Kinetics and structural features of dimeric glutamine-dependent bacterial NAD+ synthetases suggest evolutionary adaptation to available metabolites.

PubMed

Santos, Adrian Richard Schenberger; Gerhardt, Edileusa Cristina Marques; Moure, Vivian Rotuno; Pedrosa, Fábio Oliveira; Souza, Emanuel Maltempi; Diamanti, Riccardo; Högbom, Martin; Huergo, Luciano Fernandes

2018-05-11

NADH (NAD + ) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD + also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD + biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD + synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE NH3 and octameric glutamine-dependent NadE Gln , and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE Gln in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE Gln enzymes may constitute evolutionary intermediates between dimeric NadE NH3 and octameric NadE Gln The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes. © 2018 Santos et al.

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

PubMed

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus.

PubMed

Hektor, Harm J; Kloosterman, Harm; Dijkhuizen, Lubbert

2002-12-06

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.

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.

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

PubMed

Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

2014-11-01

A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. Copyright © 2014 Elsevier B.V. All rights reserved.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

PubMed

Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

2015-08-21

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

PubMed Central

Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

2015-01-01

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes*

PubMed Central

Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.; Jensen, Pernille Rose; Lerche, Mathilde H.

2014-01-01

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD+]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments. PMID:24302737

NAD Acts as an Integral Regulator of Multiple Defense Layers1[OPEN

PubMed Central

Patrit, Oriane; Tcherkez, Guillaume; Gakière, Bertrand

2016-01-01

Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea. Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens. PMID:27621425

Dynamics of NAD-metabolism: everything but constant.

PubMed

Opitz, Christiane A; Heiland, Ines

2015-12-01

NAD, as well as its phosphorylated form, NADP, are best known as electron carriers and co-substrates of various redox reactions. As such they participate in approximately one quarter of all reactions listed in the reaction database KEGG. In metabolic pathway analysis, the total amount of NAD is usually assumed to be constant. That means that changes in the redox state might be considered, but concentration changes of the NAD moiety are usually neglected. However, a growing number of NAD-consuming reactions have been identified, showing that this assumption does not hold true in general. NAD-consuming reactions are common characteristics of NAD(+)-dependent signalling pathways and include mono- and poly-ADP-ribosylation of proteins, NAD(+)-dependent deacetylation by sirtuins and the formation of messenger molecules such as cyclic ADP-ribose (cADPR) and nicotinic acid (NA)-ADP (NAADP). NAD-consuming reactions are thus involved in major signalling and gene regulation pathways such as DNA-repair or regulation of enzymes central in metabolism. All known NAD(+)-dependent signalling processes include the release of nicotinamide (Nam). Thus cellular NAD pools need to be constantly replenished, mostly by recycling Nam to NAD(+). This process is, among others, regulated by the circadian clock, causing complex dynamic changes in NAD concentration. As disturbances in NAD homoeostasis are associated with a large number of diseases ranging from cancer to diabetes, it is important to better understand the dynamics of NAD metabolism to develop efficient pharmacological invention strategies to target this pathway. © 2015 Authors; published by Portland Press Limited.

NAD Acts as an Integral Regulator of Multiple Defense Layers.

PubMed

Pétriacq, Pierre; Ton, Jurriaan; Patrit, Oriane; Tcherkez, Guillaume; Gakière, Bertrand

2016-11-01

Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens. © 2016 American Society of Plant Biologists. All Rights Reserved.

Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

PubMed Central

Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

2014-01-01

ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

Glucose consumption rate critically depends on redox state in Corynebacterium glutamicum under oxygen deprivation.

PubMed

Tsuge, Yota; Uematsu, Kimio; Yamamoto, Shogo; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

2015-07-01

Rapid sugar consumption is important for the microbial production of chemicals and fuels. Here, we show that overexpression of the NADH dehydrogenase gene (ndh) increased glucose consumption rate in Corynebacterium glutamicum under oxygen-deprived conditions through investigating the relationship between the glucose consumption rate and intracellular NADH/NAD(+) ratio in various mutant strains. The NADH/NAD(+) ratio was strongly repressed under oxygen deprivation when glucose consumption was accelerated by the addition of pyruvate or sodium hydrogen carbonate. Overexpression of the ndh gene in the wild-type strain under oxygen deprivation decreased the NADH/NAD(+) ratio from 0.32 to 0.13, whereas the glucose consumption rate increased by 27%. Similarly, in phosphoenolpyruvate carboxylase gene (ppc)- or malate dehydrogenase gene (mdh)-deficient strains, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.66 to 0.37 and 2.20 to 0.57, respectively, whereas the glucose consumption rate increased by 57 and 330%, respectively. However, in a lactate dehydrogenase gene (L-ldhA)-deficient strain, although the NADH/NAD(+) ratio decreased from 5.62 to 1.13, the glucose consumption rate was not markedly altered. In a tailored D-lactate-producing strain, which lacked ppc and L-ldhA genes, but expressed D-ldhA from Lactobacillus delbrueckii, overexpression of the ndh gene decreased the NADH/NAD(+) ratio from 1.77 to 0.56, and increased the glucose consumption rate by 50%. Overall, the glucose consumption rate was found to be inversely proportional to the NADH/NAD(+) ratio in C. glutamicum cultured under oxygen deprivation. These findings could provide an option to increase the productivity of chemicals and fuels under oxygen deprivation.

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

PubMed Central

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

2016-01-01

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

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

Effects of temperature changes on maize production in Mozambique

USGS Publications Warehouse

Harrison, L.; Michaelsen, J.; Funk, Chris; Husak, G.

2011-01-01

We examined intraseasonal changes in maize phenology and heat stress exposure over the 1979-2008 period, using Mozambique meteorological station data and maize growth requirements in a growing degree-day model. Identifying historical effects of warming on maize growth is particularly important in Mozambique because national food security is highly dependent on domestic food production, most of which is grown in already warm to hot environments. Warming temperatures speed plant development, shortening the length of growth periods necessary for optimum plant and grain size. This faster phenological development also alters the timing of maximum plant water demand. In hot growing environments, temperature increases during maize pollination threaten to make midseason crop failure the norm. In addition to creating a harsher thermal environment, we find that early season temperature increases have caused the maize reproductive period to start earlier, increasing the risk of heat and water stress. Declines in time to maize maturation suggest that, independent of effects to water availability, yield potential is becoming increasingly limited by warming itself. Regional variations in effects are a function of the timing and magnitude of temperature increases and growing season characteristics. Continuation of current climatic trends could induce substantial yield losses in some locations. Farmers could avoid some losses through simple changes to planting dates and maize varietal types.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

NASA Astrophysics Data System (ADS)

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-05-01

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

PubMed Central

Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

2015-01-01

Abstract. Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs. PMID:25688541

Isonicotinamide Enhances Sir2 Protein-mediated Silencing and Longevity in Yeast by Raising Intracellular NAD+ Concentration*

PubMed Central

McClure, Julie M.; Wierman, Margaret B.; Maqani, Nazif; Smith, Jeffrey S.

2012-01-01

Sirtuins are an evolutionarily conserved family of NAD+-dependent protein deacetylases that function in the regulation of gene transcription, cellular metabolism, and aging. Their activity requires the maintenance of an adequate intracellular NAD+ concentration through the combined action of NAD+ biosynthesis and salvage pathways. Nicotinamide (NAM) is a key NAD+ precursor that is also a byproduct and feedback inhibitor of the deacetylation reaction. In Saccharomyces cerevisiae, the nicotinamidase Pnc1 converts NAM to nicotinic acid (NA), which is then used as a substrate by the NAD+ salvage pathway enzyme NA phosphoribosyltransferase (Npt1). Isonicotinamide (INAM) is an isostere of NAM that stimulates yeast Sir2 deacetylase activity in vitro by alleviating the NAM inhibition. In this study, we determined that INAM stimulates Sir2 through an additional mechanism in vivo, which involves elevation of the intracellular NAD+ concentration. INAM enhanced normal silencing at the rDNA locus but only partially suppressed the silencing defects of an npt1Δ mutant. Yeast cells grown in media lacking NA had a short replicative life span, which was extended by INAM in a SIR2-dependent manner and correlated with increased NAD+. The INAM-induced increase in NAD+ was strongly dependent on Pnc1 and Npt1, suggesting that INAM increases flux through the NAD+ salvage pathway. Part of this effect was mediated by the NR salvage pathways, which generate NAM as a product and require Pnc1 to produce NAD+. We also provide evidence suggesting that INAM influences the expression of multiple NAD+ biosynthesis and salvage pathways to promote homeostasis during stationary phase. PMID:22539348

Expression of the human NAD(P)-metabolizing ectoenzyme CD38 compromises systemic acquired resistance in Arabidopsis.

PubMed

Zhang, Xudong; Mou, Zhonglin

2012-09-01

Plant systemic acquired resistance (SAR) is a long-lasting, broad-spectrum immune response that is mounted after primary pathogen infection. Although SAR has been extensively researched, the molecular mechanisms underlying its activation have not been completely understood. We have previously shown that the electron carrier NAD(P) leaks into the plant extracellular compartment upon pathogen attack and that exogenous NAD(P) activates defense gene expression and disease resistance in local treated leaves, suggesting that extracellular NAD(P) [eNAD(P)] might function as a signal molecule activating plant immune responses. To further establish the function of eNAD(P) in plant immunity, we tested the effect of exogenous NAD(P) on resistance gene-mediated hypersensitive response (HR) and SAR. We found that exogenous NAD(P) completely suppresses HR-mediated cell death but does not affect HR-mediated disease resistance. Local application of exogenous NAD(P) is unable to induce SAR in distal tissues, indicating that eNAD(P) is not a sufficient signal for SAR activation. Using transgenic Arabidopsis plants expressing the human NAD(P)-metabolizing ectoenzyme CD38, we demonstrated that altering eNAD(P) concentration or signaling compromises biological induction of SAR. This result suggests that eNAD(P) may play a critical signaling role in activation of SAR.

Thermotropism by primary roots of maize

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

Fortin, M.-C.; Poff, K.L.

1990-05-01

Sensing in the roots of higher plants has long been recognized to be restricted mainly to gravitropism and thigmotropism. However, root responses to temperature gradients have not been extensively studied. We have designed experiments under controlled conditions to test if and how root direction of maize can be altered by thermal gradients perpendicular to the gravity vector. Primary roots of maize grown on agar plates exhibit positive thermotropism (curvature toward the warmer temperature) when exposed to gradients of 0.5 to 4.2{degree}C cm{sup {minus}1}. The extent of thermotropism depends on the temperature gradient and the temperature at which the root ismore » placed within the gradient. The curvature cannot be accounted for by differential growth as a direct effect of temperature on each side of the root.« less

ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling.

PubMed

Weckwerth, Philipp; Ehlert, Britta; Romeis, Tina

2015-03-01

Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold stress response. Based upon in silico analysis of the Z. mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 was sufficient to mediate calcium independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize. © 2014 John Wiley & Sons Ltd.

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.

Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1.

PubMed

Wu, Tung-Yun; Chen, Chang-Ting; Liu, Jessica Tse-Jin; Bogorad, Igor W; Damoiseaux, Robert; Liao, James C

2016-06-01

Methanol utilization by methylotrophic or non-methylotrophic organisms is the first step toward methanol bioconversion to higher carbon-chain chemicals. Methanol oxidation using NAD-dependent methanol dehydrogenase (Mdh) is of particular interest because it uses NAD(+) as the electron carrier. To our knowledge, only a limited number of NAD-dependent Mdhs have been reported. The most studied is the Bacillus methanolicus Mdh, which exhibits low enzyme specificity to methanol and is dependent on an endogenous activator protein (ACT). In this work, we characterized and engineered a group III NAD-dependent alcohol dehydrogenase (Mdh2) from Cupriavidus necator N-1 (previously designated as Ralstonia eutropha). This enzyme is the first NAD-dependent Mdh characterized from a Gram-negative, mesophilic, non-methylotrophic organism with a significant activity towards methanol. Interestingly, unlike previously reported Mdhs, Mdh2 does not require activation by known activators such as B. methanolicus ACT and Escherichia coli Nudix hydrolase NudF, or putative native C. necator activators in the Nudix family under mesophilic conditions. This enzyme exhibited higher or comparable activity and affinity toward methanol relative to the B. methanolicus Mdh with or without ACT in a wide range of temperatures. Furthermore, using directed molecular evolution, we engineered a variant (CT4-1) of Mdh2 that showed a 6-fold higher K cat/K m for methanol and 10-fold lower K cat/K m for n-butanol. Thus, CT4-1 represents an NAD-dependent Mdh with much improved catalytic efficiency and specificity toward methanol compared with the existing NAD-dependent Mdhs with or without ACT activation.

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.

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.

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.

Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

PubMed Central

Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

2013-01-01

Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of endogenous vascular antioxidant defences. PMID:22817606

Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the 3D organisation of the A. thaliana genome

PubMed Central

Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Saez-Vasquez, Julio

2017-01-01

The nucleolus is the site of ribosomal RNA (rRNA) gene transcription, rRNA processing and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli by Fluorescence Activated Cell Sorting (FACS) and identified Nucleolus-Associated Chromatin Domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein, NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions and mostly inactive protein-coding genes. However, NADs also include active ribosomal RNA genes, and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. PMID:27477271

Plastidial NAD-Dependent Malate Dehydrogenase: A Moonlighting Protein Involved in Early Chloroplast Development Through its Interaction with an FtsH12-FtsHi Protease Complex.

PubMed

Schreier, Tina B; Antoine, Cléry; Schläfli, Michael; Galbier, Florian; Stadler, Martha; Demarsy, Emilie; Albertini, Daniele; Maier, Benjamin A; Kessler, Felix; Hörtensteiner, Stefan; Zeeman, Samuel C; Kötting, Oliver

2018-06-22

Malate dehydrogenases (MDH) convert malate to oxaloacetate using NAD(H) or NADP(H) as a cofactor. Arabidopsis thaliana mutants lacking plastidial NAD-dependent MDH (pdnad-mdh) are embryo-lethal, and constitutive silencing (miR-mdh-1) causes a pale, dwarfed phenotype. The reason for these severe phenotypes is unknown. Here, we rescued the embryo lethality of pdnad-mdh via embryo-specific expression of pdNAD-MDH. Rescued seedlings developed white leaves with aberrant chloroplasts and failed to reproduce. Inducible silencing of pdNAD-MDH at the rosette stage also resulted in white newly emerging leaves. These data suggest that pdNAD-MDH is important for early plastid development, which is consistent with the reductions in major plastidial galactolipid, carotenoid and protochlorophyllide levels in miR-mdh-1 seedlings. Surprisingly, the targeting of other NAD-dependent MDH isoforms to the plastid did not complement the embryo lethality of pdnad-mdh, while expression of enzymatically inactive pdNAD-MDH did. These complemented plants grew indistinguishably from the wild type. Both active and inactive forms of pdNAD-MDH interact with a heteromeric AAA-ATPase complex at the inner membrane of the chloroplast envelope. Silencing the expression of FtsH12, a key member of this complex, resulted in a phenotype that strongly resembles miR-mdh-1. We propose that pdNAD-MDH is essential for chloroplast development due to its moonlighting role in stabilizing FtsH12, distinct from its enzymatic function. © 2018 American Society of Plant Biologists. All rights reserved.

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha.

PubMed

Gertzberg, Nancy; Neumann, Paul; Rizzo, Victor; Johnson, Arnold

2004-01-01

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.

Adenosine mimetics as inhibitors of NAD+-dependent histone deacetylases, from kinase to sirtuin inhibition.

PubMed

Trapp, Johannes; Jochum, Anne; Meier, Rene; Saunders, Laura; Marshall, Brett; Kunick, Conrad; Verdin, Eric; Goekjian, Peter; Sippl, Wolfgang; Jung, Manfred

2006-12-14

NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.

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.

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

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

NASA Astrophysics Data System (ADS)

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-05

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

Effect of phorbol derivatives and staurosporine on gravitropic response of primary root of maize

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

Mulkey, T.J.; Kim, S.Y.; Lee, J.S.

1991-05-01

Time-lapse videography and computer-based, video image digitization were used to examine the effects of phorbol derivatives (phorbol 12-myristate 13-acetate, TPA; phorbol 12-myristate 13-acetate 4-O-methyl ether, mTPA) and staurosporine on the kinetics of gravicurvature of primary roots of maize (Zea mays L., Pioneer 3343 and Golden Cross Bantam). Pretreatment of roots with TPA (3 hr, 1 {mu}M) decreases the time lag prior to induction of positive gravicurvature in horizontally-oriented roots by > 60%. The rate of curvature is not significantly different than the rate observed in control roots. Wrongway curvature which is observed in 30-40% of control roots is not observedmore » in TPA-pretreated roots. Oscillatory movements observed in control roots after completion of gravitropic reorientation is completely dampened in TPA-pretreated roots. Pretreatment of roots with mTPA(3hr,1{mu}M), the inactive analog of TPA, does not significantly alter the kinetics of gravicurvature of primary roots of maize. Staurosporine (10{sup {minus}8}M), a microbial alkaloid which has been reported to have antifungal activity and to inhibit phospholipid/Ca{sup ++} dependent protein kinase, completely inhibits TPA-induced alteration of the kinetics of gravitropism. DAG (1-oleoyl-2-acetyl-rac-glycerol), a synthetic diglyceride activator of protein kinase C, exhibits similar activity to TPA. TPA-induced alterations in tissue response to auxin are presented.« less

Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase.

PubMed

Fornalé, Silvia; Capellades, Montserrat; Encina, Antonio; Wang, Kan; Irar, Sami; Lapierre, Catherine; Ruel, Katia; Joseleau, Jean-Paul; Berenguer, Jordi; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David

2012-07-01

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

[Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

PubMed

Voloshchuk, O N; Kopylchuk, G P

2016-01-01

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages

PubMed Central

Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A.

2016-01-01

Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species. PMID:26896851

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

PubMed Central

Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

2014-01-01

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. PMID:24654730

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

PubMed

Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

2014-06-01

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Vitamin C prevents zidovudine-induced NAD(P)H oxidase activation and hypertension in the rat.

PubMed

Papparella, Italia; Ceolotto, Giulio; Berto, Laura; Cavalli, Maurizio; Bova, Sergio; Cargnelli, Gabriella; Ruga, Ezia; Milanesi, Ornella; Franco, Lorenzo; Mazzoni, Martina; Petrelli, Lucia; Nussdorfer, Gastone G; Semplicini, Andrea

2007-01-15

Cardiovascular risk is increased among HIV-infected patients receiving antiretroviral therapy due to the development of hypertension and metabolic abnormalities. In this study, we investigated the effects of long-term treatment with zidovudine (AZT) and vitamin C, alone and in combination, on blood pressure and on the chain of events linking oxidative stress to cardiac damage in the rat. Six adult Wistar Kyoto rats received AZT (1 mg/ml) in the drinking water for 8 months, six vitamin C (10 g/kg of food) and AZT, six vitamin C alone, and six served as controls. AZT increased systolic blood pressure, expression of gp91(phox) and p47(phox) subunits of NAD(P)H oxidase, and protein kinase C (PKC) delta activation and reduced antioxidant power of plasma and cardiac homogenates. AZT also caused morphological alterations in cardiac myocyte mitochondria, indicative of functional damage. All of these effects were prevented by vitamin C. Chronic AZT administration increases blood pressure and promotes cardiovascular damage through a NAD(P)H oxidase-dependent mechanism that involves PKC delta. Vitamin C antagonizes these adverse effects of AZT in the cardiovascular system.

