Hung; Lai, Chih-Hsien; Wu, You-Xiang; Wu, Cian-Yi; Chen, Hsiang-Yi; Huang, Hong-Yu; Wu, Tong-Yu

    2017-01-01

    We proposed the flexible arrayed lactate biosensor based on immobilizing l-lactate dehydrogenase (LDH) and nicotinamide adenine dinucleotide (NAD+) on nickel oxide (NiO) film, and which the average sensitivity could be enhanced by using graphene oxide (GO) and magnetic beads (MBs). By using GO and MBs, it exhibits excellent sensitivity (45.397 mV/mM) with a linearity of 0.992 in a range of 0.2 mM to 3 mM. According to the results of electrochemical impedance spectroscopy (EIS), the electron transfer resistance of LDH-NAD+-MBs/GPTS/GO/NiO film was smaller than those of LDH-NAD+/GPTS/GO/NiO film and LDH-NAD+/GPTS/NiO film, and it presented the outstanding electron transfer ability. After that, the limit of detection, anti-interference effect and bending test were also investigated. PMID:28704960

  1. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

    PubMed

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  2. Biosensor reveals multiple sources for mitochondrial NAD⁺.

    PubMed

    Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

    2016-06-17

    Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.

  3. Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding.

    PubMed

    Salmas, Ramin Ekhteiari; Mestanoglu, Mert; Unlu, Ayhan; Yurtsever, Mine; Durdagi, Serdar

    2016-11-01

    Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.

  4. Characterization of 4 New Mutations in the CYBB Gene in 10 Iranian Families With X-linked Chronic Granulomatous Disease.

    PubMed

    Teimourian, Shahram; Sazgara, Faezeh; de Boer, Martin; van Leeuwen, Karin; Roos, Dirk; Lashkary, Sharzad; Chavoshzadeh, Zahra; Nabavi, Mohammad; Bemanian, Mohammad Hassan; Isaian, Anna

    2018-04-26

    Chronic granulomatous disease (CGD) is an inherited disease of the innate immune system that results from defects in 1 of the 5 subunits of nicotinamide adenine dinucleotide phosphate oxidase complex and leads to life-threatening infections with granuloma formation. During 3 years of study, we recognized 10 male patients with X-linked CGD from a tertiary referral center for immune deficiencies in Iran. The CGD patients were diagnosed according to clinical features and biochemical tests, including nitroblue tetrazolium and dihydrorhodamine-1, 2, 3 tests, performed on patients and their mothers. In all patients, Western blot analysis showed a gp91 phenotype. Mutation screening by single strand conformation polymorphism and multiplex ligation-dependent probe amplification analysis of the CYBB gene encoding gp91, followed by sequencing, showed 9 different mutations, 4 of them novel as far as we know.

  5. Tricarboxylic acid cycle without malate dehydrogenase in Streptomyces coelicolor M-145.

    PubMed

    Takahashi-Íñiguez, Tóshiko; Barrios-Hernández, Joana; Rodríguez-Maldonado, Marion; Flores, María Elena

    2018-06-23

    The oxidation of malate to oxaloacetate is catalysed only by a nicotinamide adenine dinucleotide-dependent malate dehydrogenase encoded by SCO4827 in Streptomyces coelicolor. A mutant lacking the malate dehydrogenase gene was isolated and no enzymatic activity was detected. As expected, the ∆mdh mutant was unable to grow on malate as the sole carbon source. However, the mutant grew less in minimal medium with glucose and there was a delay of 36 h. The same behaviour was observed when the mutant was grown on minimal medium with casamino acids or glycerol. For unknown reasons, the mutant was not able to grow in YEME medium with glucose. The deficiency of malate dehydrogenase affected the expression of the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes, decreasing the expression of both genes by approximately two- to threefold.

  6. Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

    NASA Technical Reports Server (NTRS)

    Sleeper, H. L.; Lohrmann, R.; Orgel, L. E.

    1978-01-01

    Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as p(n)ApA in excellent yield (greater than or equal to 80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5'-phosphorimidazolides.

  7. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kolodkin-Gal, I; Elsholz, AKW; Muth, C

    2013-04-29

    Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showedmore » that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.« less

  8. Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

    PubMed Central

    Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

    2013-01-01

    Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

  9. NAMPT/PBEF1 enzymatic activity is indispensable for myeloma cell growth and osteoclast activity

    PubMed Central

    Venkateshaiah, Sathisha Upparahalli; Khan, Sharmin; Ling, Wen; Bam, Rakesh; Li, Xin; van Rhee, Frits; Usmani, Saad; Barlogie, Bart; Epstein, Joshua; Yaccoby, Shmuel

    2015-01-01

    Multiple myeloma (MM) cells typically grow in focal lesions, stimulating osteoclasts that destroy bone and support MM. Osteoclasts and MM cells are hypermetabolic. The coenzyme nicotinamide adenine dinucleotide (NAD+) is not only essential for cellular metabolism; it also affects activity of NAD-dependent enzymes, such as PARP-1 and SIRT-1. Nicotinamide phos-phoribosyltransferase (NAMPT/PBEF/visfatin, encoded by PBEF1) is a rate-limiting enzyme in NAD+ biosynthesis from nicotinamide. Coculture of primary MM cells with osteoclasts induced PBEF1 upregulation in both cell types. PBEF1 expression was higher in experimental myelomatous bones than in nonmyelomatous bone and higher in MM patients’ plasma cells than in healthy donors’ counterparts. APO866 is a specific PBEF1 inhibitor known to deplete cellular NAD+, APO866 at low nanomolar concentrations inhibited growth of primary MM cells or MM cell lines cultured alone or cocultured with osteoclasts and induced apoptosis in these cells. PBEF1 activity and NAD+ content were reduced in MM cells by APO866, resulting in lower activity of PARP-1 and SIRT-1. The inhibitory effect of APO866 on MM cell growth was abrogated by supplementation of extracellular NAD+ or NAM. APO866 inhibited NF-κB activity in osteoclast precursors and suppressed osteoclast formation and activity. PBEF1 knockdown similarly inhibited MM cell growth and osteoclast formation. In the SCID-rab model, APO866 inhibited growth of primary MM and H929 cells and prevented bone disease. These findings indicate that MM cells and osteoclasts are highly sensitive to NAD+ depletion and that PBEF1 inhibition represents a novel approach to target cellular metabolism and inhibit PARP-1 and bone disease in MM. PMID:23435312

  10. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex...: (a) The additive is the product of the controlled reaction between ascorbic acid and nicotinamide...

  11. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex...: (a) The additive is the product of the controlled reaction between ascorbic acid and nicotinamide...

  12. Recycling nicotinamide. The transition-state structure of human nicotinamide phosphoribosyltransferase

    PubMed Central

    Burgos, Emmanuel S.; Vetticatt, Mathew J.; Schramm, Vern L.

    2013-01-01

    Human nicotinamide phosphoribosyltransferase (NAMPT) replenishes the NAD pool and controls the activities of sirtuins (SIRT), mono- and poly-(ADP-ribose) polymerases (PARP) and NAD nucleosidase (CD38). The nature of the enzymatic transition-state (TS) is central to understanding the function of NAMPT. We determined the TS structure for pyrophosphorolysis of nicotinamide mononucleotide (NMN) by kinetic isotope effects (KIEs). With the natural substrates, NMN and pyrophosphate (PPi), the intrinsic KIEs of [1′-14C], [1-15N], [1′-3H] and [2′-3H] are 1.047, 1.029, 1.154 and 1.093, respectively. A unique quantum computational approach was used for TS analysis that included structural elements of the catalytic site. Without constraints (e.g. imposed torsion angles), the theoretical and experimental data are in good agreement. The quantum-mechanical calculations incorporated a crucial catalytic site residue (D313), two magnesium atoms and coordinated water molecules. The transition state model predicts primary 14C, α-secondary 3H, β-secondary 3H and primary 15N KIE close to the experimental values. The analysis reveals significant ribocation character at the TS. The attacking PPi nucleophile is weakly interacting (rC-O = 2.60 Å) and the N-ribosidic C1′-N bond is highly elongated at the TS (rC-N = 2.35 Å), consistent with an ANDN mechanism. Together with the crystal structure of the NMN•PPi•Mg2•enzyme complex, the reaction coordinate is defined. The enzyme holds the nucleophile and leaving group in relatively fixed positions to create a reaction coordinate with C1′-anomeric migration from nicotinamide to the PPi. The transition state is reached by a 0.85 Å migration of C1′. PMID:23373462

  13. 21 CFR 172.315 - Nicotinamide-ascorbic acid complex.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.315 Nicotinamide-ascorbic acid complex. Nicotinamide-ascorbic acid complex may be safely used in accordance with the following prescribed conditions: (a) The additive is the product of...

  14. Nicotinamide megadosing increases hepatic poly(ADP-ribose) levels in choline-deficient rats.

    PubMed

    ApSimon, M M; Rawling, J M; Kirkland, J B

    1995-07-01

    Previous work in our laboratory has shown that dietary megadoses of nicotinamide, used in the prevention of diabetes, cause increases in hepatic poly(ADP-ribose). Poly(ADP-ribose) is synthesized from NAD+ by a nuclear enzyme, poly(ADP-ribose)polymerase, which is activated by DNA strand breaks. The nicotinamide-induced increase in poly(ADP-ribose) could result from an increase in substrate, NAD+, or the induction of strand breaks in DNA. Strand breaks may result from the depletion of single carbon groups, through the excretion of methylated derivatives of nicotinamide. To differentiate between these mechanisms, a 3 x 3 factorial experiment was conducted in which rats were fed diets containing various supplements of choline bitartrate (0, 2, 20 g/kg diet) and nicotinamide (0, 1, 2 g/kg diet). At the conclusion of treatments, blood NAD+ and liver lipid, NAD+ and poly(ADP-ribose) levels were determined. Choline deficiency caused the characteristic accumulation of fat in the liver at all levels of nicotinamide. In choline deficient rats, nicotinamide supplements further increased liver lipid concentration. Blood and liver NAD+ concentrations were increased by nicotinamide supplementation, irrespective of choline status. In contrast, liver poly(ADP-ribose) levels were increased by nicotinamide supplementation only in choline deficient rats. These results show that nicotinamide-induced increases in poly(ADP-ribose) levels appear to be dependent on decreased methyl donor status and suggest that adequate choline status is important for preventing some deleterious effects of nicotinamide treatment.

  15. Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH oxidase/ROS/Akt/NF‑κB pathway under high glucose conditions.

    PubMed

    Sun, Li; Li, Weiping; Li, Weizu; Xiong, Li; Li, Guiping; Ma, Rong

    2014-07-01

    Glomerular hypertrophy and hyperfiltration are the two major pathological characteristics of the early stages of diabetic nephropathy (DN), which are respectively related to mesangial cell (MC) proliferation and a decrease in calcium influx conducted by canonical transient receptor potential cation channel 6 (TRPC6). The marked increase in the production of reactive oxygen species (ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of ROS production in MCs. Astragaloside IV (AS‑IV) is an active ingredient of Radix Astragali which has a potent antioxidative effect. In this study, we aimed to investigate whether high glucose (HG)‑induced NADPH oxidase activation and ROS production contribute to MC proliferation and the downregulation of TRPC6 expression; we also wished to determine the effects of AS‑IV on MCs under HG conditions. Using a human glomerular mesangial cell line, we found that treatment with AS‑IV for 48 h markedly attenuated HG‑induced proliferation and the hypertrophy of MCs in a dose‑dependent manner. The intracellular ROS level was also markedly reduced following treatment with AS‑IV. In addition, the enhanced activity of NADPH oxidase and the expression level of NADPH oxidase 4 (Nox4) protein were decreased. Treatment with AS‑IV also inhibited the phosphorylation level of Akt and IκBα in the MCs. In addition, TRPC6 protein expression and the intracellular free calcium concentration were also markedly reduced following treatment with AS‑IV under HG conditions. These results suggest that AS‑IV inhibits HG‑induced mesangial cell proliferation and glomerular contractile dysfunction through the NADPH oxidase/ROS/Akt/nuclear factor‑κB (NF‑κB) pathway, providing a new perspective for the clinical treatment of DN.

  16. Sequence variants in the bovine silent information regulator 6, their linkage and their associations with body measurements and carcass quality traits in Qinchuan cattle.