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

Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

PubMed

Pontvianne, Frédéric; Carpentier, Marie-Christine; Durut, Nathalie; Pavlištová, Veronika; Jaške, Karin; Schořová, Šárka; Parrinello, Hugues; Rohmer, Marine; Pikaard, Craig S; Fojtová, Miloslava; Fajkus, Jiří; Sáez-Vásquez, Julio

2016-08-09

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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.

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 Biology, Inc.

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

Enhancement of NAD+-dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells

PubMed Central

Fang, Mingzhu; Guo, Wei-Ren; Park, Youngil; Kang, Hwan-Goo; Zarbl, Helmut

2015-01-01

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis. PMID:26544624

Tributyltin induces mitochondrial fission through NAD-IDH dependent mitofusin degradation in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Nakano, Mizuho; Sekino, Yuko; Kanda, Yasunari

2015-08-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT acts at the nanomolar level through genomic pathways via the peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR). We recently reported that TBT inhibits cell growth and the ATP content in the human embryonic carcinoma cell line NT2/D1 via a non-genomic pathway involving NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which metabolizes isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial NAD-IDH and energy production. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusins 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess cytotoxicity associated with metal exposure.

Chronic alcohol binging injures the liver and other organs by reducing NAD⁺ levels required for sirtuin's deacetylase activity.

PubMed

French, Samuel W

2016-04-01

NAD(+) levels are markedly reduced when blood alcohol levels are high during binge drinking. This causes liver injury to occur because the enzymes that require NAD(+) as a cofactor such as the sirtuin de-acetylases cannot de-acetylate acetylated proteins such as acetylated histones. This prevents the epigenetic changes that regulate metabolic processes and which prevent organ injury such as fatty liver in response to alcohol abuse. Hyper acetylation of numerous regulatory proteins develops. Systemic multi-organ injury occurs when NAD(+) is reduced. For instance the Circadian clock is altered if NAD(+) is not available. Cell cycle arrest occurs due to up regulation of cell cycle inhibitors leading to DNA damage, mutations, apoptosis and tumorigenesis. NAD(+) is linked to aging in the regulation of telomere stability. NAD(+) is required for mitochondrial renewal. Alcohol dehydrogenase is present in every visceral organ in the body so that there is a systemic reduction of NAD(+) levels in all of these organs during binge drinking. Copyright © 2016. Published by Elsevier Inc.

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility.

PubMed

Chiang, Shian-Huey; Harrington, W Wallace; Luo, Guizhen; Milliken, Naphtali O; Ulrich, John C; Chen, Jing; Rajpal, Deepak K; Qian, Ying; Carpenter, Tiffany; Murray, Rusty; Geske, Robert S; Stimpson, Stephen A; Kramer, Henning F; Haffner, Curt D; Becherer, J David; Preugschat, Frank; Billin, Andrew N

2015-01-01

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

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.

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.

Extracellular NAD+ Suppresses Adrenergic Effects in the Atrial Myocardium of Rats during the Early Postnatal Ontogeny.

PubMed

Pustovit, K B; Ivanova, A D; Kuz'min, V S

2018-05-01

The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD + against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD + , ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD + against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of I CaL , which can be the main mechanism of NAD + action on rat heart.

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

PubMed Central

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-01-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action. PMID:25092173

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

PubMed Central

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

2014-01-01

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

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.

Transcripts of the NADH-dehydrogenase subunit 3 gene are differentially edited in Oenothera mitochondria.

PubMed Central

Schuster, W; Wissinger, B; Unseld, M; Brennicke, A

1990-01-01

A number of cytosines are altered to be recognized as uridines in transcripts of the nad3 locus in mitochondria of the higher plant Oenothera. Such nucleotide modifications can be found at 16 different sites within the nad3 coding region. Most of these alterations in the mRNA sequence change codon identities to specify amino acids better conserved in evolution. Individual cDNA clones differ in their degree of editing at five nucleotide positions, three of which are silent, while two lead to codon alterations specifying different amino acids. None of the cDNA clones analysed is maximally edited at all possible sites, suggesting slow processing or lowered stringency of editing at these nucleotides. Differentially edited transcripts could be editing intermediates or could code for differing polypeptides. Two edited nucleotides in an open reading frame located upstream of nad3 change two amino acids in the deduced polypeptide. Part of the well-conserved ribosomal protein gene rps12 also encoded downstream of nad3 in other plants, is lost in Oenothera mitochondria by recombination events. The functional rps12 protein must be imported from the cytoplasm since the deleted sequences of this gene are not found in the Oenothera mitochondrial genome. The pseudogene sequence is not edited at any nucleotide position. Images Fig. 3. Fig. 4. Fig. 7. PMID:1688531

NAD+-Dependent Activation of Sirt1 Corrects the Phenotype in a Mouse Model of Mitochondrial Disease

PubMed Central

Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A.; Li, Wei; Leoni, Valerio; Schon, Eric A.; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

2014-01-01

Summary Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD+-dependent protein deacetylase. As NAD+ boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD+ precursor, or reduction of NAD+ consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. PMID:24814483

Steady-state kinetic mechanism of the NADP+- and NAD+-dependent reactions catalysed by betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

PubMed Central

Velasco-García, R; González-Segura, L; Muñoz-Clares, R A

2000-01-01

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NADP(H). In Pseudomonas aeruginosa this reaction is a compulsory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. The kinetic mechanisms of the NAD(+)- and NADP(+)-dependent reactions were examined by steady-state kinetic methods and by dinucleotide binding experiments. The double-reciprocal patterns obtained for initial velocity with NAD(P)(+) and for product and dead-end inhibition establish that both mechanisms are steady-state random. However, quantitative analysis of the inhibitions, and comparison with binding data, suggest a preferred route of addition of substrates and release of products in which NAD(P)(+) binds first and NAD(P)H leaves last, particularly in the NADP(+)-dependent reaction. Abortive binding of the dinucleotides, or their analogue ADP, in the betaine aldehyde site was inferred from total substrate inhibition by the dinucleotides, and parabolic inhibition by NADH and ADP. A weak partial uncompetitive substrate inhibition by the aldehyde was observed only in the NADP(+)-dependent reaction. The kinetics of P. aeruginosa BADH is very similar to that of glucose-6-phosphate dehydrogenase, suggesting that both enzymes fulfil a similar amphibolic metabolic role when the bacteria grow in choline and when they grow in glucose. PMID:11104673

Vitamins and aging: pathways to NAD+ synthesis.

PubMed

Denu, John M

2007-05-04

Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction.

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

PubMed

Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

2014-07-01

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Role of NAD+ and mitochondrial sirtuins in cardiac and renal diseases

PubMed Central

Hershberger, Kathleen A.; Martin, Angelical S.; Hirschey, Matthew D.

2017-01-01

The coenzyme nicotinamide adenine dinucleotide (NAD+) has key roles in the regulation of redox status and energy metabolism. NAD+ depletion is emerging as a major contributor to the pathogenesis of cardiac and renal diseases and NAD+ repletion strategies have shown therapeutic potential as a means to restore healthy metabolism and physiological function. The pleotropic roles of NAD+ enable several possible avenues by which repletion of this coenzyme could have therapeutic efficacy. In particular, NAD+ functions as a co-substrate in deacylation reactions carried out by the sirtuin family of enzymes. These NAD+-dependent deacylases control several aspects of metabolism and a wealth of data suggests that boosting sirtuin activity via NAD+ supplementation might be a promising therapy for cardiac and renal pathologies. This Review summarizes the role of NAD+ metabolism in the heart and kidney, and highlights the mitochondrial sirtuins as mediators of some of the beneficial effects of NAD+-boosting therapies in preclinical animal models. We surmise that modulating the NAD+–sirtuin axis is a clinically relevant approach to develop new therapies for cardiac and renal diseases. PMID:28163307

Role of NAD+ and mitochondrial sirtuins in cardiac and renal diseases.

PubMed

Hershberger, Kathleen A; Martin, Angelical S; Hirschey, Matthew D

2017-04-01

The coenzyme nicotinamide adenine dinucleotide (NAD + ) has key roles in the regulation of redox status and energy metabolism. NAD + depletion is emerging as a major contributor to the pathogenesis of cardiac and renal diseases and NAD + repletion strategies have shown therapeutic potential as a means to restore healthy metabolism and physiological function. The pleotropic roles of NAD + enable several possible avenues by which repletion of this coenzyme could have therapeutic efficacy. In particular, NAD + functions as a co-substrate in deacylation reactions carried out by the sirtuin family of enzymes. These NAD + -dependent deacylases control several aspects of metabolism and a wealth of data suggests that boosting sirtuin activity via NAD + supplementation might be a promising therapy for cardiac and renal pathologies. This Review summarizes the role of NAD + metabolism in the heart and kidney, and highlights the mitochondrial sirtuins as mediators of some of the beneficial effects of NAD + -boosting therapies in preclinical animal models. We surmise that modulating the NAD + -sirtuin axis is a clinically relevant approach to develop new therapies for cardiac and renal diseases.

Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

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

Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika

2005-12-30

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzymemore » inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.« less

The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

PubMed

Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pirini, Maurizio; Pagliarani, Alessandra

2018-01-26

The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus.

PubMed

Romkina, Anastasia Y; Kiriukhin, Michael Y

2017-01-01

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 μM and 184 μM respectively, while the Km values for DL-isocitrate were 9.0 μM (NAD+), 8.0 μM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae*

PubMed Central

Kato, Michiko; Lin, Su-Ju

2014-01-01

NAD+ is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD+ pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD+ metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD+ levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD+ homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD+ metabolism in higher eukaryotes. PMID:24759102

The human NAD metabolome: Functions, metabolism and compartmentalization

PubMed Central

Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

2015-01-01

Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

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.

NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

PubMed

Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

2014-06-03

Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Maize and resistant starch enriched breads reduce postprandial glycemic responses in rats.

PubMed

Brites, Carla M; Trigo, Maria J; Carrapiço, Belmira; Alviña, Marcela; Bessa, Rui J

2011-04-01

White wheat bread is a poor source of dietary fiber, typically containing less than 2%. A demand exists for the development of breads with starch that is slowly digestible or partially resistant to the digestive process. The utilization of maize flour and resistant starch is expected to reduce the release and absorption of glucose and, hence, lower the glycemic index of bread. This study was undertaken to investigate the hypothesis that a diet of maize bread, as produced and consumed in Portugal, would have beneficial metabolic effects on rats compared to white wheat bread. We also hypothesized that the effect of resistant starch on glycemic response could be altered by the use of different formulations and breadmaking processes for wheat and maize breads. Resistant starch (RS) was incorporated into formulations of breads at 20% of the inclusion rate of wheat and maize flours. Assays were conducted with male Wistar rats (n = 36), divided into four groups and fed either wheat bread, RS-wheat bread, maize bread, and RS-maize bread to evaluate feed intake, body weight gain, fecal pH, and postprandial blood glucose response (glycemic response). Blood triglycerides, total cholesterol concentrations, and liver weights were also determined. The maize bread group presented higher body weight gain and cholesterol level, lower fecal pH, and postprandial blood glucose response than the wheat bread group. The RS-wheat bread group showed significant reductions in feed intake, fecal pH, postprandial blood glucose response, and total cholesterol. The RS-maize group displayed significant reductions of body weight gain, fecal pH, and total cholesterol levels; however, for the glycemic response, only a reduction in fasting level was observed. These results suggest that maize bread has a lower glycemic index than wheat bread, and the magnitude of the effect of RS on glycemic response depends of type of bread. Copyright © 2011 Elsevier Inc. All rights reserved.

Elevated microRNA-34a in obesity reduces NAD+ levels and SIRT1 activity by directly targeting NAMPT.

PubMed

Choi, Sung-E; Fu, Ting; Seok, Sunmi; Kim, Dong-Hyun; Yu, Eunkyung; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Kemper, Byron; Kemper, Jongsook Kim

2013-12-01

SIRT1 is an NAD(+)-dependent deacetylase that is implicated in prevention of many age-related diseases including metabolic disorders. As SIRT1 deacetylase activity is dependent on NAD(+) levels and the development of compounds that directly activate SIRT1 has been controversial, indirectly activating SIRT1 through enhancing NAD(+) bioavailability has received increasing attention. NAD(+) levels are reduced in obesity and the aged, but the underlying mechanisms remain unclear. We recently showed that hepatic microRNA-34a (miR-34a), which is elevated in obesity, directly targets and decreases SIRT1 expression. Here, we further show that miR-34a reduces NAD(+) levels and SIRT1 activity by targeting NAMPT, the rate-limiting enzyme for NAD(+) biosynthesis. A functional binding site for miR-34a is present in the 3' UTR of NAMPT mRNA. Hepatic overexpression of miR-34a reduced NAMPT/NAD(+) levels, increased acetylation of the SIRT1 target transcriptional regulators, PGC-1α, SREBP-1c, FXR, and NF-κB, and resulted in obesity-mimetic outcomes. The decreased NAMPT/NAD(+) levels were independent of miR-34a effects on SIRT1 levels as they were also observed in SIRT1 liver-specific knockout mice. Further, the miR-34a-mediated decreases were reversed by treatment with the NAD(+) intermediate, nicotinamide mononucleotide. Conversely, antagonism of miR-34a in diet-induced obese mice restored NAMPT/NAD(+) levels and alleviated steatosis, inflammation, and glucose intolerance. Anti-miR-34a-mediated increases in NAD(+) levels were attenuated when NAMPT was downregulated. Our findings reveal a novel function of miR-34a in reducing both SIRT1 expression and activity in obesity. The miR-34a/NAMPT axis presents a potential target for treating obesity- and aging-related diseases involving SIRT1 dysfunction like steatosis and type 2 diabetes. © 2013 the Anatomical Society and John Wiley & Sons Ltd.

Normalization of NAD+ Redox Balance as a Therapy for Heart Failure.

PubMed

Lee, Chi Fung; Chavez, Juan D; Garcia-Menendez, Lorena; Choi, Yongseon; Roe, Nathan D; Chiao, Ying Ann; Edgar, John S; Goo, Young Ah; Goodlett, David R; Bruce, James E; Tian, Rong

2016-09-20

Impairments of mitochondrial function in the heart are linked intricately to the development of heart failure, but there is no therapy for mitochondrial dysfunction. We assessed the reduced/oxidized ratio of nicotinamide adenine dinucleotide (NADH/NAD(+) ratio) and protein acetylation in the failing heart. Proteome and acetylome analyses were followed by docking calculation, mutagenesis, and mitochondrial calcium uptake assays to determine the functional role of specific acetylation sites. The therapeutic effects of normalizing mitochondrial protein acetylation by expanding the NAD(+) pool also were tested. Increased NADH/NAD(+) and protein hyperacetylation, previously observed in genetic models of defective mitochondrial function, also are present in human failing hearts as well as in mouse hearts with pathologic hypertrophy. Elevation of NAD(+) levels by stimulating the NAD(+) salvage pathway suppressed mitochondrial protein hyperacetylation and cardiac hypertrophy, and improved cardiac function in responses to stresses. Acetylome analysis identified a subpopulation of mitochondrial proteins that was sensitive to changes in the NADH/NAD(+) ratio. Hyperacetylation of mitochondrial malate-aspartate shuttle proteins impaired the transport and oxidation of cytosolic NADH in the mitochondria, resulting in altered cytosolic redox state and energy deficiency. Furthermore, acetylation of oligomycin-sensitive conferring protein at lysine-70 in adenosine triphosphate synthase complex promoted its interaction with cyclophilin D, and sensitized the opening of mitochondrial permeability transition pore. Both could be alleviated by normalizing the NAD(+) redox balance either genetically or pharmacologically. We show that mitochondrial protein hyperacetylation due to NAD(+) redox imbalance contributes to the pathologic remodeling of the heart via 2 distinct mechanisms. Our preclinical data demonstrate a clear benefit of normalizing NADH/NAD(+) imbalance in the failing hearts. These findings have a high translational potential as the pharmacologic strategy of increasing NAD(+) precursors are feasible in humans. © 2016 American Heart Association, Inc.

Morphological, Physiological and Biochemical Impact of Ink Industry Effluent on Germination of Maize (Zea mays), Barley (Hordeum vulgare) and Sorghum (Sorghum bicolor).

PubMed

Zayneb, Chaâbene; Lamia, Khanous; Olfa, Ellouze; Naïma, Jebahi; Grubb, C Douglas; Bassem, Khemakhem; Hafedh, Mejdoub; Amine, Elleuch

2015-11-01

The present study focuses on effects of untreated and treated ink industry wastewater on germination of maize, barley and sorghum. Wastewater had a high chemical oxygen demand (COD) and metal content compared to treated effluent. Germination decreased with increasing COD concentration. Speed of germination also followed the same trend, except for maize seeds exposed to untreated effluent (E), which germinated slightly faster than controls. These alterations of seedling development were mirrored by changes in soluble protein content. E exerted a positive effect on soluble protein content and maximum levels occurred after 10 days with treated effluent using coagulation/flocculation (TEc/f) process and treated effluent using combined process (coagulation/flocculation/biosorption) (TEc/f/b). Likewise, activity of α-amylase was influenced by effluent composition. Its expression depended on the species, exposure time and applied treatment. Nevertheless, current results indicated TEc/f/b had no observable toxic effects on germination and could be a beneficial alternative resource to irrigation water.

The Effects of B, K10, and AR Chromosomes on the Resistance of Maize to Viral Infection

PubMed Central

McGirr, Scott C.; Endrizzi, J. E.

1978-01-01

Studies were conducted to determine if accessory (B) chromosomes, the abnormal tenth (K10) chromosome or the aberrant ratio (AR) phenomenon of maize (Zea mays L.) affect the resistance of the plants to viral infection. Genetically similar stocks of maize with and without these elements were compared to determine what effect they would have on the plants response to Brome Mosaic Virus (BMV), Maize Dwarf Mosaic Virus (MDMV), Wheat Streak Mosaic Virus (WSMV) and Barley Stripe Mosaic Virus (BSMV).—The test results with BSMV were not found to be conclusive. With BMV and MDMV, neither the B orK10 chromosomes were found to alter infections; however, these chromosomes were found to affect the resistance of the plants to WSMV infection. The B chromosomes were found to delay the onset of leaf necrosis by 15%, while the K10 chromosome was found to increase the susceptibility to necrosis by 100%. The AR phenomenon was not found to alter the resistance of maize to BMV infection. However, it was found to increase the susceptibility of maize to MDMV infection by 36% and to decrease the susceptibility of maize to WSMV infection by 92%. PMID:17248865

The Effects of B, K10, and AR Chromosomes on the Resistance of Maize to Viral Infection.

PubMed

McGirr, S C; Endrizzi, J E

1978-10-01

Studies were conducted to determine if accessory (B) chromosomes, the abnormal tenth (K10) chromosome or the aberrant ratio (AR) phenomenon of maize (Zea mays L.) affect the resistance of the plants to viral infection. Genetically similar stocks of maize with and without these elements were compared to determine what effect they would have on the plants response to Brome Mosaic Virus (BMV), Maize Dwarf Mosaic Virus (MDMV), Wheat Streak Mosaic Virus (WSMV) and Barley Stripe Mosaic Virus (BSMV).-The test results with BSMV were not found to be conclusive. With BMV and MDMV, neither the B orK10 chromosomes were found to alter infections; however, these chromosomes were found to affect the resistance of the plants to WSMV infection. The B chromosomes were found to delay the onset of leaf necrosis by 15%, while the K10 chromosome was found to increase the susceptibility to necrosis by 100%. The AR phenomenon was not found to alter the resistance of maize to BMV infection. However, it was found to increase the susceptibility of maize to MDMV infection by 36% and to decrease the susceptibility of maize to WSMV infection by 92%.

THC reduces the anticipatory nucleus accumbens response to reward in subjects with a nicotine addiction

PubMed Central

Jansma, J M; van Hell, H H; Vanderschuren, L J M J; Bossong, M G; Jager, G; Kahn, R S; Ramsey, N F

2013-01-01

Recent evidence has implicated the endocannabinoid (eCB) system in nicotine addiction. The eCB system also has an important role in reward mechanisms, and nicotine addiction has been associated with aberrant reward processing. Motivated by this evidence, we tested the hypothesis that eCB modulation of reward processing is altered in subjects with a nicotine addiction (NAD). For this purpose, we compared reward-related activity in NAD with healthy controls (HC) in a pharmacological magnetic resonance imaging (MRI) study using Δ9-tetrahydrocannabinol (THC) administration to challenge the eCB system. Eleven HC and 10 NAD participated in a 3-T functional MRI (fMRI) study with a double-blind, cross-over, placebo-controlled design, using a Monetary Incentive Delay (MID) paradigm with three reward levels. Reward activity in the nucleus accumbens (NAcc) and caudate putamen during anticipation and feedback of reward was compared after THC and placebo. fMRI results indicated a significant reduction of reward anticipation activity in the NAcc in NAD after THC administration, which was not present in HC. This is indicated by a significant group by drug by reward interaction. Our data show that THC significantly reduces the NAcc response to monetary reward anticipation in NAD. These results suggest that nicotine addiction is associated with altered eCB modulation of reward processing in the NAcc. This study adds important human data to existing evidence implicating the eCB system in nicotine addiction. PMID:23443360

Mitochondrial nad2 gene is co-transcripted with CMS-associated orfB gene in cytoplasmic male-sterile stem mustard (Brassica juncea).

PubMed

Yang, Jing-Hua; Zhang, Ming-Fang; Yu, Jing-Quan

2009-02-01

The transcriptional patterns of mitochondrial respiratory related genes were investigated in cytoplasmic male-sterile and fertile maintainer lines of stem mustard, Brassica juncea. There were numerous differences in nad2 (subunit 2 of NADH dehydrogenase) between stem mustard CMS and its maintainer line. One novel open reading frame, hereafter named orfB gene, was located at the downstream of mitochondrial nad2 gene in the CMS. The novel orfB gene had high similarity with YMF19 family protein, orfB in Raphanus sativus, Helianthus annuus, Nicotiana tabacum and Beta vulgaris, orfB-CMS in Daucus carota, atp8 gene in Arabidopsis thaliana, 5' flanking of orf224 in B. napus (nap CMS) and 5' flanking of orf220 gene in CMS Brassica juncea. Three copies probed by specific fragment (amplified by primers of nad2F and nad2R from CMS) were found in the CMS line following Southern blotting digested with HindIII, but only a single copy in its maintainer line. Meanwhile, two transcripts were shown in the CMS line following Northern blotting while only one transcript was detected in the maintainer line, which were probed by specific fragment (amplified by primers of nad2F and nad2R from CMS). Meanwhile, the expression of nad2 gene was reduced in CMS bud compared to that in its maintainer line. We thus suggested that nad2 gene may be co-transcripted with CMS-associated orfB gene in the CMS. In addition, the specific fragment that was amplified by primers of nad2F and nad2R just spanned partial sequences of nad2 gene and orfB gene. Such alterations in the nad2 gene would impact the activity of NADH dehydrogenase, and subsequently signaling, inducing the expression of nuclear genes involved in male sterility in this type of cytoplasmic male sterility.

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.

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

Boosting NAD to spare hearing.

PubMed

Brenner, Charles

2014-12-02

Ex vivo experiments have strangely shown that inhibition or stimulation of NAD metabolism can be neuroprotective. In this issue of Cell Metabolism, Brown et al. (2014) demonstrate that cochlear NAD is diminished by deafening noise but protected by nicotinamide riboside or WldS mutation. Hearing protection by nicotinamide riboside depends on Sirt3. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and genetic characterization of maize cell wall variation for improved biorefinery feedstock characteristics

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

Pauly, Markus; Hake, Sarah

2013-10-31

The objectives of this program are to 1) characterize novel maize mutants with altered cell walls for enhanced biorefinery characteristics and 2) find quantitative trait loci (QTLs) related to biorefinery characteristics by taking advantage of the genetic diversity of maize. As a result a novel non-transgenic maize plant (cal1) has been identified, whose stover (leaves and stalk) contain more glucan in their walls leading to a higher saccharification yield, when subjected to a standard enzymatic digestion cocktail. Stacking this trait with altered lignin mutants yielded evene higher saccharification yields. Cal-1 mutants do not show a loss of kernel and ormore » biomass yield when grown in the field . Hence, cal1 biomass provides an excellent feedstock for the biofuel industry.« less

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

PubMed Central

Nikiforov, Andrey; Dölle, Christian; Niere, Marc; Ziegler, Mathias

2011-01-01

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule. PMID:21504897

Analysis of DNA methylation of maize in response to osmotic and salt stress based on methylation-sensitive amplified polymorphism.

PubMed

Tan, Ming-pu

2010-01-01

Water stress is known to alter cytosine methylation, which generally represses transcription. However, little is known about the role of methylation alteration in maize under osmotic stress. Here, methylation-sensitive amplified polymorphism (MSAP) was used to screen PEG- or NaCl-induced methylation alteration in maize seedlings. The sequences of 25 differentially amplified fragments relevant to stress were successfully obtained. Two stress-specific fragments from leaves, LP166 and LPS911, shown to be homologous to retrotransposon Gag-Pol protein genes, suggested that osmotic stress-induced methylation of retrotransposons. Three MSAP fragments, representing drought-induced or salt-induced methylation in leaves, were homologous to a maize aluminum-induced transporter. Besides these, heat shock protein HSP82, Poly [ADP-ribose] polymerase 2, Lipoxygenase, casein kinase (CK2), and dehydration-responsive element-binding (DREB) factor were also homologs of MSAP sequences from salt-treated roots. One MSAP fragment amplified from salt-treated roots, designated RS39, was homologous to the first intron of maize protein phosphatase 2C (zmPP2C), whereas - LS103, absent from salt-treated leaves, was homologous to maize glutathione S-transferases (zmGST). Expression analysis showed that salt-induced intron methylation of root zmPP2C significantly downregulated its expression, while salt-induced demethylation of leaf zmGST weakly upregulated its expression. The results suggested that salinity-induced methylation downregulated zmPP2C expression, a negative regulator of the stress response, while salinity-induced demethylation upregulated zmGST expression, a positive effecter of the stress response. Altered methylation, in response to stress, might also be involved in stress acclimation. Copyright 2009 Elsevier Masson SAS. All rights reserved.

The complete mitochondrial genome of the American black flour beetle Tribolium audax (Coleoptera: Tenebrionidae).

PubMed

Ou, Jing; Liu, Jin-Bo; Yao, Fu-Jiao; Wang, Xin-Guo; Wei, Zhao-Ming

2016-01-01

Flour beetles of the genus Tribolium are all pests of stored products and cause severe economic losses every year. The American black flour beetle Tribolium audax is one of the important pest species of flour beetle, and it is also an important quarantine insect. Here we sequenced and characterized the complete mitochondrial genome of T. audax, which was intercepted by Huangpu Custom in maize from America. The complete circular mitochondrial genome (mitogenome) of T. audax was 15,924 bp in length, containing 37 typical coding genes and one non-coding AT-rich region. The mitogenome of T. audax exhibits a gene arrangement and content identical to the most common type in insects. All protein coding genes (PCGs) are start with a typical ATN initiation codon, except for the cox1, which use AAC as its start codon instead of ATN. Eleven genes use standard complete termination codon (nine TAA, two TAG), whereas the nad4 and nad5 genes end with single T. Except for trnS1 (AGN), all tRNA genes display typical secondary cloverleaf structures as those of other insects. The sizes of the large and small ribosomal RNA genes are 1288 and 780 bp, respectively. The AT content of the AT-rich region is 81.36%. The 5 bp conserved motif TACTA was found in the intergenic region between trnS2 (UCN) and nad1.

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism.

PubMed

James, Emma L; Lane, James A E; Michalek, Ryan D; Karoly, Edward D; Parkinson, E Kenneth

2016-12-07

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism

PubMed Central

James, Emma L.; Lane, James A. E.; Michalek, Ryan D.; Karoly, Edward D.; Parkinson, E. Kenneth

2016-01-01

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease. PMID:27924925

NAD(+)- dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10

USDA-ARS?s Scientific Manuscript database

A member of the sirtuin family of NAD (+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated ...

Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase.

PubMed

Weckbecker, Andrea; Hummel, Werner

2004-11-01

Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD+ and NADPH. The pnta and pntb genes encoding for the alpha- and beta-subunits were cloned and co-expressed with NADP+-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12 h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.

Key role of an ADP - ribose - dependent transcriptional regulator of NAD metabolism for fitness and virulence of Pseudomonas aeruginosa.

PubMed

Okon, Elza; Dethlefsen, Sarah; Pelnikevich, Anna; Barneveld, Andrea van; Munder, Antje; Tümmler, Burkhard

2017-01-01

NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control. Copyright © 2016 Elsevier GmbH. All rights reserved.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

PubMed

Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

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

PubMed

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

2017-10-30

Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD + ) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD + -dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism. Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD + /NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD + /NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD + /NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols. The ratio of NAD + /NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.

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

PubMed

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

2017-12-01

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

YCL047C/POF1 is a novel nicotinamide mononucleotide adenylyltransferase (NMNAT) in Saccharomyces cerevisiae.

PubMed

Kato, Michiko; Lin, Su-Ju

2014-05-30

NAD(+) is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD(+) pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD(+) metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD(+) levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD(+) homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutant maize variety containing the glt1-1 allele

DOEpatents

Nelson, Oliver E.; Pan, David

1994-01-01

A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

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.

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway1[W][OA

PubMed Central

Hasnain, Ghulam; Frelin, Océane; Roje, Sanja; Ellens, Kenneth W.; Ali, Kashif; Guan, Jiahn-Chou; Garrett, Timothy J.; de Crécy-Lagard, Valérie; Gregory, Jesse F.; McCarty, Donald R.; Hanson, Andrew D.

2013-01-01

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction. PMID:23150645

A conjugated fatty acid present at high levels in bitter melon seed favorably affects lipid metabolism in hepatocytes by increasing NAD(+)/NADH ratio and activating PPARα, AMPK and SIRT1 signaling pathway.

PubMed

Chen, Gou-Chun; Su, Hui-Min; Lin, Yu-Shun; Tsou, Po-Yen; Chyuan, Jong-Ho; Chao, Pei-Min

2016-07-01

α-Eleostearic acid (α-ESA), or the cis-9, trans-11, trans-13 isomer of conjugated linolenic acid, is a special fatty acid present at high levels in bitter melon seed oil. The aim of this study was to examine the effect of α-ESA on hepatic lipid metabolism. Using H4IIEC3 hepatoma cell line, we showed that α-ESA significantly lowered intracellular triglyceride accumulation compared to α-linolenic acid (LN), used as a fatty acid control, in a dose- and time-dependent manner. The effects of α-ESA on enzyme activities and mRNA profiles in H4IIEC3 cells suggested that enhanced fatty acid oxidation and lowered lipogenesis were involved in α-ESA-mediated triglyceride lowering effects. In addition, α-ESA triggered AMP-activated protein kinase (AMPK) activation without altering sirtuin 1 (SIRT1) protein levels. When cells were treated with vehicle control (VC), LN alone (LN; 100μmol/L) or in combination with α-ESA (LN+α-ESA; 75+25μmol/L) for 24h, acetylation of forkhead box protein O1 was decreased, while the NAD(+)/NADH ratio, mRNA levels of NAMPT and PTGR1 and enzyme activity of nicotinamide phosphoribosyltransferase were increased by LN+α-ESA treatment compared to treatment with LN alone, suggesting that α-ESA activates SIRT1 by increasing NAD(+) synthesis and NAD(P)H consumption. The antisteatosis effect of α-ESA was confirmed in mice treated with a high-sucrose diet supplemented with 1% α-ESA for 5weeks. We conclude that α-ESA favorably affects hepatic lipid metabolism by increasing cellular NAD(+)/NADH ratio and activating PPARα, AMPK and SIRT1 signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

The potential regulatory roles of NAD(+) and its metabolism in autophagy.

PubMed

Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

2016-04-01

(Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular, biochemical, and functional characterization of a Nudix hydrolase protein that stimulates the activity of a nicotinoprotein alcohol dehydrogenase.

PubMed

Kloosterman, Harm; Vrijbloed, Jan W; Dijkhuizen, Lubbert

2002-09-20

The cytoplasmic coenzyme NAD(+)-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicus during growth on C(1)-C(4) primary alcohols is a decameric protein with 1 Zn(2+)-ion and 1-2 Mg(2+)-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg(2+)-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD(+)-dependent activity of MDH with C(1)-C(4) primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg(2+)-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. The act gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD(+) ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation.

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

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

Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa

Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligandmore » complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.« less

Crosstalk between poly(ADP-ribose) polymerase and sirtuin enzymes

PubMed Central

Cantó, Carles; Sauve, Anthony A.; Bai, Peter

2013-01-01

Poly(ADP-ribose) polymerases (PARPs) are NAD+ dependent enzymes that were identified as DNA repair proteins, however, today it seems clear that PARPs are responsible for a plethora of biological functions. Sirtuins (SIRTs) are NAD+-dependent deacetylase enzymes involved in the same biological processes as PARPs raising the question whether PARP and SIRT enzymes may interact with each other in physiological and pathophysiological conditions. Hereby we review the current understanding of the SIRT-PARP interplay in regard to the biochemical nature of the interaction (competition for the common NAD+ substrate, mutual posttranslational modifications and direct transcriptional effects) and the physiological, or pathophysiological consequences of the interactions (metabolic events, oxidative stress response, genomic stability and ageing). Finally, we give an overview of the possibilities of pharmacological intervention to modulate PARP and SIRT enzymes either directly, or through modulating NAD+ homeostasis. PMID:23357756

Mutant maize variety containing the glt1-1 allele

DOEpatents

Nelson, O.E.; Pan, D.

1994-07-19

A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

The auxin-deficient defective kernel18 (dek18) mutation alters the expression of seed-specific biosynthethic genes in maize

USDA-ARS?s Scientific Manuscript database

The dek18 mutant of maize has decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology in maize seed development. Seeds of the dek18 mutants are smaller compared to wild type seeds and the vegetative d...

Monitoring Mitochondrial Electron Fluxes Using NAD(P)H-Flavoprotein Fluorometry Reveals Complex Action of Isoflurane on Cardiomyocytes

PubMed Central

Sedlic, Filip; Pravdic, Danijel; Hirata, Naoyuki; Mio, Yasushi; Sepac, Ana; Camara, Amadou K.; Wakatsuki, Tetsuro; Bosnjak, Zeljko J.; Bienengraeber, Martin

2010-01-01

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(H)P and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes. PMID:20646994

Uridine monophosphate synthetase enables eukaryotic de novo NAD+ biosynthesis from quinolinic acid.

PubMed

McReynolds, Melanie R; Wang, Wenqing; Holleran, Lauren M; Hanna-Rose, Wendy

2017-07-07

NAD + biosynthesis is an attractive and promising therapeutic target for influencing health span and obesity-related phenotypes as well as tumor growth. Full and effective use of this target for therapeutic benefit requires a complete understanding of NAD + biosynthetic pathways. Here, we report a previously unrecognized role for a conserved phosphoribosyltransferase in NAD + biosynthesis. Because a required quinolinic acid phosphoribosyltransferase (QPRTase) is not encoded in its genome, Caenorhabditis elegans are reported to lack a de novo NAD + biosynthetic pathway. However, all the genes of the kynurenine pathway required for quinolinic acid (QA) production from tryptophan are present. Thus, we investigated the presence of de novo NAD + biosynthesis in this organism. By combining isotope-tracing and genetic experiments, we have demonstrated the presence of an intact de novo biosynthesis pathway for NAD + from tryptophan via QA, highlighting the functional conservation of this important biosynthetic activity. Supplementation with kynurenine pathway intermediates also boosted NAD + levels and partially reversed NAD + -dependent phenotypes caused by mutation of pnc-1 , which encodes a nicotinamidase required for NAD + salvage biosynthesis, demonstrating contribution of de novo synthesis to NAD + homeostasis. By investigating candidate phosphoribosyltransferase genes in the genome, we determined that the conserved uridine monophosphate phosphoribosyltransferase (UMPS), which acts in pyrimidine biosynthesis, is required for NAD + biosynthesis in place of the missing QPRTase. We suggest that similar underground metabolic activity of UMPS may function in other organisms. This mechanism for NAD + biosynthesis creates novel possibilities for manipulating NAD + biosynthetic pathways, which is key for the future of therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism

PubMed Central

Gossmann, Toni I.; Ziegler, Mathias

2014-01-01

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification. PMID:25084685

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism.

PubMed

Gossmann, Toni I; Ziegler, Mathias

2014-11-01

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38

PubMed Central

Escande, Carlos; Nin, Veronica; Price, Nathan L.; Capellini, Verena; Gomes, Ana P.; Barbosa, Maria Thereza; O’Neil, Luke; White, Thomas A.; Sinclair, David A.; Chini, Eduardo N.

2013-01-01

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD+ metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD+ levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD+ase in mammals. Moreover, CD38 knockout mice have higher NAD+ levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD+ levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD+ levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD+ levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD+-dependent pathways. PMID:23172919

Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome.