    PubMed

    Gui, Linsheng; Jiang, Bijie; Zhang, Yaran; Zan, Linsen

    2015-03-15

    Silent information regulator 6 (SIRT6) belongs to the family of class III nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays an essential role in DNA repair and metabolism. This study was conducted to detect potential polymorphisms of the bovine SIRT6 gene and explore their relationships with body measurement and carcass quality in Qinchuan cattle. Four sequence variants (SVs) were identified in intron 6, exon 7, exon 9, and 3' UTR, via sequencing technology conducted in 468 individual Qinchuan cattle. Eleven different haplotypes were identified, of which two major haplotypes had a frequency of 45.7% (-CACT-) and 14.8% (-CGTC-). Three SVs (SV2, SV3 and SV4) were significantly associated with some of the body measurements and carcass quality traits (P<0.05 or P<0.01), and the H2H7 (CC-GA-TT-TC) diplotype had better performance than other combinations. Our results suggest that some polymorphisms in SIRT6 are associated with production traits and may be used as candidates for marker-assisted selection (MAS) and management in beef cattle breeding programs. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. NADPH oxidase-mediated redox signaling promotes oxidative stress resistance and longevity through memo-1 in C. elegans

    PubMed Central

    Ewald, Collin Yvès; Hourihan, John M; Bland, Monet S; Obieglo, Carolin; Katic, Iskra; Moronetti Mazzeo, Lorenza E; Alcedo, Joy; Blackwell, T Keith; Hynes, Nancy E

    2017-01-01

    Transient increases in mitochondrially-derived reactive oxygen species (ROS) activate an adaptive stress response to promote longevity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce ROS locally in response to various stimuli, and thereby regulate many cellular processes, but their role in aging remains unexplored. Here, we identified the C. elegans orthologue of mammalian mediator of ErbB2-driven cell motility, MEMO-1, as a protein that inhibits BLI-3/NADPH oxidase. MEMO-1 is complexed with RHO-1/RhoA/GTPase and loss of memo-1 results in an enhanced interaction of RHO-1 with BLI-3/NADPH oxidase, thereby stimulating ROS production that signal via p38 MAP kinase to the transcription factor SKN-1/NRF1,2,3 to promote stress resistance and longevity. Either loss of memo-1 or increasing BLI-3/NADPH oxidase activity by overexpression is sufficient to increase lifespan. Together, these findings demonstrate that NADPH oxidase-induced redox signaling initiates a transcriptional response that protects the cell and organism, and can promote both stress resistance and longevity. DOI: http://dx.doi.org/10.7554/eLife.19493.001 PMID:28085666

  18. Development of quantum dots-based biosensor towards on-farm detection of subclinical ketosis.

    PubMed

    Weng, Xuan; Chen, Longyan; Neethirajan, Suresh; Duffield, Todd

    2015-10-15

    Early detection of dairy animal health issues allows the producer or veterinarian to intervene before the animals' production levels, or even survival, is threatened. An increased concentration of β-hydroxybutyrate (βHBA) is a key biomarker for diagnosis of subclinical ketosis (SCK), and provides information on the health stress in cows well before any external symptoms are observable. In this study, quantum dots (QDs) modified with cofactor nicotinamide adenine dinucleotide (NAD(+)) were prepared for the sensing event, by which the βHBA concentration in the cow's blood and milk samples was determined via fluorescence analysis of the functionalized QDs. The detection was performed on a custom designed microfluidic platform combining with a low cost and miniaturized optical sensor. The sensing mechanism was first validated by a microplate reader method and then applied to the microfluidic platform. Standard βHBA solution, βHBA in blood and milk samples from cows were successfully measured by this novel technology with a detection limit at a level of 35 µM. Side by side comparison of the developed microfluidic biosensor with a commercial kit presented its good performance. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Aluminum and its effect in the equilibrium between folded/unfolded conformation of NADH.

    PubMed

    Formoso, Elena; Mujika, Jon I; Grabowski, Slawomir J; Lopez, Xabier

    2015-11-01

    Nicotinamide adenine dinucleotide (NADH) is one of the most abundant cofactor employed by proteins and enzymes. The molecule is formed by two nucleotides that can lead to two main conformations: folded/closed and unfolded/open. Experimentally, it has been determined that the closed form is about 2 kcal/mol more stable than the open formed. Computationally, a correct description of the NADH unfolding process is challenging due to different reasons: 1) The unfolding process shows a very low energy difference between the two conformations 2) The molecule can form a high number of internal hydrogen bond interactions 3) Subtle effects such as dispersion may be important. In order to tackle all these effects, we have employed a number of different state of the art computational techniques, including: a) well-tempered metadynamics, b) geometry optimizations, and c) Quantum Theory of Atoms in Molecules (QTAIM) calculations, to investigate the conformational change of NADH in solution and interacting with aluminum. All the results indicate that aluminum indeed favors the closed conformation of NADH, due mainly to the formation of a more rigid structure through key hydrogen bond interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Dunnione ameliorates cisplatin ototoxicity through modulation of NAD(+) metabolism.

    PubMed

    Kim, Hyung-Jin; Pandit, Arpana; Oh, Gi-Su; Shen, AiHua; Lee, Su-Bin; Khadka, Dipendra; Lee, SeungHoon; Shim, Hyeok; Yang, Sei-Hoon; Cho, Eun-Young; Kwak, Tae Hwan; Choe, Seong-Kyu; Park, Raekil; So, Hong-Seob

    2016-03-01

    Ototoxicity is an important issue in patients receiving cisplatin chemotherapy. Numerous studies have demonstrated that cisplatin-induced ototoxicity is related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as an important regulator of energy metabolism and cellular homeostasis. Here, we demonstrate that the levels and activities of sirtuin-1 (SIRT1) are suppressed by the reduction of intracellular NAD(+) levels in cisplatin-mediated ototoxicity. We provide evidence that the decreases in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) polymerase-1 (PARP-1) activation and microRNA-34a levels through cisplatin-mediated p53 activation aggravate the associated ototoxicity. Furthermore, we show that the induction of cellular NAD(+) levels using dunnione, which targets intracellular NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET).

    PubMed

    Cazelles, R; Lalaoui, N; Hartmann, T; Leimkühler, S; Wollenberger, U; Antonietti, M; Cosnier, S

    2016-11-15

    Direct electron transfer (DET) to proteins is of considerable interest for the development of biosensors and bioelectrocatalysts. While protein structure is mainly used as a method of attaching the protein to the electrode surface, we employed bioinformatics analysis to predict the suitable orientation of the enzymes to promote DET. Structure similarity and secondary structure prediction were combined underlying localized amino-acids able to direct one of the enzyme's electron relays toward the electrode surface by creating a suitable bioelectrocatalytic nanostructure. The electro-polymerization of pyrene pyrrole onto a fluorine-doped tin oxide (FTO) electrode allowed the targeted orientation of the formate dehydrogenase enzyme from Rhodobacter capsulatus (RcFDH) by means of hydrophobic interactions. Its electron relays were directed to the FTO surface, thus promoting DET. The reduction of nicotinamide adenine dinucleotide (NAD(+)) generating a maximum current density of 1μAcm(-2) with 10mM NAD(+) leads to a turnover number of 0.09electron/s/molRcFDH. This work represents a practical approach to evaluate electrode surface modification strategies in order to create valuable bioelectrocatalysts. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Single-walled carbon nanotubes covalently functionalized with polytyrosine: A new material for the development of NADH-based biosensors.

    PubMed

    Eguílaz, Marcos; Gutierrez, Fabiana; González-Domínguez, Jose Miguel; Martínez, María T; Rivas, Gustavo

    2016-12-15

    We report for the first time the use of single-walled carbon nanotubes (SWCNT) covalently functionalized with polytyrosine (Polytyr) (SWCNT-Polytyr) as a new electrode material for the development of nicotinamide adenine dinucleotide (NADH)-based biosensors. The oxidation of glassy carbon electrodes (GCE) modified with SWCNT-Polytyr at potentials high enough to oxidize the tyrosine residues have allowed the electrooxidation of NADH at low potentials due to the catalytic activity of the quinones generated from the primary oxidation of tyrosine without any additional redox mediator. The amperometric detection of NADH at 0.200V showed a sensitivity of (217±3)µAmM(-1)cm(-2) and a detection limit of 7.9nM. The excellent electrocatalytic activity of SWCNT-Polytyr towards NADH oxidation has also made possible the development of a sensitive ethanol biosensor through the immobilization of alcohol dehydrogenase (ADH) via Nafion entrapment, with excellent analytical characteristics (sensitivity of (5.8±0.1)µAmM(-1)cm(-2), detection limit of 0.67µM) and very successful application for the quantification of ethanol in different commercial beverages. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Biofuel cell backpacked insect and its application to wireless sensing.

    PubMed

    Shoji, Kan; Akiyama, Yoshitake; Suzuki, Masato; Nakamura, Nobuhumi; Ohno, Hiroyuki; Morishima, Keisuke

    2016-04-15

    This study investigated an enzymatic biofuel cell (BFC) which can be backpacked by cockroaches. The BFC generates electric power from trehalose in insect hemolymph by the trehalase and glucose dehydrogenase (GDH) reaction systems which dehydrogenate β-glucose obtained by hydrolyzing trehalose. First, an insect-mountable BFC (imBFC) was designed and fabricated with a 3D printer. The electrochemical reaction of anode-modified poly-L-lysine, vitamin K3, diaphorase, nicotinamide adenine dinucleotide, GDH and poly(sodium 4-styrenesulfonate) in the imBFC was evaluated and an oxidation current of 1.18 mAcm(-2) (at +0.6 V vs. Ag|AgCl) was observed. Then, the performance of the imBFC was evaluated and a maximum power output of 333 μW (285 μW cm(-)(2)) (at 0.5 V) was obtained. Furthermore, driving of both an LED device and a wireless temperature and humidity sensor device were powered by the imBFC. These results indicate that the imBFC has sufficient potential as a battery for novel ubiquitous robots such as insect cyborgs. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  5. NADPH OXIDASE: STRUCTURE AND ACTIVATION MECHANISMS (REVIEW). NOTE I.

    PubMed

    Filip-Ciubotaru, Florina; Manciuc, Carmen; Stoleriu, Gabriela; Foia, Liliana

    2016-01-01

    NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase), with its generically termed NOX isoforms, is the major source of ROS (reactive oxigen species) in biological systems. ROS are small oxygen-derived molecules with an important role in various biological processes (physiological or pathological). If under physiological conditions some processes are beneficial and necessary for life, under pathophysiological conditions they are noxious, harmful. NADPH oxidases are present in phagocytes and in a wide variety of nonphagocytic cells. The enzyme generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling them to molecular oxygen to produce superoxide anion, a reactive free-radical. Structurally, NADPH oxidase is a multicomponent enzyme which includes two integral membrane proteins, glycoprotein gp9 1 Phox and adaptor protein p22(phox), which together form the heterodimeric flavocytochrome b558 that constitutes the core of the enzyme. During the resting state, the multidomain regulatory subunits p40P(phox), p47(phox), p67(Phox) are located in the cytosol organized as a complex. The activation of phagocytic NADPH oxidase occurs through a complex series of protein interactions.

  6. Structural Basis of Substrate Recognition in Human Nicotinamide N-Methyltransferase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Peng, Yi; Sartini, Davide; Pozzi, Valentina

    2012-05-02

    Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7 {angstrom} resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles inmore » nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k{sub cat}/K{sub m} decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K{sub m} for both NCA and AdoMet and modestly decreased k{sub cat}. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K{sub m}(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.« less

  7. Multiple Animal Studies for Medical Chemical Defense Program in Soldier/ Patient Decontamination and Drug Development on Task Order 84-6: Pyruvate Dehydrogenase System for Determining the Effectiveness of Arsenic Antidotes

    DTIC Science & Technology

    1988-03-11

    adenine dinucleotide FAD = flavin-adenine dinucleotide iipS2 = lipoic acid lip(SH)2 = dihydrolipoic acid CoA = coenzyme A. SHepatic PDH complex activity...tissues has yet to be fully characterized, but it probably involves arsenic binding to the lipoic acid and dithiol moieties of the complex (Fluharty...covalently bound lipoic acid substrate of dihydrolipoyl transacetylase is greater per mole of L and CVAA than for sodium arsenite. This is possible

  8. Immobilization of flavin adenine dinucleotide (FAD) onto carbon cloth and its application as working electrode in an electroenzymatic bioreactor.

    PubMed

    Jayabalan, R; Sathishkumar, M; Jeong, E S; Mun, S P; Yun, S E

    2012-11-01

    A high porosity carbon cloth with immobilized FAD was employed as working electrode in electrochemical NADH-regeneration procedure. Carbon cloth was oxidized with hot acids to create surface carboxyl group and then coupled by adenine amino group of FAD with carbodiimide in the presence of N-hydroxysulfosuccinimide. The bioelectrocatalytic NADH-regeneration was coupled to the conversion of achiral substrate pyruvate into chiral product l-lactate by l-lactate dehydrogenase (l-LDH) within the same reactor. The conversion was completed at 96h in bioreactor with FAD-modified carbon cloth, resulting in about 6mM of l-lactate from 10mM of pyruvate. While with bare carbon cloth, the yield at 120h was around 5mM. Immobilized FAD on the surface of carbon cloth electrode facilitated it to carry electrons from electrode to electron transfer enzymes; thereby NADH-regeneration was accelerated to drive the enzymatic reaction efficiently. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    PubMed

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

    2008-03-28

    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

  10. Ultra-performance liquid chromatography tandem mass-spectrometry (uplc-ms/ms) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

    USDA-ARS?s Scientific Manuscript database

    A novel, rapid and sensitive Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin a...

  11. Silver-induced reconstruction of an adeninate-based metal-organic framework for encapsulation of luminescent adenine-stabilized silver clusters.

    PubMed

    Jonckheere, Dries; Coutino-Gonzalez, Eduardo; Baekelant, Wouter; Bueken, Bart; Reinsch, Helge; Stassen, Ivo; Fenwick, Oliver; Richard, Fanny; Samorì, Paolo; Ameloot, Rob; Hofkens, Johan; Roeffaers, Maarten B J; De Vos, Dirk E

    2016-05-21

    Bright luminescent silver-adenine species were successfully stabilized in the pores of the MOF-69A (zinc biphenyldicarboxylate) metal-organic framework, starting from the intrinsically blue luminescent bio-MOF-1 (zinc adeninate 4,4'-biphenyldicarboxylate). Bio-MOF-1 is transformed to the MOF-69A framework by selectively leaching structural adenine linkers from the original framework using silver nitrate solutions in aqueous ethanol. Simultaneously, bright blue-green luminescent silver-adenine clusters are formed inside the pores of the recrystallized MOF-69A matrix in high local concentrations. The structural transition and concurrent changes in optical properties were characterized using a range of structural, physicochemical and spectroscopic techniques (steady-state and time-resolved luminescence, quantum yield determination, fluorescence microscopy). The presented results open new avenues for exploring the use of MOFs containing luminescent silver clusters for solid-state lighting and sensor applications.

  12. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

    PubMed

    Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

    2016-01-05

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. CpG Dinucleotide Frequencies Reveal the Role of Host Methylation Capabilities in Parvovirus Evolution

    PubMed Central

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James

    2013-01-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to “fractional” methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses. PMID:24109231

  14. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

    PubMed

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  15. Enzymological Basis for Growth Inhibition by l-Phenylalanine in the Cyanobacterium Synechocystis sp. 29108

    PubMed Central

    Hall, Geraldine C.; Jensen, Roy A.

    1980-01-01

    loss in allosteric sensitivity of 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase was accompanied by a threefold increase in specific activity. This could suggest that existence of a modest degree of repression control (autogenous) over 3-deoxy-d-arabinoheptulosonate synthase, although other explanations are possible. Specific activities of chorismate mutase, prephenate dehydratase, shikimate/nicotinamide adenine dinucleotide phosphate dehydrogenase, and arogenate/nicotinamide adenine dinucleotide phosphate dehydrogenase in mutant Phe r19 were identical with those of the wild type. PMID:6108316

  16. Inhibition of miR-25 aggravates diabetic peripheral neuropathy.

    PubMed

    Zhang, Yanzhuo; Song, Chunyu; Liu, Jing; Bi, Yonghong; Li, Hao

    2018-06-05

    The hyperglycemia-induced enhanced oxidative stress is a key factor of diabetic peripheral neuropathy implicated in the pathogenesis of diabetic neuropathy, and microRNA may be involved, playing promotion or protection roles. In this study, we aimed to investigate the function of miR-25 during the development of oxidative/nitrative stress and in subsequent neurological problems. We detected the oxidative stress effects and expression of miR-25 on sciatic nerves from db/db diabetic model mice and analyzed the expression of related genes by qPCR and Western blotting. Interestingly, we observed increased reactive oxygen species (ROS) and Nox4 expression in db/db mice accompanied with reduced miR-25. MiR-25 inhibitor treatment increased nicotinamide adenine dinucleotide phosphate activity in Schwann cells, whereas miR-25 precursor overexpression led to opposite results. MiR-25 precursor reduced the activation of protein kinase C and decreased Nox4 expression at both mRNA and protein levels. Advanced glycation endproducts (AGEs) and the receptor for advanced glycation endproducts (RAGE) were increased in the serum and in the peripheral nerves obtained from diabetic mice, and miR-25 inhibitor treatment in Schwann cells from wt mice led to the same effect. However, miR-25 precursor transfection reduced AGEs and RAGE, and further reduced inflammatory factors that contribute to the pathological process of peripheral nerves. These findings, for the first time, indicate that miR-25 acts as a protection factor in diabetic neuropathy by downregulating AGE-RAGE and reducing nicotinamide adenine dinucleotide phosphate oxidase. miR-25 reduced protein kinase C-α phosphorylation to produce less reactive oxygen species in diabetic peripheral nerves, and therefore it played an important role in the regulation of oxidative/nitrative stress and in consequent neurological dysfunction.This is an open-access article distributed under the terms of the Creative Commons Attribution

  17. Leg ischaemia before circulatory arrest alters brain leucocyte count and respiratory chain redox state.

    PubMed

    Yannopoulos, Fredrik S; Arvola, Oiva; Haapanen, Henri; Herajärvi, Johanna; Miinalainen, Ilkka; Jensen, Hanna; Kiviluoma, Kai; Juvonen, Tatu

    2014-03-01

    Remote ischaemic preconditioning and its neuroprotective abilities are currently under investigation and the method has shown significant effects in several small and large animal studies. In our previous studies, leucocyte filtration during cardiopulmonary bypass reduced cerebrocortical adherent leucocyte count and mitigated cerebral damage after hypothermic circulatory arrest (HCA) in piglets. This study aimed to obtain and assess direct visual data of leucocyte behaviour in cerebral vessels after hypothermic circulatory arrest following remote ischaemic preconditioning. Twelve native stock piglets were randomized into a remote ischaemic preconditioning group (n = 6) and a control group (n = 6). The intervention group underwent hind-leg ischaemia, whereas the control group received a sham-treatment before a 60-min period of hypothermic circulatory arrest. An intravital microscope was used to obtain measurements from the cerebrocortical vessel in vivo. It included three sets of filters: a violet filter to visualize microvascular perfusion and vessel diameter, a green filter for visualization of rhodamine-labelled leucocytes and an ultraviolet filter for reduced nicotinamide adenine dinucleotide (NADH) analysis. The final magnification on the microscope was 400. After the experiment, cerebral and cerebellar biopsies were collected and analysed with transmission electron microscope by a blinded analyst. In the transmission electron microscope analysis, the entire intervention group had normal, unaffected rough endoplasmic reticulum's in their cerebellar tissue, whereas the control group had a mean score of 1.06 (standard deviation 0.41) (P = 0.026). The measured amount of adherent leucocytes was lower in the remote ischaemic preconditioning group. The difference was statistically significant at 5, 15 and 45 min after circulatory arrest. Statistically significant differences were seen also in the recovery phase at 90 and 120 min after reperfusion. Nicotinamide

  18. Colitis susceptibility in p47(phox-/-) mice is mediated by the microbiome.

    PubMed

    Falcone, E Liana; Abusleme, Loreto; Swamydas, Muthulekha; Lionakis, Michail S; Ding, Li; Hsu, Amy P; Zelazny, Adrian M; Moutsopoulos, Niki M; Kuhns, Douglas B; Deming, Clay; Quiñones, Mariam; Segre, Julia A; Bryant, Clare E; Holland, Steven M

    2016-04-05

    Chronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91(phox) (a.k.a. Nox2), p47(phox), p67(phox), p22(phox), p40(phox)) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored. Although CGD mice do not spontaneously develop colitis, we demonstrate that p47(phox-/-) mice have increased susceptibility to dextran sodium sulfate colitis in association with a distinct colonic transcript and microbiome signature. Neither restoring NOX2 reactive oxygen species production nor normalizing the microbiome using cohoused adult p47(phox-/-) with B6Tac (wild type) mice reversed this phenotype. However, breeding p47(phox+/-) mice and standardizing the microflora between littermate p47(phox-/-) and B6Tac mice from birth significantly reduced dextran sodium sulfate colitis susceptibility in p47(phox-/-) mice. We found similarly decreased colitis susceptibility in littermate p47(phox-/-) and B6Tac mice treated with Citrobacter rodentium. Our findings suggest that the microbiome signature established at birth may play a bigger role than phagocyte-derived reactive oxygen species in mediating colitis susceptibility in CGD mice. These data further support bacteria-related disease in CGD colitis.

  19. Isolation rates, serovars, and toxin genotypes of nicotinamide adenine dinucleotide-independent Actinobacillus pleuropneumoniae among pigs suffering from pleuropneumonia in Spain.

    PubMed

    Maldonado, Jaime; Valls, Laura; Martínez, Eva; Riera, Pere

    2009-11-01

    Actinobacillus pleuropneumoniae is the etiologic agent of swine pleuropneumonia, a major production-limiting disease in the pig industry. In the current study, 2,171 lung specimens obtained from pigs housed in 870 Spanish pig farms in regions of substantial pig production were examined. Conventional microbiology, coupled with species-specific polymerase chain reaction, identified 127 biovar 2 isolates, accounting for 25.3% of all A. pleuropneumoniae (n = 502) detected. Most isolates (79%) were recovered as pure primary cultures or as the predominant bacteria from lungs exhibiting lesions typical of acute swine pleuropneumonia. Coagglutination testing identified the isolates as belonging to serovars 2 (4.7%), 4 (4.7%), 7 (68.5%), and 11 (1.6%); however, 26 isolates were nontypeable. All biovar 2 isolates showed genes of the apxII operon alone, which encodes the corresponding ApxII exotoxin, leading to a different gene pattern for isolates in serovars 2, 4, and 11 compared with those of biovar 1. From this survey, it can be concluded that A. pleuropneumoniae biovar 2 infections are common in pigs in Spain, and they may be a common cause of respiratory disease in swine.

  20. Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

    We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.

  1. Nicotinamide riboside phosphorylase from beef liver: purification and characterization.

    PubMed

    Imai, T; Anderson, B M

    1987-04-01

    Nicotinamide riboside phosphorylase (NR phosphorylase) from beef liver has been purified to apparent homogeneity at 300-fold purification with a 35% yield. Kinetic constants for the enzyme-catalyzed phosphorolysis were as follows Knicotinamide riboside, 2.5 +/- 0.4 mM; Kinorganic phosphate, 0.50 +/- 0.12 mM; Vmax, 410 +/- 30 X 10(-6) mol min-1 mg protein-1, respectively. The molecular weights of the native enzyme and subunit structure were determined to be 131,000 and 32,000, respectively, suggesting the beef liver NR phosphorylase to be tetrameric in structure and consistent with the presence of identical subunits. The amino acid composition was shown to be very similar to that reported for human erythrocyte purine-nucleoside phosphorylase but differing considerably from that found for rat liver purine-nucleoside phosphorylase. In addition to catalytic activity with nicotinamide riboside, the beef liver enzyme catalyzed a phosphorolytic reaction with inosine and guanosine exhibiting activity ratios, nicotinamide riboside:inosine: guanosine of 1.00:0.35:0.29, respectively. These ratios of activity remained constant throughout purification of the beef liver enzyme and no separation of these activities was detected. Phosphorolysis of nicotinamide riboside was inhibited competitively by inosine (Ki = 75 microM) and guanosine (Ki = 75 microM). Identical rates of thermal denaturation of the beef liver enzyme were observed when determined for the phosphorolysis of either nicotinamide riboside or inosine. These observations coupled with studies of pH and specific buffer effects indicate the phosphorolysis of nicotinamide riboside, inosine, and guanosine to be catalyzed by the same enzyme.

  2. The Importance of NADPH Oxidases and Redox Signaling in Angiogenesis

    PubMed Central

    Prieto-Bermejo, Rodrigo; Hernández-Hernández, Angel

    2017-01-01

    Eukaryotic cells have to cope with the constant generation of reactive oxygen species (ROS). Although the excessive production of ROS might be deleterious for cell biology, there is a plethora of evidence showing that moderate levels of ROS are important for the control of cell signaling and gene expression. The family of the nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases or Nox) has evolved to produce ROS in response to different signals; therefore, they fulfil a central role in the control of redox signaling. The role of NADPH oxidases in vascular physiology has been a field of intense study over the last two decades. In this review we will briefly analyze how ROS can regulate signaling and gene expression. We will address the implication of NADPH oxidases and redox signaling in angiogenesis, and finally, the therapeutic possibilities derived from this knowledge will be discussed. PMID:28505091

  3. Acridone-based inhibitors of inosine 5'-monophosphate dehydrogenase: discovery and SAR leading to the identification of N-(2-(6-(4-ethylpiperazin-1-yl)pyridin-3-yl)propan-2-yl)-2- fluoro-9-oxo-9,10-dihydroacridine-3-carboxamide (BMS-566419).

    PubMed

    Watterson, Scott H; Chen, Ping; Zhao, Yufen; Gu, Henry H; Dhar, T G Murali; Xiao, Zili; Ballentine, Shelley K; Shen, Zhongqi; Fleener, Catherine A; Rouleau, Katherine A; Obermeier, Mary; Yang, Zheng; McIntyre, Kim W; Shuster, David J; Witmer, Mark; Dambach, Donna; Chao, Sam; Mathur, Arvind; Chen, Bang-Chi; Barrish, Joel C; Robl, Jeffrey A; Townsend, Robert; Iwanowicz, Edwin J

    2007-07-26

    Inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanosine nucleotides, catalyzes the irreversible nicotinamide-adenine dinucleotide dependent oxidation of inosine-5'-monophosphate to xanthosine-5'-monophosphate. Mycophenolate Mofetil (MMF), a prodrug of mycophenolic acid, has clinical utility for the treatment of transplant rejection based on its inhibition of IMPDH. The overall clinical benefit of MMF is limited by what is generally believed to be compound-based, dose-limiting gastrointestinal (GI) toxicity that is related to its specific pharmacokinetic characteristics. Thus, development of an IMPDH inhibitor with a novel structure and a different pharmacokinetic profile may reduce the likelihood of GI toxicity and allow for increased efficacy. This article will detail the discovery and SAR leading to a novel and potent acridone-based IMPDH inhibitor 4m and its efficacy and GI tolerability when administered orally in a rat adjuvant arthritis model.