PubMed

Escande, Carlos; Nin, Veronica; Price, Nathan L; Capellini, Verena; Gomes, Ana P; Barbosa, Maria Thereza; O'Neil, Luke; White, Thomas A; Sinclair, David A; Chini, Eduardo N

2013-04-01

Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD(+) metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD(+) levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD(+)ase in mammals. Moreover, CD38 knockout mice have higher NAD(+) levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD(+) levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD(+) levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD(+) levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD(+)-dependent pathways.

Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants.

PubMed Central

Hove-Jensen, B

1996-01-01

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon. PMID:8550505

Root Type-Specific Reprogramming of Maize Pericycle Transcriptomes by Local High Nitrate Results in Disparate Lateral Root Branching Patterns1[OPEN

PubMed Central

Lithio, Andrew

2016-01-01

The adaptability of root system architecture to unevenly distributed mineral nutrients in soil is a key determinant of plant performance. The molecular mechanisms underlying nitrate dependent plasticity of lateral root branching across the different root types of maize are only poorly understood. In this study, detailed morphological and anatomical analyses together with cell type-specific transcriptome profiling experiments combining laser capture microdissection with RNA-seq were performed to unravel the molecular signatures of lateral root formation in primary, seminal, crown, and brace roots of maize (Zea mays) upon local high nitrate stimulation. The four maize root types displayed divergent branching patterns of lateral roots upon local high nitrate stimulation. In particular, brace roots displayed an exceptional architectural plasticity compared to other root types. Transcriptome profiling revealed root type-specific transcriptomic reprogramming of pericycle cells upon local high nitrate stimulation. The alteration of the transcriptomic landscape of brace root pericycle cells in response to local high nitrate stimulation was most significant. Root type-specific transcriptome diversity in response to local high nitrate highlighted differences in the functional adaptability and systemic shoot nitrogen starvation response during development. Integration of morphological, anatomical, and transcriptomic data resulted in a framework underscoring similarity and diversity among root types grown in heterogeneous nitrate environments. PMID:26811190

Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth

DOE PAGES

Dever, Louisa V.; Boxall, Susanna F.; Knerova, Jana; ...

2014-11-05

Here, mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO 2 fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO 2 fixation. In well-watered conditions, these lines fixed all of theirmore » CO 2 in the light; they thus performed C 3 photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO 2 fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.« less

Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth

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

Dever, Louisa V.; Boxall, Susanna F.; Knerova, Jana

Here, mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO 2 fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO 2 fixation. In well-watered conditions, these lines fixed all of theirmore » CO 2 in the light; they thus performed C 3 photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO 2 fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock.« less

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes.

PubMed

Heyno, Eiri; Mary, Véronique; Schopfer, Peter; Krieger-Liszkay, Anja

2011-07-01

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet induced obesity

PubMed Central

Cantó, Carles; Houtkooper, Riekelt H.; Pirinen, Eija; Youn, Dou Y.; Oosterveer, Maaike H.; Cen, Yana; Fernandez-Marcos, Pablo J.; Yamamoto, Hiroyasu; Andreux, Pénélope A.; Cettour-Rose, Philippe; Gademann, Karl; Rinsch, Chris; Schoonjans, Kristina; Sauve, Anthony A.; Auwerx, Johan

2013-01-01

SUMMARY As NAD+ is a rate-limiting co-substrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38 —both NAD+ consumers— increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing and replicative lifespan in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin, NR, could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function. PMID:22682224

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

PubMed Central

Fouquerel, Elise; Sobol, Robert W.

2014-01-01

Nicotinamide adenine dinucleotide, NAD+, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD+ and NADH). NAD+ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalysed by the sirtuins (SIRTs) which use NAD+ as a substrate. In this review, we highlight the significance of NAD+ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked and dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischemia/reperfusion. PMID:25283336

Dietary proanthocyanidins boost hepatic NAD(+) metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats.

PubMed

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-04-22

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD(+)) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD(+) precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD(+). Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD(+) availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD(+) levels.

Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats

PubMed Central

Aragonès, Gerard; Suárez, Manuel; Ardid-Ruiz, Andrea; Vinaixa, Maria; Rodríguez, Miguel A.; Correig, Xavier; Arola, Lluís; Bladé, Cinta

2016-01-01

Proanthocyanidins (PACs) have been reported to modulate multiple targets by simultaneously controlling many pivotal metabolic pathways in the liver. However, the precise mechanism of PAC action on the regulation of the genes that control hepatic metabolism remains to be clarified. Accordingly, we used a metabolomic approach combining both nuclear magnetic resonance and mass spectrometry analysis to evaluate the changes induced by different doses of grape-seed PACs in the liver of healthy rats. Here, we report that PACs significantly increased the hepatic nicotinamide adenine dinucleotide (NAD+) content in a dose-dependent manner by specifically modulating the hepatic concentrations of the major NAD+ precursors as well as the mRNA levels of the genes that encode the enzymes involved in the cellular metabolism of NAD+. Notably, Sirtuin 1 (Sirt1) gene expression was also significantly up-regulated in a dose-response pattern. The increase in both the NAD+ availability and Sirt1 mRNA levels, in turn, resulted in the hepatic activation of SIRT1, which was significantly associated with improved protection against hepatic triglyceride accumulation. Our data clearly indicates that PAC consumption could be a valid tool to enhance hepatic SIRT1 activity through the modulation of NAD+ levels. PMID:27102823

Disruption of Pyridine Nucleotide Redox Status During Oxidative Challenge at Normal and Low-Glucose States: Implications for Cellular Adenosine Triphosphate, Mitochondrial Respiratory Activity, and Reducing Capacity in Colon Epithelial Cells

PubMed Central

Circu, Magdalena L.; Maloney, Ronald E.

2011-01-01

Abstract We recently demonstrated that menadione (MQ), a redox cycling quinone, mediated the loss of mitochondrial glutathione/glutathione disulfide redox balance. In this study, we showed that MQ significantly disrupted cellular pyridine nucleotide (NAD+/NADH, NADP+/NADPH) redox balance that compromised cellular ATP, mitochondrial respiratory activity, and NADPH-dependent reducing capacity in colonic epithelial cells, a scenario that was exaggerated by low glucose. In the cytosol, MQ induced NAD+ loss concurrent with increased NADP+ and NAD kinase activity, but decreased NADPH. In the mitochondria, NADH loss occurred in conjunction with increased nicotinamide nucleotide transhydrogenase activity and NADP+, and decreased NADPH. These results are consistent with cytosolic NAD+-to-NADP+ and mitochondrial NADH-to-NADPH shifts, but compromised NADPH availability. Thus, despite the sacrifice of NAD+/NADH in favor of NADPH generation, steady-state NADPH levels were not maintained during MQ challenge. Impairments of cellular bioenergetics were evidenced by ATP losses and increased mitochondrial O2 dependence of pyridine nucleotide oxidation–reduction; half-maximal oxidation (P50) was 10-fold higher in low glucose, which was lowered by glutamate or succinate supplementation. This exaggerated O2 dependence is consistent with increased O2 diversion to nonmitochondrial O2 consumption by MQ-semiquinone redox cycling secondary to decreased NADPH-dependent MQ detoxication at low glucose, a situation that was corrected by glucose-sparing mitochondrial substrates. Antioxid. Redox Signal. 14, 2151–2162. PMID:21083422

The End of the Line: Can Ferredoxin and Ferredoxin NADP(H) Oxidoreductase Determine the Fate of Photosynthetic Electrons?

PubMed Central

Goss, Tatjana; Hanke, Guy

2014-01-01

At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems. PMID:24678667

Isolation and functional characterisation of two new bZIP maize regulators of the ABA responsive gene rab28.

PubMed

Nieva, Claudia; Busk, Peter K; Domínguez-Puigjaner, Eva; Lumbreras, Victoria; Testillano, Pilar S; Risueño, Maria-Carmen; Pagès, Montserrat

2005-08-01

The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma

PubMed Central

Ju, Huai-Qiang; Ying, Haoqiang; Tian, Tian; Ling, Jianhua; Fu, Jie; Lu, Yu; Wu, Min; Yang, Lifeng; Achreja, Abhinav; Chen, Gang; Zhuang, Zhuonan; Wang, Huamin; Nagrath, Deepak; Yao, Jun; Hung, Mien-Chie; DePinho, Ronald A.; Huang, Peng; Xu, Rui-Hua; Chiao, Paul J.

2017-01-01

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC. PMID:28232723

Metabolic responses induced by DNA damage and poly (ADP-ribose) polymerase (PARP) inhibition in MCF-7 cells

PubMed Central

Bhute, Vijesh J.; Palecek, Sean P.

2015-01-01

Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage. PMID:26478723

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

Improved strategies for electrochemical 1,4-NAD(P)H2 regeneration: A new era of bioreactors for industrial biocatalysis.

PubMed

Morrison, Clifford S; Armiger, William B; Dodds, David R; Dordick, Jonathan S; Koffas, Mattheos A G

Industrial enzymatic reactions requiring 1,4-NAD(P)H 2 to perform redox transformations often require convoluted coupled enzyme regeneration systems to regenerate 1,4-NAD(P)H 2 from NAD(P) and recycle the cofactor for as many turnovers as possible. Renewed interest in recycling the cofactor via electrochemical means is motivated by the low cost of performing electrochemical reactions, easy monitoring of the reaction progress, and straightforward product recovery. However, electrochemical cofactor regeneration methods invariably produce adventitious reduced cofactor side products which result in unproductive loss of input NAD(P). We review various literature strategies for mitigating adventitious product formation by electrochemical cofactor regeneration systems, and offer insight as to how a successful electrochemical bioreactor system could be constructed to engineer efficient 1,4-NAD(P)H 2 -dependent enzyme reactions of interest to the industrial biocatalysis community. Copyright © 2017 Elsevier Inc. All rights reserved.

Nutritive value of maize silage in relation to dairy cow performance and milk quality.

PubMed

Khan, Nazir A; Yu, Peiqiang; Ali, Mubarak; Cone, John W; Hendriks, Wouter H

2015-01-01

Maize silage has become the major forage component in the ration of dairy cows over the last few decades. This review provides information on the mean content and variability in chemical composition, fatty acid (FA) profile and ensiling quality of maize silages, and discusses the major factors which cause these variations. In addition, the effect of the broad range in chemical composition of maize silages on the total tract digestibility of dietary nutrients, milk production and milk composition of dairy cows is quantified and discussed. Finally, the optimum inclusion level of maize silage in the ration of dairy cows for milk production and composition is reviewed. The data showed that the nutritive value of maize silages is highly variable and that most of this variation is caused by large differences in maturity at harvest. Maize silages ensiled at a very early stage (dry matter (DM) < 250 g kg(-1)) were particularly low in starch content and starch/neutral detergent fibre (NDF) ratio, and resulted in a lower DM intake (DMI), milk yield and milk protein content. The DMI, milk yield and milk protein content increased with advancing maturity, reaching an optimum level for maize silages ensiled at DM contents of 300-350 g kg(-1), and then declined slightly at further maturity beyond 350 g kg(-1). The increases in milk (R(2) = 0.599) and protein (R(2) = 0.605) yields with maturity of maize silages were positively related to the increase in starch/NDF ratio of the maize silages. On average, the inclusion of maize silage in grass silage-based diets improved the forage DMI by 2 kg d(-1), milk yield by 1.9 kg d(-1) and milk protein content by 1.2 g kg(-1). Further comparisons showed that, in terms of milk and milk constituent yields, the optimum grass/maize silage ratio depends on the quality of both the grass and maize silages. Replacement of grass silage with maize silage in the ration, as well as an increasing maturity of the maize silages, altered the milk FA profile of the dairy cows, notably, the concentration of the cis-unsaturated FAs, C18:3n-3 and n-3/n-6 ratio decreased in milk fat. Despite variation in nutritive value, maize silage is rich in metabolizable energy and supports higher DMI and milk yield. Harvesting maize silages at a DM content between 300 and 350 g kg(-1) and feeding in combination with grass silage results in a higher milk yield of dairy cows. © 2014 Society of Chemical Industry.

Correlation between calmodulin activity and gravitropic sensitivity in primary roots of maize

NASA Technical Reports Server (NTRS)

Stinemetz, C. L.; Kuzmanoff, K. M.; Evans, M. L.; Jarrett, H. W.

1987-01-01

Recent evidence indicates a role for calcium and calmodulin in the gravitropic response of primary roots of maize (Zea mays, L.). We examined this possibility by testing the relationship between calmodulin activity and gravitropic sensitivity in roots of the maize cultivars Merit and B73 x Missouri 17. Roots of the Merit cultivar require light to the gravitropically competent. The gravitropic response of the Missouri cultivar is independent of light. The occurrence of calmodulin in primary roots of these maize cultivars was tested by affinity gel chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with bovine brain calmodulin as standard. The distribution of calmodulin activity was measured using both the phosphodiesterase and NAD kinase assays for calmodulin. These assays were performed on whole tissue segments, crude extracts, and purified extracts. In light-grown seedlings of the Merit cultivar or in either dark- or light-grown seedlings of the Missouri cultivar, calmodulin activity per millimeter of root tissue was about 4-fold higher in the apical millimeter than in the subtending 3 millimeters. Calmodulin activity was very low in the apical millimeter of roots of dark-grown (gravitropically nonresponsive) seedlings of the Merit cultivar. Upon illumination, the calmodulin activity in the apical millimeter increased to a level comparable to that of light-grown seedlings and the roots became gravitropically competent. The time course of the development of gravitropic sensitivity following illumination paralleled the time course of the increase in calmodulin activity in the apical millimeter of the root. The results are consistent with the suggestion that calmodulin plays an important role in the gravitropic response of roots.

Stereospecificity of NAD+/NADH Reactions: A Project Experiment for Advanced Undergraduates.

ERIC Educational Resources Information Center

Lowrey, Jonathan S.; And Others

1981-01-01

Presents background information, materials needed, and experimental procedures to study enzymes dependent on pyridine nucleotide coenzymes (NAD/NADH). The experiments, suitable for advanced organic or biochemistry courses, require approximately 10-15 hours to complete. (SK)

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

PubMed Central

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

1981-01-01

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

Membraneless enzymatic ethanol/O2 fuel cell: Transitioning from an air-breathing Pt-based cathode to a bilirubin oxidase-based biocathode

NASA Astrophysics Data System (ADS)

Aquino Neto, Sidney; Milton, Ross D.; Hickey, David P.; De Andrade, Adalgisa R.; Minteer, Shelley D.

2016-08-01

The bioelectrooxidation of ethanol was investigated in a fully enzymatic membraneless ethanol/O2 biofuel cell assembly using hybrid bioanodes containing multi-walled carbon nanotube (MWCNT)-decorated gold metallic nanoparticles with either a pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) enzyme or a nicotinamide adenine dinucleotide (NAD+)-dependent ADH enzyme. The biofuel cell anode was prepared with the PQQ-dependent enzyme and designed using either a direct electron transfer (DET) architecture or via a mediated electron transfer (MET) configuration through a redox polymer, 1,1‧-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI). In the case of the bioanode containing the NAD+-dependent enzyme, only the mediated electron transfer mechanism was employed using an electropolymerized methylene green film to regenerate the NAD+ cofactor. Regardless of the enzyme being employed at the anode, a bilirubin oxidase-based biocathode prepared within a DET architecture afforded efficient electrocatalytic oxygen reduction in an ethanol/O2 biofuel cell. The power curves showed that DET-based bioanodes via the PQQ-dependent ADH still lack high current densities, whereas the MET architecture furnished maximum power density values as high as 226 ± 21 μW cm-2. Considering the complete membraneless enzymatic biofuel cell with the NAD+-dependent ADH-based bioanode, power densities as high as 111 ± 14 μW cm-2 were obtained. This shows the advantage of PQQ-dependent ADH for membraneless ethanol/O2 biofuel cell applications.

From Cholesterogenesis to Steroidogenesis: Role of Riboflavin and Flavoenzymes in the Biosynthesis of Vitamin D12

PubMed Central

Pinto, John T.; Cooper, Arthur J. L.

2014-01-01

Flavin-dependent monooxygenases and oxidoreductases are located at critical branch points in the biosynthesis and metabolism of cholesterol and vitamin D. These flavoproteins function as obligatory intermediates that accept 2 electrons from NAD(P)H with subsequent 1-electron transfers to a variety of cytochrome P450 (CYP) heme proteins within the mitochondria matrix (type I) and the (microsomal) endoplasmic reticulum (type II). The mode of electron transfer in these systems differs slightly in the number and form of the flavin prosthetic moiety. In the type I mitochondrial system, FAD-adrenodoxin reductase interfaces with adrenodoxin before electron transfer to CYP heme proteins. In the microsomal type II system, a diflavin (FAD/FMN)-dependent cytochrome P450 oxidoreductase [NAD(P)H-cytochrome P450 reductase (CPR)] donates electrons to a multitude of heme oxygenases. Both flavoenzyme complexes exhibit a commonality of function with all CYP enzymes and are crucial for maintaining a balance of cholesterol and vitamin D metabolites. Deficits in riboflavin availability, imbalances in the intracellular ratio of FAD to FMN, and mutations that affect flavin binding domains and/or interactions with client proteins result in marked structural alterations within the skeletal and central nervous systems similar to those of disorders (inborn errors) in the biosynthetic pathways that lead to cholesterol, steroid hormones, and vitamin D and their metabolites. Studies of riboflavin deficiency during embryonic development demonstrate congenital malformations similar to those associated with genetic alterations of the flavoenzymes in these pathways. Overall, a deeper understanding of the role of riboflavin in these pathways may prove essential to targeted therapeutic designs aimed at cholesterol and vitamin D metabolism. PMID:24618756

Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

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

Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.

2008-08-01

Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

Humoral and cellular immune response in Wistar Han RCC rats fed two genetically modified maize MON810 varieties for 90 days (EU 7th Framework Programme project GRACE).

PubMed

Tulinská, Jana; Adel-Patient, Karine; Bernard, Hervé; Líšková, Aurélia; Kuricová, Miroslava; Ilavská, Silvia; Horváthová, Mira; Kebis, Anton; Rollerová, Eva; Babincová, Júlia; Aláčová, Radka; Wal, Jean-Michel; Schmidt, Kerstin; Schmidtke, Jörg; Schmidt, Paul; Kohl, Christian; Wilhelm, Ralf; Schiemann, Joachim; Steinberg, Pablo

2018-07-01

The genetically modified maize event MON810 expresses a Bacillus thuringiensis-derived gene, which encodes the insecticidal protein Cry1Ab to control some lepidopteran insect pests such as the European corn borer. It has been claimed that the immune system may be affected following the oral/intragastric administration of the MON810 maize in various different animal species. In the frame of the EU-funded project GRACE, two 90-day feeding trials, the so-called studies D and E, were performed to analyze the humoral and cellular immune responses of male and female Wistar Han RCC rats fed the MON810 maize. A MON810 maize variety of Monsanto was used in the study D and a MON810 maize variety of Pioneer Hi-Bred was used in the study E. The total as well as the maize protein- and Cry1Ab-serum-specific IgG, IgM, IgA and IgE levels, the proliferative activity of the lymphocytes, the phagocytic activity of the granulocytes and monocytes, the respiratory burst of the phagocytes, a phenotypic analysis of spleen, thymus and lymph node cells as well as the in vitro production of cytokines by spleen cells were analyzed. No specific Cry1Ab immune response was observed in MON810 rats, and anti-maize protein antibody responses were similar in MON810 and control rats. Single parameters were sporadically altered in rats fed the MON810 maize when compared to control rats, but these alterations are considered to be of no immunotoxicological significance.