  4. Ligand-based virtual screening and inductive learning for identification of SIRT1 inhibitors in natural products.

    PubMed

    Sun, Yunan; Zhou, Hui; Zhu, Hongmei; Leung, Siu-wai

    2016-01-25

    Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase, and its dysregulation can lead to ageing, diabetes, and cancer. From 346 experimentally confirmed SIRT1 inhibitors, an inhibitor structure pattern was generated by inductive logic programming (ILP) with DMax Chemistry Assistant software. The pattern contained amide, amine, and hetero-aromatic five-membered rings, each of which had a hetero-atom and an unsubstituted atom at a distance of 2. According to this pattern, a ligand-based virtual screening of 1 444 880 active compounds from Chinese herbs identified 12 compounds as inhibitors of SIRT1. Three compounds (ZINC08790006, ZINC08792229, and ZINC08792355) had high affinity (-7.3, -7.8, and -8.6 kcal/mol, respectively) for SIRT1 as estimated by molecular docking software AutoDock Vina. This study demonstrated a use of ILP and background knowledge in machine learning to facilitate virtual screening.

  5. Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes.

    PubMed

    Lin, Richen; Cheng, Jun; Ding, Lingkan; Song, Wenlu; Liu, Min; Zhou, Junhu; Cen, Kefa

    2016-05-01

    Ferric oxide nanoparticles (FONPs) were used to facilitate dark hydrogen fermentation using Enterobacter aerogenes. The hydrogen yield of glucose increased from 164.5±2.29 to 192.4±1.14mL/g when FONPs concentration increased from 0 to 200mg/L. SEM images of E. aerogenes demonstrated the existence of bacterial nanowire among cells, suggesting FONPs served as electron conduits to enhance electron transfer. TEM showed cellular internalization of FONPs, indicating hydrogenase synthesis and activity was potentially promoted due to the released iron element. When further increasing FONPs concentration to 400mg/L, the hydrogen yield of glucose decreased to 147.2±2.54mL/g. Soluble metabolic products revealed FONPs enhanced acetate pathway of hydrogen production, but weakened ethanol pathway. This shift of metabolic pathways allowed more nicotinamide adenine dinucleotide for reducing proton to hydrogen. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Exploring NAD+ metabolism in host-pathogen interactions.

    PubMed

    Mesquita, Inês; Varela, Patrícia; Belinha, Ana; Gaifem, Joana; Laforge, Mireille; Vergnes, Baptiste; Estaquier, Jérôme; Silvestre, Ricardo

    2016-03-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a vital molecule found in all living cells. NAD(+) intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD(+) metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD(+) metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD(+) homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD(+) or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD(+) metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases.

  7. NAD+ protects against EAE by regulating CD4+ T-cell differentiation

    PubMed Central

    Tullius, Stefan G.; Biefer, Hector Rodriguez Cetina; Li, Suyan; Trachtenberg, Alexander J.; Edtinger, Karoline; Quante, Markus; Krenzien, Felix; Uehara, Hirofumi; Yang, Xiaoyong; Kissick, Haydn T.; Kuo, Winston P.; Ghiran, Ionita; de la Fuente, Miguel A.; Arredouani, Mohamed S.; Camacho, Virginia; Tigges, John C.; Toxavidis, Vasilis; El Fatimy, Rachid; Smith, Brian D.; Vasudevan, Anju; ElKhal, Abdallah

    2014-01-01

    CD4+ T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD+) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4+IFNγ+IL-10+ T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD+ regulates CD4+ T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD+, the frequency of T-bet−/− CD4+IFNγ+ T cells was twofold higher than wild-type CD4+ T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4+ T-cell differentiation and demonstrate that NAD+ may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases. PMID:25290058

  8. Tryptophan as key biomarker to detect gastrointestinal tract cancer using non-negative biochemical analysis of native fluorescence and Stokes Shift spectroscopy

    NASA Astrophysics Data System (ADS)

    Wang, Leana; Zhou, Yan; Liu, Cheng-hui; Zhou, Lixin; He, Yong; Pu, Yang; Nguyen, Thien An; Alfano, Robert R.

    2015-03-01

    The objective of this study was to find out the emission spectral fingerprints for discrimination of human colorectal and gastric cancer from normal tissue in vitro by applying native fluorescence. The native fluorescence (NFL) and Stokes shift spectra of seventy-two human cancerous and normal colorectal (colon, rectum) and gastric tissues were analyzed using three selected excitation wavelengths (e.g. 300 nm, 320 nm and 340 nm). Three distinct biomarkers, tryptophan, collagen and reduced nicotinamide adenine dinucleotide hydrate (NADH), were found in the samples of cancerous and normal tissues from eighteen subjects. The spectral profiles of tryptophan exhibited a sharp peak in cancerous colon tissues under a 300 nm excitation when compared with normal tissues. The changes in compositions of tryptophan, collagen, and NADH were found between colon cancer and normal tissues under an excitation of 300 nm by the non-negative basic biochemical component analysis (BBCA) model.

  9. More to NAD+ than meets the eye: A regulator of metabolic pools and gene expression in Arabidopsis.

    PubMed

    Gakière, Bertrand; Fernie, Alisdair R; Pétriacq, Pierre

    2018-01-05

    Since its discovery more than a century ago, nicotinamide adenine dinucleotide (NAD + ) is recognised as a fascinating cornerstone of cellular metabolism. This ubiquitous energy cofactor plays vital roles in metabolic pathways and regulatory processes, a fact emphasised by the essentiality of a balanced NAD + metabolism for normal plant growth and development. Research on the role of NAD in plants has been predominantly carried out in the model plant Arabidopsis thaliana (Arabidopsis) with emphasis on the redox properties and cellular signalling functions of the metabolite. This review examines the current state of knowledge concerning how NAD can regulate both metabolic pools and gene expression in Arabidopsis. Particular focus is placed on recent studies highlighting the complexity of metabolic regulations involving NAD, more particularly in the mitochondrial compartment, and of signalling roles with respect to interactions with environmental fluctuations most specifically those involving plant immunity. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Ligand-based virtual screening and inductive learning for identification of SIRT1 inhibitors in natural products

    NASA Astrophysics Data System (ADS)

    Sun, Yunan; Zhou, Hui; Zhu, Hongmei; Leung, Siu-Wai

    2016-01-01

    Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase, and its dysregulation can lead to ageing, diabetes, and cancer. From 346 experimentally confirmed SIRT1 inhibitors, an inhibitor structure pattern was generated by inductive logic programming (ILP) with DMax Chemistry Assistant software. The pattern contained amide, amine, and hetero-aromatic five-membered rings, each of which had a hetero-atom and an unsubstituted atom at a distance of 2. According to this pattern, a ligand-based virtual screening of 1 444 880 active compounds from Chinese herbs identified 12 compounds as inhibitors of SIRT1. Three compounds (ZINC08790006, ZINC08792229, and ZINC08792355) had high affinity (-7.3, -7.8, and -8.6 kcal/mol, respectively) for SIRT1 as estimated by molecular docking software AutoDock Vina. This study demonstrated a use of ILP and background knowledge in machine learning to facilitate virtual screening.

  11. Transport of adenine, hypoxanthine and uracil into Escherichia coli.

    PubMed Central

    Burton, K

    1977-01-01

    Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases. PMID:413544

  12. Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA.

    PubMed

    Huang, Ke-Jing; Niu, De-Jun; Sun, Jun-Yong; Han, Cong-Hui; Wu, Zhi-Wei; Li, Yan-Li; Xiong, Xiao-Qin

    2011-02-01

    A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334V. The detection limit for individual determination of guanine and adenine was 5.0×10(-8)M and 2.5×10(-8)M (S/N=3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G+C)/(A+T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus

    PubMed Central

    Sugiyama, Kou-ichi

    2011-01-01

    Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh promoted the activities of glyceraldehyde-3-phosephate dehydrogenase (G-3-PD), nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase (NAD-ICDH), succinate dehydrogenase (SDH) and cytochrome-c oxidase (Cyt-c OD) in seedlings. Moreover, ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint. PMID:21900743

  14. An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma.

    PubMed

    Gujar, Amit D; Le, Son; Mao, Diane D; Dadey, David Y A; Turski, Alice; Sasaki, Yo; Aum, Diane; Luo, Jingqin; Dahiya, Sonika; Yuan, Liya; Rich, Keith M; Milbrandt, Jeffrey; Hallahan, Dennis E; Yano, Hiroko; Tran, David D; Kim, Albert H

    2016-12-20

    Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD + ). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD + synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD + -dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD + metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.

  15. Upregulation of mitochondrial NAD+ levels impairs the clonogenicity of SSEA1+ glioblastoma tumor-initiating cells.

    PubMed

    Son, Myung Jin; Ryu, Jae-Sung; Kim, Jae Yun; Kwon, Youjeong; Chung, Kyung-Sook; Mun, Seon Ju; Cho, Yee Sook

    2017-06-09

    Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD + ) metabolism. However, the functional role of NAD + metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD + levels affect the characteristics of glioma-driven SSEA1 + TICs, including clonogenic growth potential. An increase in the mitochondrial NAD + levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD + levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.

  16. NAD+ Supplementation Attenuates Methylmercury Dopaminergic and Mitochondrial Toxicity in Caenorhabditis Elegans

    PubMed Central

    Caito, Samuel W.; Aschner, Michael

    2016-01-01

    Methylmercury (MeHg) is a neurotoxic contaminant of our fish supply that has been linked to dopaminergic (DAergic) dysfunction that characterizes Parkinson’s disease. We have previously shown that MeHg causes both morphological and behavioral changes in the Caenorhabditis elegans DAergic neurons that are associated with oxidative stress. We were therefore interested in whether the redox sensitive cofactor nicotinamide adenine dinucleotide (NAD+) may be affected by MeHg and whether supplementation of NAD + may prevent MeHg-induced toxicities. Worms treated with MeHg showed depletion in cellular NAD + levels, which was prevented by NAD + supplementation prior to MeHg treatment. NAD + supplementation also prevented DAergic neurodegeneration and deficits in DAergic-dependent behavior upon MeHg exposure. In a mutant worm line that cannot synthesize NAD + from nicotinamide, MeHg lethality and DAergic behavioral deficits were more sensitive to MeHg than wildtype worms, demonstrating the importance of NAD + in MeHg toxicity. In wildtype worms, NAD + supplementation provided protection from MeHg-induced oxidative stress and mitochondrial dysfunction. These data show the importance of NAD + levels in the response to MeHg exposure. NAD + supplementation may be beneficial for MeHg-induced toxicities and preventing cellular damage involved in Parkinson’s disease. PMID:26865665

  17. Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

    PubMed

    Hsu, Chia George; Burkholder, Thomas J

    2016-12-01

    The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.

  18. Molecular Links between Caloric Restriction and Sir2/SIRT1 Activation

    PubMed Central

    2014-01-01

    Ageing is the most significant risk factor for a range of prevalent diseases, including cancer, cardiovascular disease, and diabetes. Accordingly, interventions are needed for delaying or preventing disorders associated with the ageing process, i.e., promotion of healthy ageing. Calorie restriction is the only nongenetic and the most robust approach to slow the process of ageing in evolutionarily divergent species, ranging from yeasts, worms, and flies to mammals. Although it has been known for more than 80 years that calorie restriction increases lifespan, a mechanistic understanding of this phenomenon remains elusive. Yeast silent information regulator 2 (Sir2), the founding member of the sirtuin family of protein deacetylases, and its mammalian homologue Sir2-like protein 1 (SIRT1), have been suggested to promote survival and longevity of organisms. SIRT1 exerts protective effects against a number of age-associated disorders. Caloric restriction increases both Sir2 and SIRT1 activity. This review focuses on the mechanistic insights between caloric restriction and Sir2/SIRT1 activation. A number of molecular links, including nicotinamide adenine dinucleotide, nicotinamide, biotin, and related metabolites, are suggested to be the most important conduits mediating caloric restriction-induced Sir2/SIRT1 activation and lifespan extension. PMID:25349818

  19. Preparation, purification and analyses of thirteen alkali-stable dinucleotides from ribonucleic acid

    PubMed Central

    Trim, A. R.; Parker, Janet E.

    1970-01-01

    Of the 16 alkali-stable dinucleotides known to be obtained by hydrolysis of commercial yeast RNA with alkali, 13 were prepared in quantities of the order of 10mg or more. The samples, with only one exception, contain at least 90% of dinucleotide, and spectroscopic constants and nucleotide-sequence determinations, although not conclusive, indicate a high degree of purity of these products. The small dinucleotide fraction in 150g of RNA hydrolysed with alkali (1–2% of the total nucleotides) was separated from the mononucleotides by stepwise ion-exchange chromatography on DEAE-cellulose columns and resolved into seven fractions containing from one to four different dinucleotides by electrophoresis on paper at pH3.0. These fractions were resolved into their constituent dinucleotides by chromatography in ammonium sulphate. Contamination of the products by impurities from the paper was minimized by washing it before using it for chromatography or electrophoresis and, by using a thick grade of paper (Whatman no. 17), it was possible to handle and purify relatively large quantities of nucleotides. PMID:5435489

  20. ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

    PubMed Central

    Stine, G. J.