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

PubMed

Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering. Copyright © 2017 American Society for Microbiology.

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.

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

PubMed

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

2017-09-01

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

Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

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

Han, Seungil; Chang, Jeanne S.; Griffor, Matt

DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2more » tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.« less

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation

PubMed Central

Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

2014-01-01

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-l-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., 1994), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis. PMID:24286468

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

PubMed Central

Aguilar-Arnal, Lorena; Ranjit, Suman; Stringari, Chiara; Orozco-Solis, Ricardo; Gratton, Enrico; Sassone-Corsi, Paolo

2016-01-01

Sirtuin 1 (SIRT1) is an NAD+-dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD+ levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD+ levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD+ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD+ metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism. PMID:27791113

Permeabilization of fungal hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process.

PubMed

van der Weerden, Nicole L; Hancock, Robert E W; Anderson, Marilyn A

2010-11-26

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.

NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice.

PubMed

Lin, Jonathan B; Kubota, Shunsuke; Ban, Norimitsu; Yoshida, Mitsukuni; Santeford, Andrea; Sene, Abdoulaye; Nakamura, Rei; Zapata, Nicole; Kubota, Miyuki; Tsubota, Kazuo; Yoshino, Jun; Imai, Shin-Ichiro; Apte, Rajendra S

2016-09-27

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity.

PubMed

Cantó, Carles; Houtkooper, Riekelt H; Pirinen, Eija; Youn, Dou Y; Oosterveer, Maaike H; Cen, Yana; Fernandez-Marcos, Pablo J; Yamamoto, Hiroyasu; Andreux, Pénélope A; Cettour-Rose, Philippe; Gademann, Karl; Rinsch, Chris; Schoonjans, Kristina; Sauve, Anthony A; Auwerx, Johan

2012-06-06

As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function. Copyright © 2012 Elsevier Inc. All rights reserved.

Anatomy of an engineered NAD-binding site.

PubMed Central

Mittl, P. R.; Berry, A.; Scrutton, N. S.; Perham, R. N.; Schulz, G. E.

1994-01-01

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate. PMID:7833810

Phosphorylation of the amino terminus of maize sucrose synthase in relation to membrane association and enzyme activity.

PubMed

Hardin, Shane C; Winter, Heike; Huber, Steven C

2004-04-01

Sucrose synthase (SUS) is phosphorylated on a major, amino-terminal site located at Ser-15 (S15) in the maize (Zea mays) SUS1 protein. Site- and phospho-specific antibodies against a phosphorylated S15 (pS15) peptide allowed direct analysis of S15 phosphorylation in relation to membrane association. Immunoblots of the maize leaf elongation zone, divided into 4-cm segments, demonstrated that the abundance of soluble (s-SUS) and membrane (m-SUS) SUS protein showed distinct positional profiles. The content of m-SUS was maximal in the 4- to 8-cm segment where it represented 9% of total SUS and occurred as a peripheral membrane protein. In contrast, s-SUS was highest in the 12- to 16-cm segment. Relative to s-SUS, m-SUS was hypophosphorylated at S15 in the basal 4 cm but hyperphosphorylated in apical segments. Differing capabilities of the anti-pS15 and anti-S15 peptide antibodies to immunoprecipitate SUS suggested that phosphorylation of S15, or exposure of unphosphorylated SUS to slightly acidic pH, altered the structure of the amino terminus. These structural changes were generally coincident with the increased sucrose cleavage activity that occurs at pH values below 7.5. In vitro S15 phosphorylation of the S170A SUS protein by a maize calcium-dependent protein kinase (CDPK) significantly increased sucrose cleavage activity at low pH. Collectively, the results suggest that (1) SUS membrane binding is controlled in vivo; (2) relative pS15 content of m-SUS depends on the developmental state of the organ; and (3) phosphorylation of S15 affects amino-terminal conformation in a way that may stimulate the catalytic activity of SUS and influence membrane association.

Randomly amplified polymorphic-DNA analysis for detecting genotoxic effects of Boron on maize (Zea mays L.).

PubMed

Sakcali, M Serdal; Kekec, Guzin; Uzonur, Irem; Alpsoy, Lokman; Tombuloglu, Huseyin

2015-08-01

This study was carried out to investigate the genotoxic effect of boron (B) on maize using randomly amplified polymorphic DNA (RAPD) method. Experimental design was conducted under 0, 5, 10, 25, 50, 100, 125, and 150 ppm B exposures, and physiological changes have revealed a sharp decrease in root growth rates from 28% to 85%, starting from 25 ppm to 150 ppm, respectively. RAPD-polymerase chain reaction (PCR) analysis shows that DNA alterations are clearly observed from beginning to 100 ppm. B-induced inhibition in root growth had a positive correlation with DNA alterations. Total soluble protein, root and stem lengths, and B content analysis in root and leaves encourage these results as a consequence. These preliminary findings reveal that B causes chromosomal aberration and genotoxic effects on maize. Meanwhile, usage of RAPD-PCR technique is a suitable biomarker to detect genotoxic effect of B on maize and other crops for the future. © The Author(s) 2013.

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.

[Age-related characteristics of structural support for ovarian function].

PubMed

Koval'skiĭ, G B

1984-12-01

Histoenzymological assay was used to investigate various structures of the ovaries of rats of two groups aged 3-4 and 12-14 months during estral cycle. The activity of 3 beta-, 17 beta- and 20 alpha-steroid dehydrogenases, glucose-6-phosphate dehydrogenase, NAD and NADP-diaphorases, esterase, acid and alkaline phosphatases was studied. It has been shown that transport alterations in the microcirculation including the hematofollicular barrier play, the leading part in age-dependent depression of reproductive and endocrine functions. Ageing rats demonstrated no linkage between endothelial, thecal and granular cells, which points to the injury of the histophysiological mechanisms of the follicular system integration.

Enzymatic transformation of hydrocarbons by methanotrophic organisms

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

Patel, R.N.; Hou, C.T.

Soluble methane monooxygenase from a facultative methane-utilizing organism, Methylobacterium sp. CRL-26 or R6, catalyzed the NAD(P)H-dependent epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branch-chain alkenes, alkanes (C1-C8), substituted alkanes, branch-chain alkanes, carbon monoxide, ether, cyclic and aromatic compounds. The NAD -linked dehydrogenases such as formate dehydrogenase or secondary alcohol dehydrogenase in the presence of formate or secondary alcohol, respectively, regenerated NAD/NADH required for the methane monooxygenase in a coupled enzymes reactions. Oxidation of secondary alcohols to the corresponding methylketones in methanotrophs is catalyzed by an NAD -dependent, zinc-containing, secondary alcohol hydrogenase. Primary alcohols weremore » oxidized to the corresponding aldehydes by a phenazine methosulfate-dependent, pyrollo quinoline quinone (methoxatin or PQQ) containing, methanol dehydrogenase. Oxidation of aldehydes (C1 to C10) to the corresponding carboxylic acids is catalyzed by a heme-containing aldehyde dehydrogenase. Methanotrophs have been considered potentially useful for single cell protein (SCP), amino acids, and biopolymer production at the expense of growth on cheap and readily available C1 compounds. 80 references, 1 figure, 6 tables.« less

Engineering phenolics metabolism in the grasses using transcription factors

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

Grotewold, Erich

2013-07-26

The economical competitiveness of agriculture-derived biofuels can be significantly enhanced by increasing biomass/acre yields and by furnishing the desired carbon balance for facilitating liquid fuel production (e.g., ethanol) or for high-energy solid waste availability to be used as biopower (e.g., for electricity production). Biomass production and carbon balance are tightly linked to the biosynthesis of phenolic compounds, which are found in crops and in agricultural residues either as lignins, as part of the cell wall, or as soluble phenolics which play a variety of functions in the biology of plants. The grasses, in particular maize, provide the single major sourcemore » of agricultural biomass, offering significant opportunities for increasing renewable fuel production. Our laboratory has pioneered the use of transcription factors for manipulating plant metabolic pathways, an approach that will be applied here towards altering the composition of phenolic compounds in maize. Previously, we identified a small group of ten maize R2R3-MYB transcription factors with all the characteristics of regulators of different aspects of phenolic biosynthesis. Here, we propose to investigate the participation of these R2R3-MYB factors in the regulation of soluble and insoluble maize phenolics, using a combination of over-expression and down-regulation of these transcription factors in transgenic maize cultured cells and in maize plants. Maize cells and plants altered in the activity of these regulatory proteins will be analyzed for phenolic composition by targeted metabolic profiling. Specifically, we will I) Investigate the effect of gain- and loss-of-function of a select group of R2R3-MYB transcription factors on the phenolic composition of maize plants and II) Identify the biosynthetic genes regulated by each of the selected R2R3-MYB factors. While a likely outcome of these studies are transgenic maize plants with altered phenolic composition, this research will significantly contribute to understanding how different branches of the phenolic biosynthetic grid are regulated. Given the conservation of the selected regulators in other grasses, results derived from this project are likely to provide important tools for the manipulation of phenolic compounds in other emerging biomass producers (e.g., switchgrass or miscanthus), either through conventional breeding techniques (e.g., marker-assisted breeding) or by using transgenic approaches.« less

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

PubMed

Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

2013-09-01

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Root Interactions in a Maize/Soybean Intercropping System Control Soybean Soil-Borne Disease, Red Crown Rot

PubMed Central

Gao, Xiang; Wu, Man; Xu, Ruineng; Wang, Xiurong; Pan, Ruqian; Kim, Hye-Ji; Liao, Hong

2014-01-01

Background Within-field multiple crop species intercropping is well documented and used for disease control, but the underlying mechanisms are still unclear. As roots are the primary organ for perceiving signals in the soil from neighboring plants, root behavior may play an important role in soil-borne disease control. Principal Findings In two years of field experiments, maize/soybean intercropping suppressed the occurrence of soybean red crown rot, a severe soil-borne disease caused by Cylindrocladium parasiticum (C. parasiticum). The suppressive effects decreased with increasing distance between intercropped plants under both low P and high P supply, suggesting that root interactions play a significant role independent of nutrient status. Further detailed quantitative studies revealed that the diversity and intensity of root interactions altered the expression of important soybean PR genes, as well as, the activity of corresponding enzymes in both P treatments. Furthermore, 5 phenolic acids were detected in root exudates of maize/soybean intercropped plants. Among these phenolic acids, cinnamic acid was released in significantly greater concentrations when intercropped maize with soybean compared to either crop grown in monoculture, and this spike in cinnamic acid was found dramatically constrain C. parasiticum growth in vitro. Conclusions To the best of our knowledge, this study is the first report to demonstrate that intercropping with maize can promote resistance in soybean to red crown rot in a root-dependent manner. This supports the point that intercropping may be an efficient ecological strategy to control soil-borne plant disease and should be incorporated in sustainable agricultural management practices. PMID:24810161

Rhizosphere competent Pantoea agglomerans enhances maize (Zea mays) and chickpea (Cicer arietinum L.) growth, without altering the rhizosphere functional diversity.

PubMed

Mishra, Aradhana; Chauhan, Puneet Singh; Chaudhry, Vasvi; Tripathi, Manisha; Nautiyal, Chandra Shekhar

2011-10-01

Plant growth promoting Pantoea agglomerans NBRISRM (NBRISRM) was able to produce 60.4 μg/ml indole acetic acid and solubilize 77.5 μg/ml tri-calcium phosphate under in vitro conditions. Addition of 2% NaCl (w/v) in the media induced the IAA production and phosphate solubilization by 11% and 7%, respectively. For evaluating the plant growth promotory effect of NBRISRM inoculation a micro plot trial was conducted using maize and chickpea as host plants. The results revealed significant increase in all growth parameters tested in NBRISRM inoculated maize and chickpea plants, which were further confirmed by higher macronutrients (N, P and K) accumulation as compared to un-inoculated controls. Throughout the growing season of maize and chickpea, rhizosphere population of NBRISRM were in the range 10(7)-10(8) CFU/g soil and competing with 10(7)-10(9) CFU/g soil with heterogeneous bacterial population. Functional richness, diversity, and evenness were found significantly higher in maize rhizosphere as compared to chickpea, whereas NBRISRM inoculation were not able to change it, in both crops as compared to their un-inoculated control. To the best of our knowledge this is first report where we demonstrated the effect of P. agglomerans strain for improving maize and chickpea growth without altering the functional diversity.

Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review

PubMed Central

Buckel, Wolfgang; Thauer, Rudolf K.

2018-01-01

Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0′ = −420 mV) with NADH (−320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (−10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms—in which they have been found—are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered. PMID:29593673

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.

Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

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

Aksoy, Pinar; Escande, Carlos; Seccion Biologia Celular, Facultad de Ciencias, Universidad de la Republica, Igua 4225, Montevideo

2006-10-13

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1more » enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.« less

Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil▿

PubMed Central

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-01-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids. PMID:17526698

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil.

PubMed

Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

2007-08-01

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons.

PubMed

Ghosh, Debolina; Levault, Kelsey R; Brewer, Gregory J

2014-08-01

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg-AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg-AD neurons. We also observed an age-dependent loss of gene expression of key redox-dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age-related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age-related declines in NAD(P)H. Our data indicate that in aging and more so in AD-like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

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

Skardelly, Marco, E-mail: Marco.Skardelly@med.uni-tuebingen.de; Translational Centre for Regenerative Medicine, University of Leipzig, Leipzig; Glien, Anja

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC),more » we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment.« less

Photosynthesis, growth and maize yields in the context of global change

USDA-ARS?s Scientific Manuscript database

Maize is the third most important grain crop behind wheat and rice. Global mean temperatures are rising primarily due to anthropogenic carbon dioxide emissions into the earth’s atmosphere. Warmer temperatures over major landmasses are predicted to alter precipitation patterns and to increase the f...

New Therapeutic Concept of NAD Redox Balance for Cisplatin Nephrotoxicity

PubMed Central

Oh, Gi-Su; Kim, Hyung-Jin; Shen, AiHua; Lee, Su-Bin; Yang, Sei-Hoon; Shim, Hyeok; Cho, Eun-Young; Kwon, Kang-Beom; Kwak, Tae Hwan; So, Hong-Seob

2016-01-01

Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects such as ototoxicity, nephrotoxicity, and peripheral neuropathy. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses are closely associated with cisplatin-induced nephrotoxicity; however, the precise mechanism remains unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Recent studies have demonstrated associations between disturbance in intracellular NAD+ levels and clinical progression of various diseases through the production of reactive oxygen species and inflammation. Furthermore, we demonstrated that reduction of the intracellular NAD+/NADH ratio is critically involved in cisplatin-induced kidney damage through inflammation and oxidative stress and that increase of the cellular NAD+/NADH ratio suppresses cisplatin-induced kidney damage by modulation of potential damage mediators such as oxidative stress and inflammatory responses. In this review, we describe the role of NAD+ metabolism in cisplatin-induced nephrotoxicity and discuss a potential strategy for the prevention or treatment of cisplatin-induced adverse effects with a particular focus on NAD+-dependent cellular pathways. PMID:26881219

Mutations that Allow SIR2 Orthologs to Function in a NAD+-Depleted Environment.

PubMed

Ondracek, Caitlin R; Frappier, Vincent; Ringel, Alison E; Wolberger, Cynthia; Guarente, Leonard

2017-03-07

Sirtuin enzymes depend on NAD + to catalyze protein deacetylation. Therefore, the lowering of NAD + during aging leads to decreased sirtuin activity and may speed up aging processes in laboratory animals and humans. In this study, we used a genetic screen to identify two mutations in the catalytic domain of yeast Sir2 that allow the enzyme to function in an NAD + -depleted environment. These mutant enzymes give rise to a significant increase of yeast replicative lifespan and increase deacetylation by the Sir2 ortholog, SIRT1, in mammalian cells. Our data suggest that these mutations increase the stability of the conserved catalytic sirtuin domain, thereby increasing the catalytic efficiency of the mutant enzymes. Our approach to identifying sirtuin mutants that permit function in NAD + -limited environments may inform the design of small molecules that can maintain sirtuin activity in aging organisms. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein–Protein Interactions

PubMed Central

Bartlett, Madelaine; Thompson, Beth; Brabazon, Holly; Del Gizzi, Robert; Zhang, Thompson; Whipple, Clinton

2016-01-01

Protein–protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PIL) and AP3-like (AP3L) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PIL protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PIL homodimerization is an anomaly or indicative of broader trends, we characterized PIL dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PIL homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PIL homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PIL protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PIL dimerization activity. Furthermore, ectopic expression of a Joinvillea PIL homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. PMID:26908583

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

PubMed Central

Hensgens, C M; Vonck, J; Van Beeumen, J; van Bruggen, E F; Hansen, T A

1993-01-01

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH. Images PMID:8491707

RIBEYE(B)-domain binds to lipid components of synaptic vesicles in an NAD(H)-dependent, redox-sensitive manner.

PubMed

Schwarz, Karin; Schmitz, Frank

2017-03-20

Synaptic ribbons are needed for fast and continuous exocytosis in ribbon synapses. RIBEYE is a main protein component of synaptic ribbons and is necessary to build the synaptic ribbon. RIBEYE consists of a unique A-domain and a carboxyterminal B-domain, which binds NAD(H). Within the presynaptic terminal, the synaptic ribbons are in physical contact with large numbers of synaptic vesicle (SV)s. How this physical contact between ribbons and synaptic vesicles is established at a molecular level is not well understood. In the present study, we demonstrate that the RIBEYE(B)-domain can directly interact with lipid components of SVs using two different sedimentation assays with liposomes of defined chemical composition. Similar binding results were obtained with a SV-containing membrane fraction. The binding of liposomes to RIBEYE(B) depends upon the presence of a small amount of lysophospholipids present in the liposomes. Interestingly, binding of liposomes to RIBEYE(B) depends on NAD(H) in a redox-sensitive manner. The binding is enhanced by NADH, the reduced form, and is inhibited by NAD + , the oxidized form. Lipid-mediated attachment of vesicles is probably part of a multi-step process that also involves additional, protein-dependent processes. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The significance of nitrogen limited condition in the initiation of lipid biosynthesis in Aurantiochytrium sp. SW1

NASA Astrophysics Data System (ADS)

Haladu, Zangoma Maryam; Ibrahim, Izyanti; Hamid, Aidil Abdul

2018-04-01

The manner of the onset of lipid synthesis in Aurantiochytrium sp. SW1 as well as the possible role of NAD+ dependent isocitrate dehydrogenase (NAD+: ICDH) in the initiation of lipid biosynthesis were studied. The initiation of lipid synthesis in the microalgae was not associated with the cessation of growth, but commence at the early phase of growth. Substantial amount of lipid (30 %, g/g biomass) was accumulated during the active growth phase at 48 h with growth rate decreasing from 0.11 g/L/h during active growth to 0.02 g/L/h in the limited growth phase. At that period the activity of NAD+: ICDH was still detectable although it slightly decreased to 20 nmol/min/mg in 48 h from 25 nmol/min/mg at 24 h. Analysis of ammonium sulfate fractionated of NAD+: ICDH activity showed that NAD+: ICDH was not completely dependent on adenosine monophosphate (AMP) for its activity, although the presence of AMP increased the enzyme's affinity towards its substrate (isocitrate) indicated by the low Km value of the enzyme for isocitrate. While citrate acts as inhibitor of the enzyme only at high concentration. The probable implications of these properties to the regulation of lipid are discussed.

The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes.

PubMed

Rao, Xiaolan; Dixon, Richard A

2016-01-01

As an adaptation to changing climatic conditions that caused high rates of photorespiration, C 4 plants have evolved to display higher photosynthetic efficiency than C 3 plants under elevated temperature, high light intensities, and drought. The C 4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C 4 mechanisms to concentrate CO 2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C 4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C 4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C 4 metabolic flow, C 4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C 4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes.

The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes

PubMed Central

Rao, Xiaolan; Dixon, Richard A.

2016-01-01

As an adaptation to changing climatic conditions that caused high rates of photorespiration, C4 plants have evolved to display higher photosynthetic efficiency than C3 plants under elevated temperature, high light intensities, and drought. The C4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C4 mechanisms to concentrate CO2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C4 metabolic flow, C4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes. PMID:27790235

Pyridine nucleotides in regulation of cell death and survival by redox and non-redox reactions.

PubMed

Novak Kujundžić, Renata; Žarković, Neven; Gall Trošelj, Koraljka

2014-01-01

Changes of the level and ratios of pyridine nucleotides determine metabolism- dependent cellular redox status and the activity of poly(ADP-ribose) polymerases (PARPs) and sirtuins, thereby influencing several processes closely related to cell survival and death. Pyridine nucleotides participate in numerous metabolic reactions whereby their net cellular level remains constant, but the ratios of NAD+/NADP+ and NADH/NADPH oscillate according to metabolic changes in response to diverse stress signals. In non-redox reactions, NAD+ is degraded and quickly, afterward, resynthesized in the NAD+ salvage pathway, unless overwhelming activation of PARP-1 consumes NAD+ to the point of no return, when the cell can no longer generate enough ATP to accommodate NAD+ resynthesis. The activity of PARP-1 is mandatory for the onset of cytoprotective autophagy on sublethal stress signals. It has become increasingly clear that redox status, largely influenced by the metabolism-dependent composition of the pyridine nucleotides pool, plays an important role in the synthesis of pro-apoptotic and anti-apoptotic sphingolipids. Awareness of the involvement of the prosurvival sphingolipid, sphingosine-1-phosphate, in transition from inflammation to malignant transformation has recently emerged. Here, the participation of pyridine nucleotides in redox and non-redox reactions, sphingolipid metabolism, and their role in cell fate decisions is reviewed.

Metabolic drift in the aging brain.

PubMed

Ivanisevic, Julijana; Stauch, Kelly L; Petrascheck, Michael; Benton, H Paul; Epstein, Adrian A; Fang, Mingliang; Gorantla, Santhi; Tran, Minerva; Hoang, Linh; Kurczy, Michael E; Boska, Michael D; Gendelman, Howard E; Fox, Howard S; Siuzdak, Gary

2016-05-01

Brain function is highly dependent upon controlled energy metabolism whose loss heralds cognitive impairments. This is particularly notable in the aged individuals and in age-related neurodegenerative diseases. However, how metabolic homeostasis is disrupted in the aging brain is still poorly understood. Here we performed global, metabolomic and proteomic analyses across different anatomical regions of mouse brain at different stages of its adult lifespan. Interestingly, while severe proteomic imbalance was absent, global-untargeted metabolomics revealed an energymetabolic drift or significant imbalance in core metabolite levels in aged mouse brains. Metabolic imbalance was characterized by compromised cellular energy status (NAD decline, increased AMP/ATP, purine/pyrimidine accumulation) and significantly altered oxidative phosphorylation and nucleotide biosynthesis and degradation. The central energy metabolic drift suggests a failure of the cellular machinery to restore metabostasis (metabolite homeostasis) in the aged brain and therefore an inability to respond properly to external stimuli, likely driving the alterations in signaling activity and thus in neuronal function and communication.

A comparative study of annealing of waxy, normal and high-amylose maize starches: the role of amylose molecules.

PubMed

Wang, Shujun; Wang, Jinrong; Yu, Jinglin; Wang, Shuo

2014-12-01

The effect of annealing on starch structure and functionality of three maize starches (waxy, normal and high-amylose) was investigated, with the aim of understanding the role of amylose molecules during starch annealing. Amylose content, granular morphology and crystallinity of maize starches were little affected by annealing treatment. Annealing treatment did not alter the swelling power of waxy maize starch, but reduced the swelling power of normal and high-amylose maize starches. The thermal transition temperatures were increased, and the temperature range was decreased, but the enthalpy change was not affected greatly. The pasting viscosities of normal and waxy maize starches were decreased significantly, with the pasting temperature being little affected. The in vitro digestibility of three maize starches was not affected significantly by annealing treatment. Our results demonstrated that amylose molecules play an important role in the structural reorganization of starch granules during annealing treatment. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of the Aryl Hydrocarbon Receptor Target Gene TiPARP as a Mediator of Suppression of Hepatic Gluconeogenesis by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and of Nicotinamide as a Corrective Agent for This Effect*

PubMed Central

Diani-Moore, Silvia; Ram, Payal; Li, Xintian; Mondal, Prosenjit; Youn, Dou Yeon; Sauve, Anthony A.; Rifkind, Arleen B.

2010-01-01

The environmental toxin TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin) produces diverse toxic effects including a lethal wasting syndrome whose hallmark is suppressed hepatic gluconeogenesis. All TCDD toxicities require activation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. Whereas the mechanism for AHR induction of target genes is well understood, it is not known how AHR activation produces any TCDD toxicity. This report identifies for the first time an AHR target gene, TiPARP (TCDD-inducible poly(ADP-ribose) polymerase, PARP7) that can mediate a TCDD toxicity, i.e. suppression of hepatic gluconeogenesis. TCDD suppressed hepatic glucose production, expression of key gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), and NAD+ levels, and increased PARP activity and TiPARP expression. TCDD also increased acetylation and ubiquitin-dependent proteosomal degradation of the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α), a coactivator of PEPCK and G6Pase transcription. TiPARP overexpression reproduced TCDD effects on glucose output and NAD+ levels whereas TiPARP silencing diminished them. TiPARP overexpression also increased PGC1α acetylation and decreased PGC1α levels. In contrast, silencing of cytochromes P450 (CYP) 1A, main AHR-induced genes, did not alter TCDD suppression of gluconeogenesis. The vitamin B3 constituent, nicotinamide (NAM), prevented TCDD suppression of glucose output, NAD+, and gluconeogenic genes and stabilized PGC1α. The corrective effects of NAM could be attributed to increased NAD+ levels and suppression of AHR target gene induction. The results reveal that TiPARP can mediate a TCDD effect, that the AHR is linked to PGC1α function and stability and that NAM has novel AHR antagonist activity. PMID:20876576

NAD+ administration significantly attenuates synchrotron radiation X-ray-induced DNA damage and structural alterations of rodent testes

PubMed Central

Sheng, Caibin; Chen, Heyu; Wang, Ban; Liu, Tengyuan; Hong, Yunyi; Shao, Jiaxiang; He, Xin; Ma, Yingxin; Nie, Hui; Liu, Na; Xia, Weiliang; Ying, Weihai

2012-01-01

Synchrotron radiation (SR) X-ray has great potential for its applications in medical imaging and cancer treatment. In order to apply SR X-ray in clinical settings, it is necessary to elucidate the mechanisms underlying the damaging effects of SR X-ray on normal tissues, and to search for the strategies to reduce the detrimental effects of SR X-ray on normal tissues. However, so far there has been little information on these topics. In this study we used the testes of rats as a model to characterize SR X-ray-induced tissue damage, and to test our hypothesis that NAD+ administration can prevent SR X-ray-induced injury of the testes. We first determined the effects of SR X-ray at the doses of 0, 0.5, 1.3, 4 and 40 Gy on the biochemical and structural properties of the testes one day after SR X-ray exposures. We found that 40 Gy of SR X-ray induced a massive increase in double-strand DNA damage, as assessed by both immunostaining and Western blot of phosphorylated H2AX levels, which was significantly decreased by intraperitoneally (i.p.) administered NAD+ at doses of 125 and 625 mg/kg. Forty Gy of SR X-ray can also induce marked increases in abnormal cell nuclei as well as significant decreases in the cell layers of the seminiferous tubules one day after SR X-ray exposures, which were also ameliorated by the NAD+ administration. In summary, our study has shown that SR X-ray can produce both molecular and structural alterations of the testes, which can be significantly attenuated by NAD+ administration. These results have provided not only the first evidence that SR X-ray-induced tissue damage can be ameliorated by certain approaches, but also a valuable basis for elucidating the mechanisms underlying SR X-ray-induced tissue injury. PMID:22518270

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

PubMed

Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

2014-02-01

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

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.

Protein Conformational Gating of Enzymatic Activity in Xanthine Oxidoreductase

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

Ishikita, Hiroshi; Eger, Bryan T.; Okamoto, Ken

2012-05-24

In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E{sub sq/hq}) is {approx}170 mV, a striking difference. The former greatly prefers NAD{sup +} as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD{sup +}), however, the redox potential of the electron donor FeS-IImore » is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 {angstrom} resolution crystal structures for XDH, XO, the NAD{sup +}- and NADH-complexed XDH, E{sub sq/hq} were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E{sub sq/hq} difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD{sup +} binding at XDH. Instead, the positive charge of the NAD{sup +} ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD{sup +} molecule all contribute to altering E{sub sq/hq} upon NAD{sup +} binding to XDH.« less

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.

NAD+-dependent sirtuin 1 and 6 proteins coordinate a switch from glucose to fatty acid oxidation during the acute inflammatory response.

PubMed

Liu, Tie Fu; Vachharajani, Vidula T; Yoza, Barbara K; McCall, Charles E

2012-07-27

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

Wheat glutenin alters protein body structure in maize but not levels of endogenous storage proteins

USDA-ARS?s Scientific Manuscript database

Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. They belong to three subfamilies of grasses or Poaceae. Wheat, barley, and oats belong to the subfamily Pooideae, rice to the Ehrhartoideae, and maize, millets, sugarcane, and sorghum to the Panicoid...

The ZmCLA4 gene in the qLA4-1 QTL controls leaf angle in maize (Zea mays L.).

PubMed

Zhang, J; Ku, L X; Han, Z P; Guo, S L; Liu, H J; Zhang, Z Z; Cao, L R; Cui, X J; Chen, Y H

2014-09-01

Maize architecture is a major contributing factor to their high level of productivity. Maize varieties with an erect-leaf-angle (LA) phenotype, which increases light harvesting for photosynthesis and grain-filling, have elevated grain yields. Although a large body of information is available on the map positions of quantitative trait loci (QTL) for LA, little is known about the molecular mechanism of these QTL. In this study, the ZmCLA4 gene, which is responsible for the qLA4-1 QTL associated with LA, was identified and isolated by fine mapping and positional cloning. The ZmCLA4 gene is an orthologue of LAZY1 in rice and Arabidopsis. Sequence analysis revealed two SNPs and two indel sites in ZmCLA4 between the D132 and D132-NIL inbred maize lines. Association analysis showed that C/T/mutation667 and CA/indel965 were strongly associated with LA. Subcellular localization verified the functions of a predicted transmembrane domain and a nuclear localization signal in ZmCLA4. Transgenic maize plants with a down-regulated ZmCLA4 RNAi construct and transgenic rice plants over-expressing ZmCLA4 confirmed that the ZmCLA4 gene located in the qLA4 QTL regulated LA. The allelic variants of ZmCLA4 in the D132 and D132-NIL lines exhibited significant differences in leaf angle. ZmCLA4 transcript accumulation was higher in D132-NIL than in D132 during all the developmental stages and was negatively correlated with LA. The gravitropic response was increased and cell shape and number at the leaf and stem junctions were altered in D132-NIL relative to D132. These findings suggest that ZmCLA4 plays a negative role in the control of maize LA through the alteration of mRNA accumulation, leading to altered shoot gravitropism and cell development. The cloning of the gene responsible for the qLA4-1 QTL provides information on the molecular mechanisms of LA in maize and an opportunity for the improvement of plant architecture with regard to LA through maize breeding. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions.

PubMed

Dijk, John A; Gerritse, Jan; Schraa, Gosse; Stams, Alfons J M

2004-12-01

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

A systematic proteomic analysis of NaCl-stressed germinating maize seeds.

PubMed

Meng, Ling-Bo; Chen, Yi-Bo; Lu, Tian-Cong; Wang, Yue-Feng; Qian, Chun-Rong; Yu, Yang; Ge, Xuan-Liang; Li, Xiao-Hui; Wang, Bai-Chen

2014-05-01

Salt (NaCl) is a common physiological stressor of plants. To better understand how germinating seeds respond to salt stress, we examined the changes that occurred in the proteome of maize seeds during NaCl-treated germination. Phenotypically, salt concentrations less than 0.2 M appear to delay germination, while higher concentrations disrupt development completely, leading to seed death. The identities of 96 proteins with expression levels altered by NaCl-incubation were established using 2-DE-MALDI-TOF-MS and 2-DE-MALDI-TOF-MS/MS. Of these 96 proteins, 79 were altered greater than twofold when incubated with a 0.2 M salt solution, while 51 were altered when incubated with a 0.1 M salt solution. According to their functional annotations in the Swiss-Prot protein-sequence databases, these proteins are mainly involved in seed storage, energy metabolism, stress response, and protein metabolism. Notably, the expression of proteins that respond to abscisic acid signals increased in response to salt stress. The results of this study provide important clues as to how NaCl stresses the physiology of germinating maize seeds.

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

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

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.

Temporal Shift of Circadian-Mediated Gene Expression and Carbon Fixation Contributes to Biomass Heterosis in Maize Hybrids.

PubMed

Ko, Dae Kwan; Rohozinski, Dominica; Song, Qingxin; Taylor, Samuel H; Juenger, Thomas E; Harmon, Frank G; Chen, Z Jeffrey

2016-07-01

Heterosis has been widely used in agriculture, but the molecular mechanism for this remains largely elusive. In Arabidopsis hybrids and allopolyploids, increased photosynthetic and metabolic activities are linked to altered expression of circadian clock regulators, including CIRCADIAN CLOCK ASSOCIATED1 (CCA1). It is unknown whether a similar mechanism mediates heterosis in maize hybrids. Here we report that higher levels of carbon fixation and starch accumulation in the maize hybrids are associated with altered temporal gene expression. Two maize CCA1 homologs, ZmCCA1a and ZmCCA1b, are diurnally up-regulated in the hybrids. Expressing ZmCCA1 complements the cca1 mutant phenotype in Arabidopsis, and overexpressing ZmCCA1b disrupts circadian rhythms and biomass heterosis. Furthermore, overexpressing ZmCCA1b in maize reduced chlorophyll content and plant height. Reduced height stems from reduced node elongation but not total node number in both greenhouse and field conditions. Phenotypes are less severe in the field than in the greenhouse, suggesting that enhanced light and/or metabolic activities in the field can compensate for altered circadian regulation in growth vigor. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis reveals a temporal shift of ZmCCA1-binding targets to the early morning in the hybrids, suggesting that activation of morning-phased genes in the hybrids promotes photosynthesis and growth vigor. This temporal shift of ZmCCA1-binding targets correlated with nonadditive and additive gene expression in early and late stages of seedling development. These results could guide breeding better hybrid crops to meet the growing demand in food and bioenergy.

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 development of heart failure in mice, more robustly in DCM, and partially after transverse aorta constriction, by stabilizing myocardial NAD + levels in the failing heart. Nicotinamide riboside treatment also robustly increases the myocardial levels of 3 metabolites, nicotinic acid adenine dinucleotide, methylnicotinamide, and N1-methyl-4-pyridone-5-carboxamide, that can be used as validation biomarkers for the treatment. The data show that nicotinamide riboside, the most energy-efficient among NAD precursors, could be useful for treatment of heart failure, notably in the context of DCM, a disease with few therapeutic options. © 2017 American Heart Association, Inc.

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

PubMed

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

2014-06-01

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

Complete mitochondrial genome and evolutionary analysis of Turritopsis dohrnii, the "immortal" jellyfish with a reversible life-cycle.

PubMed

Lisenkova, A A; Grigorenko, A P; Tyazhelova, T V; Andreeva, T V; Gusev, F E; Manakhov, A D; Goltsov, A Yu; Piraino, S; Miglietta, M P; Rogaev, E I

2017-02-01

Turritopsis dohrnii (Cnidaria, Hydrozoa, Hydroidolina, Anthoathecata) is the only known metazoan that is capable of reversing its life cycle via morph rejuvenation from the adult medusa stage to the juvenile polyp stage. Here, we present a complete mitochondrial (mt) genome sequence of T. dohrnii, which harbors genes for 13 proteins, two transfer RNAs, and two ribosomal RNAs. The T. dohrnii mt genome is characterized by typical features of species in the Hydroidolina subclass, such as a high A+T content (71.5%), reversed transcriptional orientation for the large rRNA subunit gene, and paucity of CGN codons. An incomplete complementary duplicate of the cox1 gene was found at the 5' end of the T. dohrnii mt chromosome, as were variable repeat regions flanking the chromosome. We identified species-specific variations (nad5, nad6, cob, and cox1 genes) and putative selective constraints (atp8, nad1, nad2, and nad5 genes) in the mt genes of T. dohrnii, and predicted alterations in tertiary structures of respiratory chain proteins (NADH4, NADH5, and COX1 proteins) of T. dohrnii. Based on comparative analyses of available hydrozoan mt genomes, we also determined the taxonomic relationships of T. dohrnii, recovering Filifera IV as a paraphyletic taxon, and assessed intraspecific diversity of various Hydrozoa species. Copyright © 2016 Elsevier Inc. All rights reserved.