    1968-01-01

    Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged. PMID:4384627

  1. Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sanders, B.; Zhao, K; Slama, J

    2007-01-01

    The Sir2 family of proteins consists of broadly conserved NAD+-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD+ at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with relatedmore » biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called 'C pocket' binding site for the nicotinamide group of NAD+. These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.« less

  2. Nicotinamide attenuates the ischemic brain injury-induced decrease of Akt activation and Bad phosphorylation.

    PubMed

    Koh, Phil-Ok

    2011-07-08

    Nicotinamide protects cortical neuronal cells against cerebral ischemic injury through activation of various cytoprotective mechanisms. Here, this study confirmed the neuroprotective effects of nicotinamide in focal cerebral ischemic injury and investigated whether nicotinamide modulates a crucial survival pathway, Akt and its downstream targets. Adult male rats were treated with vehicle or nicotinamide (500 mg/kg) 2h after the onset of middle cerebral artery occlusion (MCAO). Brains were collected 24h after MCAO and infarct volumes were analyzed. Nicotinamide significantly reduced the infarct volume in the cerebral cortex. Potential activation was measured by phosphorylation of PDK1 at Ser(241), Akt at Ser(473), and Bad at Ser(136) using Western blot analysis. Nicotinamide prevented the injury-induced decrease of pPDK1, pAkt, and pBad levels. 14-3-3 levels were not different between vehicle- and nicotinamide-treated animals. However, pBad and 14-3-3 interaction levels decreased during MCAO, but were maintained in the presence of nicotinamide, compared to levels in control animals. These findings suggest that nicotinamide attenuates cell death due to focal cerebral ischemic injury and that neuroprotective effects are mediated through the Akt signaling pathway, thus enhancing neuronal survival. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  3. New approach to biosensing of co-enzyme nicotinamide adenine dinucleotide (NADH) by incorporation of neutral red in aluminum doped nanostructured ZnO thin films.

    PubMed

    V T, Fidal; T S, Chandra

    2017-06-01

    Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000nm varying dependent on time of deposition (30 to 150min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~300nm in contrast to its absence (AZO) with ~200nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45μAcm -2 mM -1 and LoD of 22μM within the range 0.075-4mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD + dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56μAcm -2 mM -1 and LoD for lactate was 27μM in the range of 0.1-1mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader. Copyright © 2017 Elsevier B.V. All rights

  4. In silico identification of novel and selective monoamine oxidase B inhibitors.

    PubMed

    Yelekçi, Kemal; Büyüktürk, Bora; Kayrak, Nurdan

    2013-06-01

    Monoamine oxidases (MAO) A and B are flavin adenine dinucleotides containing enzymes bound to the mitochondrial outer membranes of the cells of the brain, liver, intestine, and placenta, as well as platelets. Recently, selective MAO-B inhibitors have received increasing attention due to their neuroprotective properties and the multiple roles they can play in the therapy of neurodegenerative disorders. This study was based on 10 scaffolds that were selected from more than a million lead compounds in the ZINCv12 lead library for their structural and physicochemical properties which inhibit MAO-B. Utilizing ZINC and Accelrys 3.1 fragment-based libraries, which contain about 400 thousand fragments, we generated 200 potential candidates. GOLD, LibDock, and AutoDock 4.02 were used to identify the inhibition constants and their position in the active sites of both MAO isozymes. The dispositions of the candidate molecules within the organism were checked with ADMET PSA 2D (polar surface area) against ADMET AlogP98 (the logarithm of the partition coefficient between n-octanol and water). The MAO-B inhibition activities of the candidates were compared with the properties of rasagiline which is known to be a selective inhibitor of MAO-B.

  5. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species

    PubMed Central

    Di Giallonardo, Francesca; Schlub, Timothy E.; Shi, Mang

    2017-01-01

    ABSTRACT Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide

  6. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

    PubMed

    Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

    2017-04-15

    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone. IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

  7. Molecular modeling of substrate binding in wild-type and mutant Corynebacteria 2,5-diketo-D-gluconate reductases.

    PubMed

    Khurana, S; Sanli, G; Powers, D B; Anderson, S; Blaber, M

    2000-04-01

    2,5-diketo-D-gluconic acid reductase (2,5-DKGR; E.C. 1.1.1.-) catalyzes the Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent stereo-specific reduction of 2, 5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate (2-KLG), a precursor in the industrial production of vitamin C (L-ascorbate). Microorganisms that naturally ferment D-glucose to 2,5-DKG can be genetically modified to express the gene for 2,5-DKGR, and thus directly produce vitamin C from D-glucose. Two naturally occurring variants of DKGR (DKGR A and DKGR B) have been reported. DKGR B exhibits higher specific activity toward 2,5-DKG than DKGR A; however, DKGR A exhibits a greater selectivity for this substrate and significantly higher thermal stability. Thus, a modified form of DKGR, combining desirable properties from both enzymes, would be of substantial commercial interest. In the present study we use a molecular dynamics-based approach to understand the conformational changes in DKGR A as the active site is mutated to include two active site residue changes that occur in the B form. The results indicate that the enhanced kinetic properties of the B form are due, in part, to residue substitutions in the binding pocket. These substitutions augment interactions with the substrate or alter the alignment with respect to the putative proton donor group. Proteins 2000;39:68-75. Copyright 2000 Wiley-Liss, Inc.

  8. Catalytic carbene transfer allows the direct customization of cyclic purine dinucleotides.

    PubMed

    Fei, Na; Häussinger, Daniel; Blümli, Seraina; Laventie, Benoît-Joseph; Bizzini, Lorenzo D; Zimmermann, Kaspar; Jenal, Urs; Gillingham, Dennis

    2014-08-11

    We describe a simple method for the direct modification of nucleobases in cyclic purine dinucleotides, important signalling molecules in both prokaryotes and eukaryotes. The method tolerates all members of the cyclic dinucleotide family and could be used to modulate their function or introduce useful side-chains such as fluorophores and photo-crosslinking groups.

  9. Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases.

    PubMed

    Rössl, Anthony; Bentley-DeSousa, Amanda; Tseng, Yi-Chieh; Nwosu, Christine; Downey, Michael

    2016-10-01

    Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1 Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems. Copyright © 2016 by the Genetics Society of America.

  10. Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases

    PubMed Central

    Rössl, Anthony; Bentley-DeSousa, Amanda; Tseng, Yi-Chieh; Nwosu, Christine; Downey, Michael

    2016-01-01

    Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1. Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems. PMID:27527516

  11. A comparison of adenine and some derivatives on pig isolated tracheal muscle.

    PubMed Central

    Bach-Dieterle, Y.; Holden, W. E.; Junod, A. F.

    1983-01-01

    We studied the muscle relaxation induced by adenine and several adenine derivatives in strips of tracheal smooth muscle from pigs; in addition their metabolism by the tissue was examined. Adenine relaxed tissue which was contracted by carbachol, histamine, or KCl. Adenine's potency was similar to that of adenosine and ATP (threshold about 4 X 10(-5)M). In tissues with carbachol-induced tone, the adenine effect differed from adenosine and ATP by being slower in onset and in 'washout' time. Furthermore, neither dipyridamole nor theophylline modified the response to adenine. The relationship was examined between pharmacological effects and the metabolism of [3H]-adenosine and [3H]-adenine. Both substrates were taken up by the tissue and converted to nucleotides, but relaxation correlated with nucleotide accumulation only in the case of [3H]-adenine. We conclude that the site and mechanism of adenine-induced relaxation is different from that of adenosine and ATP in porcine tracheal muscle. PMID:6571222

  12. pHP-Tethered N-Acyl Carbamate: A Photocage for Nicotinamide.

    PubMed

    Salahi, Farbod; Purohit, Vatsal; Ferraudi, Guillermo; Stauffacher, Cynthia; Wiest, Olaf; Helquist, Paul

    2018-05-04

    The synthesis of a new photocaged nicotinamide having an N-acyl carbamate linker and a p-hydroxyphenacyl (pHP) chromophore is described. The photophysical and photochemical studies showed an absorption maximum at λ = 330 nm and a quantum yield for release of 11% that are dependent upon both pH and solvent. While the acyl carbamate releases nicotinamide efficiently, a simpler amide linker was inert to photocleavage. This photocaged nicotinamide has significant advantages with respect to quantum yield, absorbance wavelength, rate of release, and solubility that make it the first practical example of a photocaged amide.

  13. Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22{sup phox} expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Chaoyun; He, Yanhao; Department of Pharmacology, Xi'an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Disease, Ministry of Education, Xi'an, Shaanxi 710061

    Intracellular reactive oxygen species (ROS) are derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Angiotensin II (Ang II) can cause endothelial dysfunction by promoting intracellular ROS generation. Safflor yellow B (SYB) effectively inhibits ROS generation by upregulating Bcl-2 expression. In this study, we examined the effects of SYB on Ang II-induced injury to human umbilical vein endothelial cells (HUVECs), and elucidated the roles of NADPH oxidase and Bcl-2. We treated cultured HUVECs with Ang II, SYB, and Bcl-2 siRNA, and determined NADPH oxidase activity and ROS levels. Furthermore, cellular and mitochondrial physiological states were evaluated, and the expression levels ofmore » target proteins were analyzed. Ang II significantly enhanced intracellular ROS levels, caused mitochondrial membrane dysfunction, and decreased cell viability, leading to apoptosis. This was associated with increased expression of AT1R and p22{sup phox}, increased NADPH oxidase activity, and an increased ratio of Bax/Bcl-2, leading to decreases in antioxidant enzyme activities, which were further strengthened after blocking Bcl-2. Compared to Ang II treatment alone, co-treatment with SYB significantly reversed HUVEC injury. Taken together, these results demonstrate that SYB could significantly protect endothelial cells from Ang II-induced cell damage, and that it does so by upregulating Bcl-2 expression and inhibiting ROS generation. - Highlights: • Angiotensin II depresses mitochondria physiological function. • Angiotensin II activates NADPH oxidase via up-regulating expresion of p22{sup phox}. • Bcl-2 plays a pivotal role in improving mitochondria function and regulates ROS level. • Inhibitor of Bcl-2 promotes angiotensin II mediated HUVEC injury. • SYB attenuates angiotensin II mediated HUVEC injury via up regulating Bcl-2 expression.« less

  14. Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

    PubMed

    Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana

    2016-01-01

    Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms. © 2015 CSIC New Phytologist © 2015 New Phytologist Trust.

  15. Role of Hyperhomocysteinemia in the Regulation of Oxidative Stress and Inflammatory Responses in the Kidney: Protective Effect of Folic Acid Supplementation

    NASA Astrophysics Data System (ADS)

    Hwang, Sun-Young

    Hyperhomocysteinemia, a condition of elevated blood homocysteine (Hcy) level, is an independent risk factor for cardiovascular disease. Folic acid supplementation can effectively reduce blood Hcy levels. Recent studies have demonstrated that hyperhomocysteinemia is also associated with kidney disease. However, the underlying mechanisms remain unclear. The overall objective of the study was to investigate the biochemical and molecular mechanisms of Hcy-induced kidney injury and the effect of folic acid supplementation on Hcy-induced kidney injury. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 12 weeks. An elevation of serum total Hcy level was observed in hyperhomocysteinemic rats. Hyperhomocysteinemia-induced superoxide anion production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation resulted in oxidative stress in the kidney. Reduction of oxidative stress by inhibiting superoxide anion production effectively ameliorated hyperhomocysteinemia-induced kidney injury. Inflammatory responses such as increased chemokine expression have been implicated as one of the mechanisms of kidney disease. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that is involved in the inflammatory response in kidney disease. Nuclear factor-kappa B (NF-kappaB) plays an important role in upregulation of MCP-1 expression. We investigated the effect of hyperhomocysteinemia on MCP-1 expression and the molecular mechanism responsible for such an effect in rat kidneys as well as in human kidney proximal tubular cells.

  16. Base Excision Repair of Tandem Modifications in a Methylated CpG Dinucleotide*

    PubMed Central

    Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S.; Beard, William A.; Wilson, Samuel H.

    2014-01-01

    Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3′-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5′-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide. PMID:24695738

  17. Nicotinamide polymeric nanoemulsified systems: a quality-by-design case study for a sustained antimicrobial activity

    PubMed Central

    Zidan, Ahmed S; Ahmed, Osama AA; Aljaeid, Bader M

    2016-01-01

    Nicotinamide, the amide form of vitamin B3, was demonstrated to combat some of the antibiotic-resistant infections that are increasingly common around the world. The objective of this study was to thoroughly understand the formulation and process variabilities affecting the preparation of nicotinamide-loaded polymeric nanoemulsified particles. The quality target product profile and critical quality attributes of the proposed product were presented. Plackett–Burman screening design was employed to screen eight variables for their influences on the formulation’s critical characteristics. The formulations were prepared by an oil-in-water emulsification followed by solvent replacement. The prepared systems were characterized by entrapment capacity (EC), entrapment efficiency (EE), particle size, polydispersity index, zeta potential, transmission electron microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, powder X-ray diffraction, in vitro drug release, and their antibacterial activity against bacterial scrums. EC, EE, particle size, polydispersity index, zeta potential, and percentage release in 24 hours were found to be in the range of 33.5%–68.8%, 53.1%–67.1%, 43.3–243.3 nm, 0.08–0.28, 9.5–53.3 mV, and 5.8%–22.4%, respectively. One-way analysis of variance and Pareto charts revealed that the experimental loadings of 2-hydroxypropyl-β-cyclodextrin and Eudragit® S100 were the most significant for their effects on nicotinamide EC and EE. Moreover, the polymeric nanoemulsified particles demonstrated a sustained release profile for nicotinamide. The Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction demonstrated a significant interaction between the drug and 2-hydroxypropyl-β-cyclodextrin that might modulate the sustained release behavior. Furthermore, the formulations provided a sustained antibacterial activity that depended on nicotinamide-loading concentration

  18. Oxidative stress as a mechanism of added sugar-induced cardiovascular disease.

    PubMed

    Prasad, Kailash; Dhar, Indu

    2014-12-01

    Added sugars comprising of table sugar, brown sugar, corn syrup, maple syrup, honey, molasses, and other sweeteners in the prepared processed foods and beverages have been implicated in the pathophysiology of cardiovascular diseases. This article deals with the reactive oxygen species (ROS) as a mechanism of sugar-induced cardiovascular diseases. There is an association between the consumption of high levels of serum glucose with cardiovascular diseases. Various sources of sugar-induced generation of ROS, including mitochondria, nicotinamide adenine dinucleotide phosphate-oxidase, advanced glycation end products, insulin, and uric acid have been discussed. The mechanism by which ROS induce the development of atherosclerosis, hypertension, peripheral vascular disease, coronary artery disease, cardiomyopathy, heart failure, and cardiac arrhythmias have been discussed in detail. In conclusion, the data suggest that added sugars induce atherosclerosis, hypertension, peripheral vascular disease, coronary artery disease, cardiomyopathy, heart failure, and cardiac arrhythmias and that these effects of added sugars are mediated through ROS.

  19. Metabolism of 4-Chloro-2-Methylphenoxyacetic Acid by Soil Bacteria

    PubMed Central

    Bollag, J.-M.; Helling, C. S.; Alexander, M.

    1967-01-01

    A microorganism capable of degrading 4-chloro-2-methylphenoxyacetic acid (MCPA) was isolated from soil and identified as Flavobacterium peregrinum. All of the chlorine of MCPA was released as chloride, and the carboxyl-carbon was converted to volatile products by growing cultures of the bacterium, but a phenol accumulated in the medium. The phenol was identified as 4-chloro-2-methylphenol on the basis of its gas chromatographic and infrared characteristics. Extracts of cells of F. peregrinum and of a phenoxyacetate-metabolizing Arthrobacter sp. dehalogenated MCPA and several catechols but not 4-chloro-2-methylanisole. The Arthrobacter sp. cell extract was fractionated, and an enzyme preparation was obtained which catalyzed the conversion of MCPA to 4-chloro-2-methylphenol. The latter compound was not metabolized unless reduced nicotinamide adenine dinucleotide phosphate was added to the fractionated extract. The phenol in turn was apparently oxidized to a catechol by components of the enzyme preparation. PMID:16349751

  20. Effect of tricarboxylic acid cycle regulator on carbon retention and organic component transformation during food waste composting.

    PubMed

    Lu, Qian; Zhao, Yue; Gao, Xintong; Wu, Junqiu; Zhou, Haixuan; Tang, Pengfei; Wei, Qingbin; Wei, Zimin

    2018-05-01

    Composting is an environment friendly method to recycling organic waste. However, with the increasing concern about greenhouse gases generated in global atmosphere, it is significant to reduce the emission of carbon dioxide (CO 2 ). This study analyzes tricarboxylic acid (TCA) cycle regulators on the effect of reducing CO 2 emission, and the relationship among organic component (OC) degradation and transformation and microorganism during composting. The results showed that adding adenosine tri-phosphate (ATP) and nicotinamide adenine dinucleotide (NADH) could enhance the transformation of OC and increase the diversity of microorganism community. Malonic acid (MA) as a competitive inhibitor could decrease the emission of CO 2 by inhibiting the TCA cycle. A structural equation model was established to explore effects of different OC and microorganism on humic acid (HA) concentration during composting. Furthermore, added MA provided an environmental benefit in reducing the greenhouse gas emission for manufacture sustainable products. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. A repeatedly refuelable mediated biofuel cell based on a hierarchical porous carbon electrode

    NASA Astrophysics Data System (ADS)

    Fujita, Shuji; Yamanoi, Shun; Murata, Kenichi; Mita, Hiroki; Samukawa, Tsunetoshi; Nakagawa, Takaaki; Sakai, Hideki; Tokita, Yuichi

    2014-05-01

    Biofuel cells that generate electricity from renewable fuels, such as carbohydrates, must be reusable through repeated refuelling, should these devices be used in consumer electronics. We demonstrate the stable generation of electricity from a glucose-powered mediated biofuel cell through multiple refuelling cycles. This refuelability is achieved by immobilizing nicotinamide adenine dinucleotide (NAD), an electron-transfer mediator, and redox enzymes in high concentrations on porous carbon particles constituting an anode while maintaining their electrochemical and enzymatic activities after the immobilization. This bioanode can be refuelled continuously for more than 60 cycles at 1.5 mA cm-2 without significant potential drop. Cells assembled with these bioanodes and bilirubin-oxidase-based biocathodes can be repeatedly used to power a portable music player at 1 mW cm-3 through 10 refuelling cycles. This study suggests that the refuelability within consumer electronics should facilitate the development of long and repeated use of the mediated biofuel cells as well as of NAD-based biosensors, bioreactors, and clinical applications.

  2. A model for evolution and regulation of nicotine biosynthesis regulon in tobacco.

    PubMed

    Kajikawa, Masataka; Sierro, Nicolas; Hashimoto, Takashi; Shoji, Tsubasa

    2017-06-03

    In tobacco, the defense alkaloid nicotine is produced in roots and accumulates mainly in leaves. Signaling mediated by jasmonates (JAs) induces the formation of nicotine via a series of structural genes that constitute a regulon and are coordinated by JA-responsive transcription factors of the ethylene response factor (ERF) family. Early steps in the pyrrolidine and pyridine biosynthesis pathways likely arose through duplication of the polyamine and nicotinamide adenine dinucleotide (NAD) biosynthetic pathways, respectively, followed by recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon. Transcriptional regulation of nicotine biosynthesis by ERF and cooperatively-acting MYC2 transcription factors is implied by the frequency of cognate cis-regulatory elements for these factors in the promoter regions of the downstream structural genes. Indeed, a mutant tobacco with low nicotine content was found to have a large chromosomal deletion in a cluster of closely related ERF genes at the nicotine-controlling NICOTINE2 (NIC2) locus.

  3. Validation of an LDH Assay for Assessing Nanoparticle Toxicity

    PubMed Central

    Han, Xianglu; Gelein, Robert; Corson, Nancy; Wade-Mercer, Pamela; Jiang, Jingkun; Biswas, Pratim; Finkelstein, Jacob N.; Elder, Alison; Oberdörster, Günter

    2014-01-01

    Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40 nm), silver (Ag-35, 35 nm; Ag-40, 40 nm), and titanium dioxide (TiO2-25, 25 nm) NPs by examining their potential to inactivate LDH and interference with β-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO2-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case. PMID:21722700

  4. Engineered yeast with a CO2-fixation pathway to improve the bio-ethanol production from xylose-mixed sugars.

    PubMed

    Li, Yun-Jie; Wang, Miao-Miao; Chen, Ya-Wei; Wang, Meng; Fan, Li-Hai; Tan, Tian-Wei

    2017-03-06

    Bio-ethanol production from lignocellulosic raw materials could serve as a sustainable potential for improving the supply of liquid fuels in face of the food-to-fuel competition and the growing energy demand. Xylose is the second abundant sugar of lignocelluloses hydrolysates, but its commercial-scale conversion to ethanol by fermentation is challenged by incomplete and inefficient utilization of xylose. Here, we use a coupled strategy of simultaneous maltose utilization and in-situ carbon dioxide (CO 2 ) fixation to achieve efficient xylose fermentation by the engineered Saccharomyces cerevisiae. Our results showed that the introduction of CO 2 as electron acceptor for nicotinamide adenine dinucleotide (NADH) oxidation increased the total ethanol productivity and yield at the expense of simultaneous maltose and xylose utilization. Our achievements present an innovative strategy using CO 2 to drive and redistribute the central pathways of xylose to desirable products and demonstrate a possible breakthrough in product yield of sugars.

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

    PubMed

    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.

  6. Growth of Pseudomonas C on C1 Compounds: Continuous Culture

    PubMed Central

    Battat, E.; Goldberg, I.; Mateles, R. I.

    1974-01-01

    Pseudomonas C was grown in continuous culture on methanol, formaldehyde, or formate as sole carbon source. On methanol μmax = 0.49/h and yield constant (Y) = 0.54; on formaldehyde and on unsupplemented media, μmax was about 0.2/h and Y was 0.15, whereas addition of p-aminobenzoic acid, folic acid, serine, or glycine to the medium raised Y to about 0.26 to 0.29, and addition of p-aminobenzoic acid, folic acid, serine, nicotinamide adenine dinucleotide, and Tween 80 raised the yield to 0.35. On formate and on unsupplemented media, μmax = 0.2/h and Y = 0.02, whereas addition of 0.1 mM p-aminobenzoic acid increased μmax to about 0.47 and Y to about 0.23. At low cell concentrations or growth rates a beneficial effect of CO2 was observed. Formaldehyde or formate, when added together with methanol, were utilized simultaneously with the methanol. PMID:4375436

  7. Leopard Skin-Like Colonic Mucosa: A Novel Endoscopic Finding of Chronic Granulomatous Disease-Associated Colitis.

    PubMed

    Obayashi, Naho; Arai, Katsuhiro; Nakano, Natsuko; Mizukami, Tomoyuki; Kawai, Toshinao; Yamamoto, Shojiro; Shimizu, Hirotaka; Nunoi, Hiroyuki; Shimizu, Toshiaki; Tang, Julian; Onodera, Masafumi

    2016-01-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes are unable to eradicate pathogens because of a deficit of nicotinamide adenine dinucleotide phosphate oxidase. Among CGD patients, ∼ 30% to 50% develop severe gastrointestinal tract symptoms. Although characteristic histologic findings of CGD-associated colitis have been reported, information on endoscopic features remained vague. A total of 8 male patients with CGD (ages 2-23 years) from 2 Japanese institutions underwent colonoscopy for the evaluation of their fever, diarrhea, bloody stool, and abdominal pain. The endoscopic and histologic findings were retrospectively reviewed. The endoscopic findings of CGD-associated colitis appeared varied. Notably, brownish dots over a yellowish edematous mucosa were observed in 3 of the 8 patients. Prominent pigment-laden macrophages were noted histologically on the mucosa. Although nonspecific endoscopic findings of CGD-associated colitis have been reported before, our observation of brownish dots spread across a yellowish edematous mucosa, termed "leopard sign," could be a unique feature of this condition.

  8. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome.

    PubMed

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V; Eisenberg, Tobias; Megalou, Evgenia; Schroeder, Sabrina; Cabrera, Sandra; Bénit, Paule; Rustin, Pierre; Criollo, Alfredo; Kepp, Oliver; Galluzzi, Lorenzo; Shen, Shensi; Malik, Shoaib Ahmad; Maiuri, Maria Chiara; Horio, Yoshiyuki; López-Otín, Carlos; Andersen, Jens S; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2011-02-21

    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

  9. Sample Handling and Chemical Kinetics in an Acoustically Levitated Drop Microreactor

    PubMed Central

    2009-01-01

    Accurate measurement of enzyme kinetics is an essential part of understanding the mechanisms of biochemical reactions. The typical means of studying such systems use stirred cuvettes, stopped-flow apparatus, microfluidic systems, or other small sample containers. These methods may prove to be problematic if reactants or products adsorb to or react with the container’s surface. As an alternative approach, we have developed an acoustically-levitated drop reactor eventually intended to study enzyme-catalyzed reaction kinetics related to free radical and oxidative stress chemistry. Microliter-scale droplet generation, reactant introduction, maintenance, and fluid removal are all important aspects in conducting reactions in a levitated drop. A three capillary bundle system has been developed to address these needs. We report kinetic measurements for both luminol chemiluminescence and the reaction of pyruvate with nicotinamide adenine dinucleotide, catalyzed by lactate dehydrogenase, to demonstrate the feasibility of using a levitated drop in conjunction with the developed capillary sample handling system as a microreactor. PMID:19769373

  10. SIRT2 and lysine fatty acylation regulate the transforming activity of K-Ras4a

    PubMed Central

    Wisner, Stephanie A; Chen, Xiao; Spiegelman, Nicole A; Linder, Maurine E

    2017-01-01

    Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation. PMID:29239724

  11. Quantification of ethanol in plasma by electrochemical detection with an unmodified screen printed carbon electrode

    NASA Astrophysics Data System (ADS)

    Tian, Gang; Zhang, Xiao-Qing; Zhu, Ming-Song; Zhang, Zhong; Shi, Zheng-Hu; Ding, Min

    2016-03-01

    Simple, rapid and accurate detection of ethanol concentration in blood is very crucial in the diagnosis and management of potential acute ethanol intoxication patients. A novel electrochemical detection method was developed for the quantification of ethanol in human plasma with disposable unmodified screen-printed carbon electrode (SPCE) without sample preparation procedure. Ethanol was detected indirectly by the reaction product of ethanol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD+). Method validation indicated good quantitation precisions with intra-day and inter-day relative standard deviations of ≤9.4% and 8.0%, respectively. Ethanol concentration in plasma is linear ranging from 0.10 to 3.20 mg/mL, and the detection limit is 40.0 μg/mL (S/N > 3). The method shows satisfactory correlation with the reference method of headspace gas chromatography in twenty human plasma samples (correlation coefficient 0.9311). The proposed method could be applied to diagnose acute ethanol toxicity or ethanol-related death.