Linking a sea level pressure anomaly dipole over North America to the central Pacific El Niño

NASA Astrophysics Data System (ADS)

Ding, Ruiqiang; Li, Jianping; Tseng, Yu-heng; Sun, Cheng; Zheng, Fei

2017-08-01

This study demonstrates the close connection between the north-south dipole pattern of sea level pressure anomalies over northeastern North America to the western tropical North Atlantic, referred to as the North American dipole (NAD), and the central Pacific (CP)-type El Niño a year later. In contrast to other ENSO precursors, such as the North Pacific Oscillation (NPO) and Pacific-North America (PNA) pattern, the NAD appears more closely related to the CP-type El Niño than to the eastern Pacific (EP)-type El Niño, indicating that the NAD may serve as a unique precursor for the CP El Niño. The wintertime NAD induces sea surface temperature anomalies in the northern tropical Atlantic (NTA), which subsequently play an important role in developing the CP El Niño-like pattern in the tropical Pacific over the course of the following year. It appears that the NAD influence on CP El Niño involves air-sea interaction over several major basins, including the subtropical/tropical Pacific and the NTA. Additional analysis indicates that the correlation of either the NAD index or the NPO index with the CP El Niño state a year later depends on the status of the other index. When the wintertime NAD index is of the opposite sign to the simultaneous NPO index, the correlation of the NAD or NPO index with the Niño4 index becomes much weaker.

Malate valves: Old shuttles with new perspectives.

PubMed

Selinski, Jennifer; Scheibe, Renate

2018-06-22

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyze the reversible interconversion of malate and oxaloacetate and their transport. Depending on the coenzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes: Activities of NAD-dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids, respectively. In addition, chloroplasts possess a NADP-dependent MDH isoform. The NADP-MDH as part of the "light malate valve" plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post-translational redox-modification mediated via the ferredoxin-thioredoxin system and fine control via the NADP + /NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD-MDH ("dark malate valve") is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, the knowledge about regulation of the other two cytosolic MDHs as well as NAD-MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria, and peroxisomes have been characterized, but not much is known about cytosolic NAD-MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange focusing on the various metabolic functions of these valves. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

A Gene-Oriented Haplotype Comparison Reveals Recently Selected Genomic Regions in Temperate and Tropical Maize Germplasm

PubMed Central

Zhang, Jie; Li, Yongxiang; Zheng, Jun; Zhang, Hongwei; Yang, Xiaohong; Wang, Jianhua; Wang, Guoying

2017-01-01

The extensive genetic variation present in maize (Zea mays) germplasm makes it possible to detect signatures of positive artificial selection that occurred during temperate and tropical maize improvement. Here we report an analysis of 532,815 polymorphisms from a maize association panel consisting of 368 diverse temperate and tropical inbred lines. We developed a gene-oriented approach adapting exonic polymorphisms to identify recently selected alleles by comparing haplotypes across the maize genome. This analysis revealed evidence of selection for more than 1100 genomic regions during recent improvement, and included regulatory genes and key genes with visible mutant phenotypes. We find that selected candidate target genes in temperate maize are enriched in biosynthetic processes, and further examination of these candidates highlights two cases, sucrose flux and oil storage, in which multiple genes in a common pathway can be cooperatively selected. Finally, based on available parallel gene expression data, we hypothesize that some genes were selected for regulatory variations, resulting in altered gene expression. PMID:28099470

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

PubMed Central

Kishko, Iryna; Harish, Balasubramanian; Zayats, Vasilina; Reha, David; Tenner, Brian; Beri, Dhananjay; Gustavsson, Tobias; Ettrich, Rüdiger; Carey, Jannette

2012-01-01

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots. PMID:22952804

The unique kinetic behavior of the very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum.

PubMed

Kawakami, Ryushi; Oyama, Masaki; Sakuraba, Haruhiko; Ohshima, Toshihisa

2010-01-01

The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum showed positive cooperativity toward alpha-ketoglutarate and NADH, and the Michaelis-Menten type toward ammonium chloride in the absence of the catalytic activator, L-aspartate. An increase in the maximum activity accompanied the decrease in the S(0.5) values for alpha-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for alpha-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mM L-aspartate.

Initial reactions involved in the dissimilation of mandelate by Rhodotorula graminis.

PubMed Central

Durham, D R

1984-01-01

Rhodotorula graminis utilized DL-mandelate, L(+)-mandelate, and D(-)-mandelate as sole sources of carbon and energy. Growth on these aromatic substrates resulted in the induction of an NAD-dependent D(-)-mandelate dehydrogenase and a dye-linked L(+)-mandelate dehydrogenase, each catalyzing the stereospecific conversion of its respective enantiomer of mandelate to benzoylformate. Benzoylformate was oxidized to benzaldehyde, which was dehydrogenated to benzoate by an NAD-dependent benzaldehyde dehydrogenase. Benzoate was further metabolized through p-hydroxybenzoate and the protocatechuate branch of the beta-ketoadipate pathway. PMID:6389497

Ethanol disrupts chondrification of the neurocranial cartilages in medaka embryos without affecting aldehyde dehydrogenase 1A2 (Aldh1A2) promoter methylation

PubMed Central

Hu, Yuhui; Willett, Kristine L.; Khan, Ikhlas A.; Scheffler, Brian E.; Dasmahapatra, Asok K.

2009-01-01

Medaka (Oryzias latipes) embryos at different developmental stages were exposed to ethanol for 48 h, then allowed to hatch. Teratogenic effects were evaluated in hatchlings after examining chondrocranial cartilage deformities. Ethanol disrupted cartilage development in medaka in a dose and developmental stage-specific manner. Compared to controls, the linear length of the neurocranium and other cartilages were reduced in ethanol-treated groups. Moreover, the chondrification in cartilages, specifically trabeculae and polar cartilages, were inhibited by ethanol. To understand the mechanism of ethanol teratogenesis, NAD+: NADH status during embryogenesis and the methylation pattern of Aldh1A2 promoter in whole embryos and adult tissues (brain, eye, heart and liver) were analyzed. Embryos 6 dpf had higher NAD+ than embryos 0 or 2 dpf. Ethanol (200 or 400 mM) was able to reduce NAD+ content in 2 and 6 dpf embryos. However, in both cases reductions were not significantly different from the controls. Moreover, no significant difference in either NADH content or in NAD+: NADH status of the ethanol-treated embryos, with regard to controls, was observed. The promoter of Aldh1A2 contains 31 CpG dinucleotides (-705 to +154, ATG = +1); none of which were methylated. Compared to controls, embryonic ethanol exposure (100 and 400 mM) was unable to alter Aldh1A2 promoter methylation in embryos or in the tissues of adults (breeding) developmentally exposed to ethanol (300 mM, 48 hpf). From these data we conclude that ethanol teratogenesis in medaka does not induce alteration in the methylation pattern of Aldh1A2 promoter, but does change cartilage development. PMID:19651241

SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.

PubMed

Schäfer, Martin; Le, Tung B K; Hearnshaw, Stephen J; Maxwell, Anthony; Challis, Gregory L; Wilkinson, Barrie; Buttner, Mark J

2015-06-19

Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone. Copyright © 2015. Published by Elsevier Ltd.

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I*

PubMed Central

Quinlan, Casey L.; Goncalves, Renata L. S.; Hey-Mogensen, Martin; Yadava, Nagendra; Bunik, Victoria I.; Brand, Martin D.

2014-01-01

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I. PMID:24515115

Atmospheric oscillations comparison on long term tropospheric delay time series derived from ray-tracing and GPS

NASA Astrophysics Data System (ADS)

Nikolaidou, Thalia; Santos, Marcelo

2017-04-01

The caused time delay induced by the atmosphere on the GNSS signals (NAD), depends primarily on the amount of atmosphere the signal traverses till it reaches to the Earth's surface and can exceed t 20 m for low elevation angles (around 3 degrees). For a particular ray i.e. satellite/quasar-antenna link, the delay depends on the atmospheric parameters of total pressure, temperature, and the partial pressure of water vapor. Because of that, numerical weather models (NWM) have already proven beneficial for atmospheric modelling and geodesy. By direct raytracing, inside NWM, the VMF1 and the University of New Brunswick VMF1 (UNB-VMF1) (Urquhart et al. 2011), access the 3D variation of the meteorological parameters that determine the delay thus being the state-the-art mapping functions used today. The raytracing procedure is capable of providing NADs delays for any point on the Earth's surface. In this study we study the impact of regional numerical weather models, with high spatial and temporal resolution, namely 25km and 6h. These models outweigh the currently used NWM by having about 2.6 times better spatial resolution. Raytracing through such NWM, using the independent raytracing algorithm develop at UNB (Nievinski, 2009), we acquire superior quality NADs with regional application. We ray-trace for the International GNSS service (IGS) network stations for a time span of 11 years. Benchmarking against the IGS troposphere product is performed to access the accuracy of our results. A periodicity analysis is conducted to examine the signature of atmospheric oscillations on the NAD time series. In order to recognize the NAD periodicities, we compared our product against the GPS-derived IGS troposphere product. Systematic effects within each single technique are identified and long-term NAD stability is accessed.

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

PubMed

Sugiya, H; Hara-Yokoyama, M; Furuyama, S

1992-03-30

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.

Dissecting the Mechanisms of Drug Resistance in BRCA1/2-Mutant Breast Cancers

DTIC Science & Technology

2017-10-01

LPS (25 mg/ml), IL-4(5 ng/ml) and RP105 (0.5 mg/ml). PAR levels in stimulated B cells were analyzed on day 3 by ELISA . *pɘ.05. 4...cells reproducibly by ELISA (Figure 1). PARPs are NAD+-dependent enzymes and thus require a source of NAD+ in all cellular compartments in which

Successive contractile periods activate mitochondria at the onset of contractions in intact rat cardiac trabeculae.

PubMed

Wüst, Rob C I; Stienen, Ger J M

2018-04-01

The rate of oxidative phosphorylation depends on the contractile activity of the heart. Cardiac mitochondrial oxidative phosphorylation is determined by free ADP concentration, mitochondrial Ca 2+ accumulation, mitochondrial enzyme activities, and Krebs cycle intermediates. The purpose of the present study was to examine the factors that limit oxidative phosphorylation upon rapid changes in contractile activity in cardiac muscle. We tested the hypotheses that prior contractile performance enhances the changes in NAD(P)H and FAD concentration upon an increase in contractile activity and that this mitochondrial "priming" depends on pyruvate dehydrogenase activity. Intact rat cardiac trabeculae were electrically stimulated at 0.5 Hz for at least 30 min. Thereafter, two equal bouts at elevated stimulation frequency of 1, 2, or 3 Hz were applied for 3 min with 3 min of 0.5-Hz stimulation in between. No discernible time delay was observed in the changes in NAD(P)H and FAD fluorescence upon rapid changes in contractile activity. The amplitudes of the rapid changes in fluorescence upon an increase in stimulation frequency (the on-transients) were smaller than upon a decrease in stimulation frequency (the off-transients). A first bout in glucose-containing superfusion solution resulted, during the second bout, in an increase in the amplitudes of the on-transients, but the off-transients remained the same. No such priming effect was observed after addition of 10 mM pyruvate. These results indicate that mitochondrial priming can be observed in cardiac muscle in situ and that pyruvate dehydrogenase activity is critically involved in the mitochondrial adaptation to increases in contractile performance. NEW & NOTEWORTHY Mitochondrial respiration increases with increased cardiac contractile activity. Similar to mitochondrial "priming" in skeletal muscle, we hypothesized that cardiac mitochondrial activity is altered upon successive bouts of contractions and depends on pyruvate dehydrogenase activity. We found altered bioenergetics upon repeated contractile periods, indicative of mitochondrial priming in rat myocardium. No effect was seen when pyruvate was added to the perfusate. As such, pyruvate dehydrogenase activity is involved in the mitochondrial adaptation to increased contractile performance.

Simulated heat waves during maize reproductive stages alter reproductive growth but have no lasting effect when applied during vegetative stages

USDA-ARS?s Scientific Manuscript database

Due to climate change, heat waves are predicted to become more frequent and severe. While long-term studies on temperature stress have been conducted on important crops such as maize (Zea mays), the immediate and or long-term effects of short duration but extreme high temperature events during key d...

Maize (Zea mays) seeds can detect above-ground weeds; thiamethoxam alters the view.

PubMed

Afifi, Maha; Lee, Elizabeth; Lukens, Lewis; Swanton, Clarence

2015-09-01

Far red light is known to penetrate soil and delay seed germination. Thiamethoxam as a seed treatment has been observed to enhance seed germination. No previous work has explored the effect of thiamethoxam on the physiological response of buried maize seed when germinating in the presence of above-ground weeds. We hypothesised that the changes in red:far red reflected from above-ground weeds would be detected by maize seed phytochrome and delay seed germination by decreasing the level of GA and increasing ABA. We further hypothesised that thiamethoxam would overcome this delay in germination. Thiamethoxam enhanced seed germination in the presence of above-ground weeds by increasing GA signalling and downregulating DELLA protein and ABA signalling genes. An increase in amylase activity and a degradation of starch were also observed. Far red reflected from the above-ground weeds was capable of penetrating below the soil surface and was detected by maize seed phytochrome. Thiamethoxam altered the effect of far red on seed germination by stimulating GA and inhibiting ABA synthesis. This is the first study to suggest that the mode of action of thiamethoxam involves both GA synthesis and ABA inhibition. © 2014 Society of Chemical Industry.

Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134

PubMed Central

Kang, ChulHee; Hayes, Robert; Sanchez, Emiliano J.; Webb, Brian N.; Li, Qunrui; Hooper, Travis; Nissen, Mark S.; Xun, Luying

2012-01-01

Summary FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn2+ coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn2+ coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD+ dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD+ to NADH that is subsequently used for furfural reduction. PMID:22081946

Carotenoid Stability during Dry Milling, Storage, and Extrusion Processing of Biofortified Maize Genotypes.

PubMed

Ortiz, Darwin; Ponrajan, Amudhan; Bonnet, Juan Pablo; Rocheford, Torbert; Ferruzzi, Mario G

2018-05-09

Translation of the breeding efforts designed to biofortify maize ( Z. mays) genotypes with higher levels of provitamin A carotenoid (pVAC) content for sub-Saharan Africa is dependent in part on the stability of carotenoids during postharvest through industrial and in-home food processing operations. The purpose of this study was to simulate production of commercial milled products by determining the impact of dry milling and extrusion processing on carotenoid stability in three higher pVAC maize genotypes (C17xDE3, Orange ISO, Hi27xCML328). Pericarp and germ removal of biofortified maize kernels resulted in ∼10% loss of total carotenoids. Separating out the maize flour fraction (<212 μm) resulted in an additional ∼15% loss of total carotenoids. Carotenoid degradation was similar across milled maize fractions. Dry-milled products of Orange ISO and Hi27xCML328 genotypes showed ∼28% pVAC loss after 90-days storage. Genotype C17xDE3, with highest levels of all- trans-β-carotene, showed a 68% pVAC loss after 90-day storage. Extrusion processing conditions were optimal at 35% extrusion moisture, producing fully cooked instant maize flours with high pVAC retention (70-93%). These results support the notion that postharvest losses in maize milled fractions may be dependent, in part, on genotype and that extrusion processing may provide an option for preserving biofortified maize products.

Malate decarboxylases: evolution and roles of NAD(P)-ME isoforms in species performing C(4) and C(3) photosynthesis.

PubMed

Maier, Alexandra; Zell, Martina B; Maurino, Veronica G

2011-05-01

In the C(4) pathway of photosynthesis two types of malate decarboxylases release CO(2) in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C(4) decarboxylases involved in C(4) photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C(4) NADP-ME is thought to have evolved from a C(3) chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C(4) NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C(4) plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C(3) ancestors. In addition to this, for the evolution of C(4) NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C(3) ancestors would have changed the expression without gene duplication.

NAD1 Controls Defense-Like Responses in Medicago truncatula Symbiotic Nitrogen Fixing Nodules Following Rhizobial Colonization in a BacA-Independent Manner

PubMed Central

Domonkos, Ágota; Kovács, Szilárd; Gombár, Anikó; Kiss, Ernő; Horváth, Beatrix; Kováts, Gyöngyi Z.; Farkas, Attila; Tóth, Mónika T.; Ayaydin, Ferhan; Bóka, Károly; Fodor, Lili; Endre, Gabriella; Kaló, Péter

2017-01-01

Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatula DNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules. PMID:29240711

NAD1 Controls Defense-Like Responses in Medicago truncatula Symbiotic Nitrogen Fixing Nodules Following Rhizobial Colonization in a BacA-Independent Manner.

PubMed

Domonkos, Ágota; Kovács, Szilárd; Gombár, Anikó; Kiss, Ernő; Horváth, Beatrix; Kováts, Gyöngyi Z; Farkas, Attila; Tóth, Mónika T; Ayaydin, Ferhan; Bóka, Károly; Fodor, Lili; Ratet, Pascal; Kereszt, Attila; Endre, Gabriella; Kaló, Péter

2017-12-14

Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatula DNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules.

[Co-adaptation of enzymatic systems of cells and blood supply in smooth muscle tumors of the corpus uteri].

PubMed

Lazaarev, A F; Avbalian, A M; Bobrov, I P; Klimachev, V V; Mischenko, E V

2008-01-01

We investigated co-adaptation of enzymatic systems of cells using data on activity of NAD(Ph)-dependent enzymes and AgNOR proteins of vascular endothelium vis-a-vis angiogenesis in benign and malignant smooth muscle tumors of the corpus uteri. Overall metabolic activity (NAD-H2 diaphorase) was found to directly correlate with angiogenesis and endothelial vessel proliferation (r = 0.76 and 0.84, respectively). SDH-regulated oxidation in the main metabolic succession of a tricarbonic acid cycle depended on blood supply and endothelial vessel proliferation (r = 0.84 and 0.92, respectively; p = 0.04). A similar relationship was shown for anaerobic glycolysis of SDH (LDH content), on the one hand, and blood supply and endothelial vessel proliferation(r = 0.57 and 0.70, respectively; p = 0.02), on the other. Hence, metabolic profile varied in unaltered myometrium and tumor with variable cellular density and peculiar extracellular matrix. The highest levels of metabolic activity with NAD(Ph)-dependent enzyme co-adaptation was observed in sarcomas which were also characterized by the highest vascular density for endothelial proliferation.

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

Proteotoxicity and the contrasting effects of oxaloacetate and glycerol on Caenorhabditis elegans life span: a role for methylglyoxal?