  12. NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

    PubMed Central

    Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

  13. Fluorescence of bioaerosols: mathematical model including primary fluorescing and absorbing molecules in bacteria.

    PubMed

    Hill, Steven C; Pan, Yong-Le; Williamson, Chatt; Santarpia, Joshua L; Hill, Hanna H

    2013-09-23

    This paper describes a mathematical model of fluorescent biological particles composed of bacteria, viruses, or proteins. The fluorescent and/or light absorbing molecules included in the model are amino acids (tryptophan, etc.); nucleic acids (DNA, RNA, etc.); coenzymes (nicotinamide adenine dinucleotides, flavins, and vitamins B₆ and K and variants of these); and dipicolinates. The concentrations, absorptivities, and fluorescence quantum yields are estimated from the literature, often with large uncertainties. The bioparticles in the model are spherical and homogeneous. Calculated fluorescence cross sections for particles excited at 266, 280, and 355 nm are compared with measured values from the literature for several bacteria, bacterial spores and albumins. The calculated 266- and 280-nm excited fluorescence is within a factor of 3.2 of the measurements for the vegetative cells and proteins, but overestimates the fluorescence of spores by a factor of 10 or more. This is the first reported modeling of the fluorescence of bioaerosols in which the primary fluorophores and absorbing molecules are included.

  14. Hydrogen isotope fractionation during lipid biosynthesis by Haloarcula marismortui

    NASA Astrophysics Data System (ADS)

    Dirghangi, Sitindra S.; Pagani, Mark

    2013-10-01

    We studied the controls on the fractionation of hydrogen isotopes during lipid biosynthesis by Haloarcula marismortui, a halophilic archaea, in pure culture experiments by varying organic substrate, the hydrogen isotope composition (D/H) of water, temperature, and salinity. Cultures were grown on three substrates: succinate, pyruvate and glycerol with known hydrogen isotope compositions, and in water with different hydrogen isotopic compositions. All culture series grown on a particular substrate show strong correlations between δDarchaeol and δDwater. However, correlations are distinctly different for cultures grown on different substrates. Our results indicate that the metabolic pathway of substrate exerts a fundamental influence on the δD value of lipids, likely by influencing the D/H composition of NADPH (nicotinamide adenine dinucleotide phosphate), the reducing agent that contributes hydrogen to carbon atoms during lipid biosynthesis. Temperature and salinity have smaller, but similar effects on δDlipid, primarily due to the way temperature and salinity influence growth rate, as well as temperature effects on the activity of enzymes.

  15. Quantification of ethanol in plasma by electrochemical detection with an unmodified screen printed carbon electrode

    PubMed Central

    Tian, Gang; Zhang, Xiao-Qing; Zhu, Ming-Song; Zhang, Zhong; Shi, Zheng-Hu; Ding, Min

    2016-01-01

    Simple, rapid and accurate detection of ethanol concentration in blood is very crucial in the diagnosis and management of potential acute ethanol intoxication patients. A novel electrochemical detection method was developed for the quantification of ethanol in human plasma with disposable unmodified screen-printed carbon electrode (SPCE) without sample preparation procedure. Ethanol was detected indirectly by the reaction product of ethanol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD+). Method validation indicated good quantitation precisions with intra-day and inter-day relative standard deviations of ≤9.4% and 8.0%, respectively. Ethanol concentration in plasma is linear ranging from 0.10 to 3.20 mg/mL, and the detection limit is 40.0 μg/mL (S/N > 3). The method shows satisfactory correlation with the reference method of headspace gas chromatography in twenty human plasma samples (correlation coefficient 0.9311). The proposed method could be applied to diagnose acute ethanol toxicity or ethanol-related death. PMID:27006081

  16. Towards two-photon excited endogenous fluorescence lifetime imaging microendoscopy

    PubMed Central

    Hage, C. H.; Leclerc, P.; Brevier, J.; Fabert, M.; Le Nézet, C.; Kudlinski, A.; Héliot, L.; Louradour, F.

    2017-01-01

    In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs). Fluorescence photons are collected by the fiber inner cladding and we show that its contribution to the impulse response function (IRF), which originates from its intermodal and chromatic dispersions, is small (< 600 ps) and stable for lengths up to 8 m and allows for short lifetime measurements. We use the phasor representation as a quick visualization tool adapted to the endoscopy speed requirements. PMID:29359093

  17. Uric acid and transforming growth factor in fructose-induced production of reactive oxygen species in skeletal muscle.

    PubMed

    Madlala, Hlengiwe P; Maarman, Gerald J; Ojuka, Edward

    2016-04-01

    The consumption of fructose, a major constituent of the modern diet, has raised increasing concern about the effects of fructose on health. Research suggests that excessive intake of fructose (>50 g/d) causes hyperuricemia, insulin resistance, mitochondrial dysfunction, de novo lipogenesis by the liver, and increased production of reactive oxygen species (ROS) in muscle. In a number of tissues, uric acid has been shown to stimulate the production of ROS via activation of transforming growth factor β1 and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4. The role of uric acid in fructose-induced production of ROS in skeletal muscle, however, has not been investigated. This review examines the evidence for fructose-induced production of ROS in skeletal muscle, highlights proposed mechanisms, and identifies gaps in current knowledge. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

    PubMed

    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.

  19. Juvenile Leigh syndrome, optic atrophy, ataxia, dystonia, and epilepsy due to T14487C mutation in the mtDNA-ND6 gene: a mitochondrial syndrome presenting from birth to adolescence.

    PubMed

    Leshinsky-Silver, Esther; Shuvalov, Ruslan; Inbar, Shani; Cohen, Sarit; Lev, Dorit; Lerman-Sagie, Tally

    2011-04-01

    An increasing number of reports describe mutations in mitochondrial DNA coding regions, especially in mitochondrial DNA- encoded nicotinamide adenine dinucleotide dehydrogenase subunit genes of the respiratory chain complex I, as causing early-onset Leigh syndrome. The authors report the molecular findings in a 24-year-old patient with juvenile-onset Leigh syndrome presenting with optic atrophy, ataxia dystonia, and epilepsy. A brain magnetic resonance imaging revealed bilateral basal ganglia and thalamic hypointensities, and a magnetic resonance spectroscopy revealed an increased lactate peak. The authors identified a T14487C change causing M63V substitution in the mitochondrial ND6 gene. The mutation was heteroplasmic in muscle and blood samples, with different mutation loads, and was absent in the patient's mother's urine and blood samples. They suggest that the T14487C mtDNA mutation should be analyzed in Leigh syndrome, presenting with optic atrophy, ataxia, dystonia, and epilepsy, regardless of age.

  20. Dinucleotide controlled null models for comparative RNA gene prediction.

    PubMed

    Gesell, Tanja; Washietl, Stefan

    2008-05-27

    Comparative prediction of RNA structures can be used to identify functional noncoding RNAs in genomic screens. It was shown recently by Babak et al. [BMC Bioinformatics. 8:33] that RNA gene prediction programs can be biased by the genomic dinucleotide content, in particular those programs using a thermodynamic folding model including stacking energies. As a consequence, there is need for dinucleotide-preserving control strategies to assess the significance of such predictions. While there have been randomization algorithms for single sequences for many years, the problem has remained challenging for multiple alignments and there is currently no algorithm available. We present a program called SISSIz that simulates multiple alignments of a given average dinucleotide content. Meeting additional requirements of an accurate null model, the randomized alignments are on average of the same sequence diversity and preserve local conservation and gap patterns. We make use of a phylogenetic substitution model that includes overlapping dependencies and site-specific rates. Using fast heuristics and a distance based approach, a tree is estimated under this model which is used to guide the simulations. The new algorithm is tested on vertebrate genomic alignments and the effect on RNA structure predictions is studied. In addition, we directly combined the new null model with the RNAalifold consensus folding algorithm giving a new variant of a thermodynamic structure based RNA gene finding program that is not biased by the dinucleotide content. SISSIz implements an efficient algorithm to randomize multiple alignments preserving dinucleotide content. It can be used to get more accurate estimates of false positive rates of existing programs, to produce negative controls for the training of machine learning based programs, or as standalone RNA gene finding program. Other applications in comparative genomics that require randomization of multiple alignments can be considered. SISSIz

  1. The Copper-nicotinamide complex: sustainable applications in coupling and cycloaddition reactions

    EPA Science Inventory

    Crystalline copper (II)-nicotinamide complex, synthesized via simple mixing of copper chloride and nicotinamide solution at room temperature, catalyzes the C-S, C-N bond forming and cycloaddition reactions under a variety of sustainable reaction conditions.

  2. Hepatic NAD(+) deficiency as a therapeutic target for non-alcoholic fatty liver disease in ageing.

    PubMed

    Zhou, Can-Can; Yang, Xi; Hua, Xia; Liu, Jian; Fan, Mao-Bing; Li, Guo-Qiang; Song, Jie; Xu, Tian-Ying; Li, Zhi-Yong; Guan, Yun-Feng; Wang, Pei; Miao, Chao-Yu

    2016-08-01

    Ageing is an important risk factor of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD(+) ), a ubiquitous coenzyme, links ageing with NAFLD. Hepatic concentrations of NAD(+) , protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD(+) biosynthesis, were compared in middle-aged and aged mice or patients. The influences of NAD(+) decline on the steatosis and steatohepatitis were evaluated in wild-type and H247A dominant-negative, enzymically-inactive NAMPT transgenic mice (DN-NAMPT) given normal or high-fat diet (HFD). Hepatic NAD(+) level decreased in aged mice and humans. NAMPT-controlled NAD(+) salvage, but not de novo biosynthesis pathway, was compromised in liver of elderly mice and humans. Given normal chow, middle-age DN-NAMPT mice displayed systemic NAD(+) reduction and had moderate NAFLD phenotypes, including lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver. All these NAFLD phenotypes, especially release of pro-inflammatory factors, Kupffer cell accumulation, monocytes infiltration, NLRP3 inflammasome pathway and hepatic fibrosis (Masson's staining and α-SMA staining), deteriorated further under HFD challenge. Oral administration of nicotinamide riboside, a natural NAD(+) precursor, completely corrected these NAFLD phenotypes induced by NAD(+) deficiency alone or HFD, whereas adenovirus-mediated SIRT1 overexpression only partially rescued these phenotypes. These results provide the first evidence that ageing-associated NAD(+) deficiency is a critical risk factor for NAFLD, and suggest that supplementation with NAD(+) substrates may be a promising therapeutic strategy to prevent and treat NAFLD. © 2016 The British Pharmacological Society.

  3. Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector.

    PubMed

    Urban, Pawel L; Goodall, David M; Bergström, Edmund T; Bruce, Neil C

    2007-08-31

    An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.

  4. Renal haemodynamics and natriuretic responses to intravenous administration of diadenosine tetraphosphate (Ap4A) and nicotinamide adenine dinucleotide (NAD) in rat.

    PubMed

    Szczepańska-Konkel, M; Langner, G; Bednarczuk, G; Stiepanow-Trzeciak, A; Jankowski, M; Angielski, S

    2003-06-01

    Effects of Ap4A and NAD--precursor of adenosine, on renal plasma flow (RPF), glomerular filtration rate (GFR) and urine excretion were determined in the anaesthetised rats. Infusion of Ap4A or NAD (i.v., bolus--1 micromol/kg followed by 10 nmol/min/kg) decreased RPF and GFR (by 30 and 40%, respectively). In spite of GFR reduction during Ap4A infusion, the significant increase in sodium excretion and urine flow was noticed: fractional sodium (FENa) and urine excretion (FEurine) rose 15-fold and 2.5-fold in comparison with the control value, respectively. In contrast to Ap4A, NAD-induced decrease in GFR was associated with parallel decrease in sodium and urine excretion, thus the FENa and FEurine did not significantly change. Pretreatment with adenosine deaminase (adenosine degrading enzyme, 2 U/min/kg) or theophylline (P1-receptors antagonist, 0.2 mmol/min/kg) ceased responses to NAD, whereas Ap4A-induced changes were not affected. Pre-treatment with suramin (P2-receptors antagonist, (i.v., bolus--12 mg/kg followed by 1.2 mg/min/kg) completely abolished the renal effects of Ap4A. We conclude that Ap4A may exert specific action on renal function. It acts different from NAD that modified renal function through its hydrolysis product--adenosine. Ap4A might reduce glomerular filtration rate and evoke natriuresis and diuresis, and its effects are probably mediated through stimulation of P2-receptors.

  5. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    PubMed

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

    PubMed

    Pustovit, K B; Kuz'min, V S; Sukhova, G S

    2015-06-01

    In rat sinoatrial node, NAD(+) (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

  7. Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable.

    PubMed

    Xiao, Yang; Kwong, Mandy; Daemen, Anneleen; Belvin, Marcia; Liang, Xiaorong; Hatzivassiliou, Georgia; O'Brien, Thomas

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.

  8. A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif.

    PubMed

    Kandimalla, Ekambar R; Bhagat, Lakshmi; Zhu, Fu-Gang; Yu, Dong; Cong, Yan-Ping; Wang, Daqing; Tang, Jimmy X; Tang, Jin-Yan; Knetter, Cathrine F; Lien, Egil; Agrawal, Sudhir

    2003-11-25

    Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-kappa B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-gamma, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

  9. Aging and stem cell therapy: AMPK as an applicable pharmacological target for rejuvenation of aged stem cells and achieving higher efficacy in stem cell therapy.

    PubMed

    Khorraminejad-Shirazi, Mohammadhossein; Farahmandnia, Mohammad; Kardeh, Bahareh; Estedlal, Alireza; Kardeh, Sina; Monabati, Ahmad

    2017-10-19

    In recent years, tissue regeneration has become a promising field for developing stem cell-based transplantation therapies for human patients. Adult stem cells are affected by the same aging mechanisms that involve somatic cells. One of the mechanisms involved in cellular aging is hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and disruption of 5' adenosine monophosphate-activated protein kinase (AMPK). Aging of stem cells results in their impaired regenerative capacity and depletion of stem cell pools in adult tissue, which results in lower efficacy of stem cell therapy. By utilizing an effective therapeutic intervention for aged stem cells, stem cell therapy can become more promising for future application. mTORC1 inhibition is a practical approach to preserve the stem cell pool. In this article, we review the dynamic interaction between sirtuin (silent mating type information regulation 2 homolog) 1, AMPK, and mTORC1. We propose that using AMPK activators such as 5-aminoimidazole-4-carboxamide ribonucleotide, A769662, metformin, and oxidized nicotinamide adenine dinucleotide (NAD + ) are practical ways to be employed for achieving better optimized results in stem cell-based transplantation therapies. Copyright © 2017 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

  10. RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

    PubMed

    Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

    2012-08-01

    A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

  11. Effect of CO2 on NADH production of denitrifying microbes via inhibiting carbon source transport and its metabolism.

    PubMed

    Wan, Rui; Chen, Yinguang; Zheng, Xiong; Su, Yinglong; Huang, Haining

    2018-06-15

    The potential effect of CO 2 on environmental microbes has drawn much attention recently. As an important section of the nitrogen cycle, biological denitrification requires electron donor to reduce nitrogen oxide. Nicotinamide adenine dinucleotide (NADH), which is formed during carbon source metabolism, is a widely reported electron donor for denitrification. Here we studied the effect of CO 2 on NADH production and carbon source utilization in the denitrifying microbe Paracoccus denitrificans. We observed that NADH level was decreased by 45.5% with the increase of CO 2 concentration from 0 to 30,000ppm, which was attributed to the significantly decreased utilization of carbon source (i.e., acetate). Further study showed that CO 2 inhibited carbon source utilization because of multiple negative influences: (1) suppressing the growth and viability of denitrifier cells, (2) weakening the driving force for carbon source transport by decreasing bacterial membrane potential, and (3) downregulating the expression of genes encoding key enzymes involved in intracellular carbon metabolism, such as citrate synthase, aconitate hydratase, isocitrate dehydrogenase, succinate dehydrogenase, and fumarate reductase. This study suggests that the inhibitory effect of CO 2 on NADH production in denitrifiers might deteriorate the denitrification performance in an elevated CO 2 climate scenario. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia

    PubMed Central

    Laidlaw, Kamilla M.E.; Berhan, Samuel; Liu, Suhu; Silvestri, Giovannino; Holyoake, Tessa L.; Frank, David A.; Aggarwal, Bharat; Bonner, Michael Y.; Perrotti, Danilo

    2016-01-01

    The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation. PMID:27438151

  13. [The relationship between oxidized form glutathione, coenzyme II and carotid artery atherosclerosis].

    PubMed

    Huang, Yan-sheng; Wang, Shu-ren; Zhi, Yan-fang; Xu, Bo-shi; Sun, Lin; Wu, Yu; Lu, Jian-min; Dai, Fu-min

    2006-06-01

    To explore the relationship between plasma redox status and atherosclerosis. IVUS was performed in common carotid in the neck of 167 patients with heart diseases. Patients were divided into three groups: plaque-forming group (A, n = 79), intima-thickening group (B, n = 52) and control group (C, n = 36). Plasma glutathione (reduced form GSH and oxidized form GSSG), nicotinamide adenine dinucleotide phosphate (reduced form NADPH and oxidized form NADP(+)), oxidized low density lipoprotein (ox-LDL) and malondialdehyde (MDA) were measured in all patients. The GSH/GSSG and NADPH/NADP(+) redox potential were calculated according to Nernst equation, and correlation analysis performed. GSH and GSH/GSSG gradually reduced and GSH/GSSG redox potential gradually increased in proportion to the thickening of artery intima (from Group C to Group A, P < 0.05). Similar but milder results were shown for NADPH and NADPH/NADP(+) redox status. The products of oxidative stress ox-LDL and MDA also increased significantly (P < 0.05) in proportion to the thickening of artery intima. GSH/GSSG redox potential is positively correlated to ox-LDL (P < 0.05). The redox status shifted to oxidizing direction in proportion to the intima thickness. The imbalance of plasma redox status deviating to oxidation might be implicated in oxidized injury of lipid, intima thickening and atherosclerosis progress.

  14. 15-Deoxy-∆12,14-PGJ 2, by activating peroxisome proliferator-activated receptor-gamma, suppresses p22phox transcription to protect brain endothelial cells against hypoxia-induced apoptosis.

    PubMed

    Wu, Jui-Sheng; Tsai, Hsin-Da; Huang, Chien-Yu; Chen, Jin-Jer; Lin, Teng-Nan

    2014-08-01

    15-Deoxy-∆(12,14)-PGJ(2) (15d-PGJ(2)) and thiazolidinedione attenuate reactive oxygen species (ROS) production via a peroxisome proliferator-activated receptor-gamma (PPAR-γ)-dependent pathway. Nonetheless, how PPAR-γ mediates ROS production to ameliorate ischemic brain injury is not clear. Recent studies indicated that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is the major source of ROS in the vascular system. In the present study, we used an in vitro oxygen-glucose deprivation and reoxygenation (hypoxia reoxygenation [HR]) paradigm to study whether PPAR-γ interacts with NADPH oxidase, thereby regulating ROS formation in cerebral endothelial cells (CECs). With pharmacological (PPAR-γ antagonist GW9662), loss-of-function (PPAR-γ siRNA), and gain-of-function (Ad-PPAR-γ) approaches, we first demonstrated that 15d-PGJ(2) protected HR-treated CECs against ROS-induced apoptosis in a PPAR-γ-dependent manner. Results of promoter and subcellular localization analyses further revealed that 15d-PGJ(2), by activating PPAR-γ, blocked HR-induced NF-κB nuclear translocation, which led to inhibited transcription of the NADPH oxidase subunit p22phox. In summary, we report a novel transrepression mechanism whereby PPAR-γ downregulates hypoxia-activated p22phox transcription and the subsequent NADPH oxidase activation, ROS formation, and CEC apoptosis.

  15. Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures.

    PubMed

    Upadhyay, Mohita; Vivekanandan, Perumal

    2015-01-01

    Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses. We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses. All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses. The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our

  16. Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs After Exposure to Hyperoxia

    PubMed Central

    Audi, Said H.; Staniszewski, Kevin S.; Haworth, Steven T.; Jacobs, Elizabeth R.; Ranji, Mahsa; Zablocki, Clement J.

    2013-01-01

    Recently, we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and oxidized form of flavin adenine dinucleotide \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$({\\rm FADH}_{2})$\\end{document} (FAD), as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this paper was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${>}{95\\%}~{\\rm O}_{2}$\\end{document} for 48 h) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, when compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change

  17. Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling

    PubMed Central

    Förstermann, Ulrich; Li, Huige

    2011-01-01

    Nitric oxide (NO) produced by the endothelium is an important protective molecule in the vasculature. It is generated by the enzyme endothelial NO synthase (eNOS). Similar to all NOS isoforms, functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH), via the flavins flavin adenine dinucleotide and flavin mononucleotide in the carboxy-terminal reductase domain, to the heme in the amino-terminal oxygenase domain. Here, the substrate L-arginine is oxidized to L-citrulline and NO. Cardiovascular risk factors such as diabetes mellitus, hypertension, hypercholesterolaemia or cigarette smoking reduce bioactive NO. These risk factors lead to an enhanced production of reactive oxygen species (ROS) in the vessel wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in the presence of cardiovascular risk factors. NADPH-oxidase-derived superoxide avidly reacts with eNOS-derived NO to form peroxynitrite (ONOO-). The essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH4) is highly sensitive to oxidation by this ONOO-. In BH4 deficiency, oxygen reduction uncouples from NO synthesis, thereby converting NOS to a superoxide-producing enzyme. Among conventional drugs, compounds interfering with the renin-angiotensin-aldosterone system and statins can reduce vascular oxidative stress and increase bioactive NO. In recent years, we have identified a number of small molecules that have the potential to prevent eNOS uncoupling and, at the same time, enhance eNOS expression. These include the protein kinase C inhibitor midostaurin, the pentacyclic triterpenoids ursolic acid and betulinic acid, the eNOS enhancing compounds AVE9488 and AVE3085, and the polyphenolic phytoalexin trans-resveratrol. Such compounds enhance NO production from eNOS also under pathophysiological conditions and may thus have therapeutic potential. PMID:21198553

  18. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  19. Optical biopsy - a new armamentarium to detect disease using light

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Alfano, Robert R.

    2015-03-01

    Optical spectroscopy has been considered a promising method for cancer detection for past thirty years because of its advantages over the conventional diagnostic methods of no tissue removal, minimal invasiveness, rapid diagnoses, less time consumption and reproducibility since the first use in 1984. It offers a new armamentarium. Human tissue is mainly composed of extracellular matrix of collagen fiber, proteins, fat, water, and epithelial cells with key molecules in different structures. Tissues contain a number of key fingerprint native endogenous fluorophore molecules, such as tryptophan, collagen, elastin, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and porphyrins. It is well known that abnormalities in metabolic activity precede the onset of a lot of main diseases: carcinoma, diabetes mellitus, atherosclerosis, Alzheimer, and Parkinson's disease, etc. Optical spectroscopy may help in detecting various disorders. Conceivably the biochemical or morphologic changes that cause the spectra variations would appear earlier than the histological aberration. Therefore, "optical biopsy" holds a great promise as clinical tool for diagnosing early stage of carcinomas and other deceases by combining with available photonic technology (e.g. optical fibers, photon detectors, spectrographs spectroscopic ratiometer, fiber-optic endomicroscope and nasopharyngoscope) for in vivo use. This paper focuses on various methods available to detect spectroscopic changes in tissues, for example to distinguish cancerous prostate tissues and/or cells from normal prostate tissues and/or cells. The methods to be described are fluorescence, stokes shift, scattering, Raman, and time-resolved spectroscopy will be reviewed. The underlying physical and biological basis for these optical approaches will be discussed with examples. The idea is to present some of the salient works to show the usefulness and methods of Optical Biopsy for cancer detection and

  20. Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders.

    PubMed

    Sakurai, Takeshi; Ramoz, Nicolas; Barreto, Marta; Gazdoiu, Mihaela; Takahashi, Nagahide; Gertner, Michael; Dorr, Nathan; Gama Sosa, Miguel A; De Gasperi, Rita; Perez, Gissel; Schmeidler, James; Mitropoulou, Vivian; Le, H Carl; Lupu, Mihaela; Hof, Patrick R; Elder, Gregory A; Buxbaum, Joseph D

    2010-05-01

    SLC25A12, a susceptibility gene for autism spectrum disorders that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate-glutamate carrier (aspartate-glutamate carrier isoform 1 [AGC1]). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate production. We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies. Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In postnatal day 13 to 14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cell-autonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetylaspartate and/or alterations in the dihydronicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide(+) ratio, resulting in myelin defects. Our data implicate AGC1 activity in myelination and in neuronal structure and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development, contributing to increased autism susceptibility. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.