PubMed

Hipkiss, Alan R

2010-10-01

Because accumulation of altered proteins is the most common biochemical symptom of aging, it is at least possible that such proteotoxicity may cause aging and influence life span. The life span of the nematode worm Caenorhabditis elegans is strongly influenced by changes in the intracellular concentration of methylglyoxal (MG), a putative source of much age-related proteotoxicity and organelle, cellular, and molecular dysfunction. Glycerol has recently been shown to shorten, whereas oxaloacetate has been found to extend, life span in C. elegans. It is suggested here that glycerol and oxaloacetate exert opposing effects on MG formation in C. elegans. It is proposed that, if not secreted by aquaporin, glycerol is converted to glycerol phosphate and then to dihydroxyacetone phosphate (DHAP) via a reaction requiring nicotinamide adenine dinucleotide (NAD(+)). This inhibits operation of the glycerol phosphate cycle in which DHAP is converted into glycerol phosphate, which concomitantly regenerates NAD(+) from NADH, thereby ensuring glycolytic oxidation of glyceraldehyde-3-phosphate (G3P). Because DHAP and G3P spontaneously decompose into MG, and NAD(+) is required for conversion of G3P into phosphoglycerate, the glycerol-induced increased DHAP formation and decreased NAD(+) availability will increase the potential for MG generation. In contrast, oxaloacetate may decrease MG generation by stimulating the operation of the malate-oxaloacetate shuttle, in which oxaloacetate is converted to malate, which regenerates NAD(+) from NADH. By the ensuing G3P oxidation, increased NAD(+) availability will decrease the potential for MG formation. It should be noted that mitochondria are involved in the operation of the above cycle/shuttles and that increased NAD(+) availability also stimulates those sirtuin activities that increase mitogenesis and mitochondrial activity via effects on signal transduction and gene expression, which frequently accompany dietary restriction-induced life span extension.

Benchmark analysis of native and artificial NAD+-dependent enzymes generated by a sequence based design method with or without phylogenetic data.

PubMed

Nakano, Shogo; Motoyama, Tomoharu; Miyashita, Yurina; Ishizuka, Yuki; Matsuo, Naoya; Tokiwa, Hiroaki; Shinoda, Suguru; Asano, Yasuhisa; Ito, Sohei

2018-05-22

The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial L-threonine 3-dehydrogenase (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1 and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD+ recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH): the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5°C higher than CnTDH, respectively, and the dissociation constants toward NAD+ of FcTDH-N1 and AncTDH were two- and seven-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.

4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

PubMed

Synesiou, Elena; Fairbanks, Lynnette D; Simmonds, H Anne; Slominska, Ewa M; Smolenski, Ryszard T; Carrey, Elizabeth A

2011-06-01

We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.

Pharmacological Inhibitors of NAD Biosynthesis as Potential An ticancer Agents.

PubMed

Lucas, Stephanie; Soave, Claire; Nabil, Ghazal; Ahmed, Zainab Sabry Othman; Chen, Guohua; El-Banna, Hossny Awad; Dou, Q Ping; Wang, Jian

2017-01-01

Alteration of cellular metabolism is a hallmark of cancer, which underlies exciting opportunities to develop effective, anti-cancer therapeutics through inhibition of cancer metabolism. Nicotinamide Adenine Dinucleotide (NAD+), an essential coenzyme of energy metabolism and a signaling molecule linking cellular energy status to a spectrum of molecular regulation, has been shown to be in high demand in a variety of cancer cells. Depletion of NAD+ by inhibition of its key biosynthetic enzymes has become an attractive strategy to target cancer. The main objective of this article is to review the recent patents which develop and implicate the chemical inhibitors of the key NAD+ biosynthetic enzymes for cancer treatment. We first discuss the biological principles of NAD+ metabolism in normal and malignant cells, with a focus on the feasibility of selectively targeting cancer cells by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT) and indoleamine/tryptophan 2,3-dioxygenases (IDO/TDO), the rate-limiting salvage and de novo NAD+ biosynthetic enzymes, respectively. We then analyze a series of recent patents on development and optimization of chemical scaffolds for inhibiting NAMPT or IDO/TDO enzymes as potential anticancer drugs. Conclusion and Results: We have reviewed 16 relevant patents published since 2015, and summarized the chemical properties, mechanisms of action and proposed applications of the patented compounds. Without a better understanding of the properties of these compounds, their utility for further optimization and clinical use is unknown. For the compounds that have been tested using cell and mouse models of cancer, results look promising and clinical trials are currently ongoing to see if these results translate to improved cancer treatments. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

NAD(P)H-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

PubMed

Petrovova, Miroslava; Tkadlec, Jan; Dvoracek, Lukas; Streitova, Eliska; Licha, Irena

2014-01-01

One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

Ecological guild and enzyme activities of rhizosphere soil microbial communities associated with Bt-maize cultivation under field conditions in North West Province of South Africa.

PubMed

van Wyk, Deidré A B; Adeleke, Rasheed; Rhode, Owen H J; Bezuidenhout, Carlos C; Mienie, Charlotte

2017-09-01

Insecticidal proteins expressed by genetically modified Bt maize may alter the enzymatic and microbial communities associated with rhizosphere soil. This study investigated the structure and enzymatic activity of rhizosphere soil microbial communities associated with field grown Bt and non-Bt maize. Rhizosphere soil samples were collected from Bt and non-Bt fields under dryland and irrigated conditions. Samples were subjected to chemical tests, enzyme analyses, and next generation sequencing. Results showed that nitrate and phosphorus concentrations were significantly higher in non-Bt maize dryland soils, while organic carbon was significantly higher in non-Bt maize irrigated field soil. Acid phosphatase and β-glucosidase activities were significantly reduced in soils under Bt maize cultivation. The species diversity differed between fields and Bt and non-Bt maize soils. Results revealed that Actinobacteria, Proteobacteria, and Acidobacteria were the dominant phyla present in these soils. Redundancy analyses indicated that some chemical properties and enzyme activities could explain differences in bacterial community structures. Variances existed in microbial community structures between Bt and non-Bt maize fields. There were also differences between the chemical and biochemical properties of rhizosphere soils under Bt and non-Bt maize cultivation. These differences could be related to agricultural practices and cultivar type. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic drift in the aging brain

PubMed Central

Ivanisevic, Julijana; Stauch, Kelly L.; Petrascheck, Michael; Benton, H. Paul; Epstein, Adrian A.; Fang, Mingliang; Gorantla, Santhi; Tran, Minerva; Hoang, Linh; Kurczy, Michael E.; Boska, Michael D.; Gendelman, Howard E.; Fox, Howard S.; Siuzdak, Gary

2016-01-01

Brain function is highly dependent upon controlled energy metabolism whose loss heralds cognitive impairments. This is particularly notable in the aged individuals and in age-related neurodegenerative diseases. However, how metabolic homeostasis is disrupted in the aging brain is still poorly understood. Here we performed global, metabolomic and proteomic analyses across different anatomical regions of mouse brain at different stages of its adult lifespan. Interestingly, while severe proteomic imbalance was absent, global-untargeted metabolomics revealed an energy metabolic drift or significant imbalance in core metabolite levels in aged mouse brains. Metabolic imbalance was characterized by compromised cellular energy status (NAD decline, increased AMP/ATP, purine/pyrimidine accumulation) and significantly altered oxidative phosphorylation and nucleotide biosynthesis and degradation. The central energy metabolic drift suggests a failure of the cellular machinery to restore metabostasis (metabolite homeostasis) in the aged brain and therefore an inability to respond properly to external stimuli, likely driving the alterations in signaling activity and thus in neuronal function and communication. PMID:27182841

Methylotrophic Bacillus methanolicus Encodes Two Chromosomal and One Plasmid Born NAD+ Dependent Methanol Dehydrogenase Paralogs with Different Catalytic and Biochemical Properties

PubMed Central

Müller, Jonas E. N.; Kupper, Christiane E.; Schneider, Olha; Vorholt, Julia A.; Ellingsen, Trond E.; Brautaset, Trygve

2013-01-01

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD+-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD+-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD+ as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation. PMID:23527128

Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties.

PubMed

Krog, Anne; Heggeset, Tonje M B; Müller, Jonas E N; Kupper, Christiane E; Schneider, Olha; Vorholt, Julia A; Ellingsen, Trond E; Brautaset, Trygve

2013-01-01

Bacillus methanolicus can utilize methanol as the sole carbon source for growth and it encodes an NAD(+)-dependent methanol dehydrogenase (Mdh), catalyzing the oxidation of methanol to formaldehyde. Recently, the genomes of the B. methanolicus strains MGA3 (ATCC53907) and PB1 (NCIMB13113) were sequenced and found to harbor three different putative Mdh encoding genes, each belonging to the type III Fe-NAD(+)-dependent alcohol dehydrogenases. In each strain, two of these genes are encoded on the chromosome and one on a plasmid; only one chromosomal act gene encoding the previously described activator protein ACT was found. The six Mdhs and the ACT proteins were produced recombinantly in Escherichia coli, purified, and characterized. All Mdhs required NAD(+) as cosubstrate, were catalytically stimulated by ACT, exhibited a broad and different substrate specificity range and displayed both dehydrogenase and reductase activities. All Mdhs catalyzed the oxidation of methanol; however the catalytic activity for methanol was considerably lower than for most other alcohols tested, suggesting that these enzymes represent a novel class of alcohol dehydrogenases. The kinetic constants for the Mdhs were comparable when acting as pure enzymes, but together with ACT the differences were more pronounced. Quantitative PCR experiments revealed major differences with respect to transcriptional regulation of the paralogous genes. Taken together our data indicate that the repertoire of methanol oxidizing enzymes in thermotolerant bacilli is larger than expected with complex mechanisms involved in their regulation.

Foraging in maize field areas: A risky business?

PubMed

Boily, Monique; Aras, Philippe; Jumarie, Catherine

2017-12-01

In Quebec, Canada, the cultivation of maize dominates the agricultural territory. This crop requires a sustained supply of fertilizers from different sources: chemical, natural or from residual materials (sludge). These amendments contain metallic trace elements, which may lead to metal-contaminated maize pollen, a possible source of prooxidants for the foraging bees. Our objective was to determine whether maize fields environment influences the oxidation processes and the accumulation of metals in bees. A few days prior to pollen shedding, beehives were installed in maize fields: one organically grown (site A) and three conventionally grown (sites B, C and D). Soil, maize pollen and bees were analyzed for metal content. Every 15days, bees were collected and analyzed for peroxidation of lipids, metallothionein-like proteins (MTLPs), proteins, retinoids and lipophilic antioxidants (carotenoids and α-tocopherol). The compound β-carotene was the most abundant in bees from all sites, followed by α-carotene, β-cryptoxanthin, α-cryptoxanthin, zeaxanthin and lutein. Retinaldehyde and retinol varied according to times and sites without demonstrating clear trends. However, significant differences between sites were noted in 13-cis-retinoic acid and two retinoic acid metabolites measured in bees, suggesting alteration in the reduction-oxidation processes. In line with these results, the level of lipid peroxidation was globally higher in sites B, C and D compared with the organic site. Higher concentrations of metals were observed in soil and pollen from the field A, but bees metal contents were equal or less than those measured in bees from other sites. Higher bee MTLP levels were measured in sites B, C and D. For most sampling times, the discriminant analysis revealed that the conditions were distinguished by the oxidation processes in bees. Our data suggest that bees foraging in conventionally grown maize fields are at risk of increased oxidative damages which can alter the fine regulation of retinoids. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-01-01

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

Alterations in brain glucose utilization accompanying elevations in blood ethanol and acetate concentrations in the rat.

PubMed

Pawlosky, Robert J; Kashiwaya, Yoshihiro; Srivastava, Shireesh; King, Michael T; Crutchfield, Calvin; Volkow, Nora; Kunos, George; Li, Ting-Kai; Veech, Richard L

2010-02-01

Previous studies in humans have shown that alcohol consumption decreased the rate of brain glucose utilization. We investigated whether the major metabolite of ethanol, acetate, could account for this observation by providing an alternate to glucose as an energy substrate for brain and the metabolic consequences of that shift. Rats were infused with solutions of sodium acetate, ethanol, or saline containing (13)C-2-glucose as a tracer elevating the blood ethanol (BEC) and blood acetate (BAcC) concentrations. After an hour, blood was sampled and the brains of animals were removed by freeze blowing. Tissue samples were analyzed for the intermediates of glucose metabolism, Krebs' cycle, acyl-coenzyme A (CoA) compounds, and amino acids. Mean peak BEC and BAcC were approximately 25 and 0.8 mM, respectively, in ethanol-infused animals. Peak blood BAcC increased to 12 mM in acetate-infused animals. Both ethanol and acetate infused animals had a lower uptake of (13)C-glucose into the brain compared to controls and the concentration of brain (13)C-glucose-6-phosphate varied inversely with the BAcC. There were higher concentrations of brain malonyl-CoA and somewhat lower levels of free Mg(2+) in ethanol-treated animals compared to saline controls. In acetate-infused animals the concentrations of brain lactate, alpha-ketoglutarate, and fumarate were higher. Moreover, the free cytosolic [NAD(+)]/[NADH] was lower, the free mitochondrial [NAD(+)]/[NADH] and [CoQ]/[CoQH(2)] were oxidized and the DeltaG' of ATP lowered by acetate infusion from -61.4 kJ to -59.9 kJ/mol. Animals with elevated levels of blood ethanol or acetate had decreased (13)C-glucose uptake into the brain. In acetate-infused animals elevated BAcC were associated with a decrease in (13)C-glucose phosphorylation. The co-ordinate decrease in free cytosolic NAD, oxidation of mitochondrial NAD and Q couples and the decrease in DeltaG' of ATP was similar to administration of uncoupling agents indicating that the metabolism of acetate in brain caused the mitochondrial voltage dependent pore to form.

Alterations in Brain Glucose Utilization Accompanying Elevations in Blood Ethanol and Acetate Concentrations in the Rat

PubMed Central

Pawlosky, Robert J.; Kashiwaya, Yoshihiro; Srivastava, Shireesh; King, Michael T.; Crutchfield, Calvin; Volkow, Nora; Kunos, George; Li, Ting-Kai; Veech, Richard L.

2010-01-01

Background Previous studies in humans have shown that alcohol consumption decreased the rate of brain glucose utilization. We investigated whether the major metabolite of ethanol, acetate, could account for this observation by providing an alternate to glucose as an energy substrate for brain and the metabolic consequences of that shift. Methods Rats were infused with solutions of sodium acetate, ethanol, or saline containing 13C-2-glucose as a tracer elevating the blood ethanol (BEC) and blood acetate (BAcC) concentrations. After an hour, blood was sampled and the brains of animals were removed by freeze blowing. Tissue samples were analyzed for the intermediates of glucose metabolism, Krebs’ cycle, acyl-coenzyme A (CoA) compounds, and amino acids. Results Mean peak BEC and BAcC were approximately 25 and 0.8 mM, respectively, in ethanol-infused animals. Peak blood BAcC increased to 12 mM in acetate-infused animals. Both ethanol and acetate infused animals had a lower uptake of 13C-glucose into the brain compared to controls and the concentration of brain 13C-glucose-6-phosphate varied inversely with the BAcC. There were higher concentrations of brain malonyl-CoA and somewhat lower levels of free Mg2+ in ethanol-treated animals compared to saline controls. In acetate-infused animals the concentrations of brain lactate, α-ketoglutarate, and fumarate were higher. Moreover, the free cytosolic [NAD+]/[NADH] was lower, the free mitochondrial [NAD+]/[NADH] and [CoQ]/[CoQH2] were oxidized and the ΔG′ of ATP lowered by acetate infusion from −61.4 kJ to −59.9 kJ/mol. Conclusions Animals with elevated levels of blood ethanol or acetate had decreased 13C-glucose uptake into the brain. In acetate-infused animals elevated BAcC were associated with a decrease in 13C-glucose phosphorylation. The co-ordinate decrease in free cytosolic NAD, oxidation of mitochondrial NAD and Q couples and the decrease in ΔG′ of ATP was similar to administration of uncoupling agents indicating that the metabolism of acetate in brain caused the mitochondrial voltage dependent pore to form. PMID:19951290

Melatonin protects chondrocytes from impairment induced by glucocorticoids via NAD+-dependent SIRT1.

PubMed

Yang, Wei; Kang, Xiaomin; Qin, Na; Li, Feng; Jin, Xinxin; Ma, Zhengmin; Qian, Zhuang; Wu, Shufang

2017-10-01

Intra-articular injection of glucocorticoids is used to relieve pain and inflammation in osteoarthritis patients, which is occasionally accompanied with the serious side effects of glucocorticoids in collagen-producing tissue. Melatonin is the major hormone released from the pineal gland and its beneficial effects on cartilage has been suggested. In the present study, we investigated the protective role of melatonin on matrix degeneration in chondrocytes induced by dexamethasone (Dex). The chondrocytes isolated from mice knee joint were treated with Dex, melatonin, EX527 and siRNA targeted for SIRT6, respectively. Dex treatment induced the loss of the extracellular matrix, NAD + /NADH ratio and NADPH concentration in chondrocytes. Melatonin alone have no effect on the quantity of proteoglycans and collagen type IIa1, however, the pretreatment of melatonin reversed the negative effects induced by Dex. Meanwhile, the significant decrease in NAD + /NADH ratio and NADPH concentration in Dex group were up-regulated by pretreatment of melatonin. Furthermore, it was revealed that inhibition of SIRT1 blocked the protective effects of melatonin. The enhancement of NAD + -dependent SIRT1 activity contributes to the chondroprotecfive effects of melatonin, which has a great benefit to prevent dexamethasone-induced chondrocytes impairment. Copyright © 2017 Elsevier Inc. All rights reserved.

Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

PubMed

Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

2009-01-02

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.

Pro-inflammatory effects of interleukin-17A on vascular smooth muscle cells involve NAD(P)H- oxidase derived reactive oxygen species.

PubMed

Pietrowski, Eweline; Bender, Bianca; Huppert, Jula; White, Robin; Luhmann, Heiko J; Kuhlmann, Christoph R W

2011-01-01

T cells are known for their contribution to the inflammatory element of atherosclerosis. Recently, it has been demonstrated that the Th17 derived cytokine IL-17 is involved in the pro-inflammatory response of vascular smooth muscle cells (VSMC). The aim of the present study was to examine whether reactive oxygen species (ROS) might be involved in this context. The effect of IL-17A on ROS generation was examined using the fluorescent dye 2'7'-dichlorodihydrofluorescein (H(2)DCF) in primary murine VSMC. IL-17A induced an increase in H(2)DCF fluorescence in VSMC, and this effect was blocked by the NAD(P)H-oxidase inhibitor apocynin and siRNA targeting Nox2. The p38-MAPK inhibitors SB203580 and SB202190 dose-dependently reduced the IL-17A induced ROS production. The IL-17A induced release of the pro-inflammatory cytokines IL-6, G-CSF, GM-CSF and MCP-1 from VSMC, as detected by the Luminex technology, was completely abolished by NAD(P)H-oxidase inhibition. Taken together, our data indicate that IL-17A causes the NAD(P)H-oxidase dependent generation of ROS leading to a pro-inflammatory activation of VSMC. Copyright © 2010 S. Karger AG, Basel.

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases.

PubMed

Li, Qunrui; Metthew Lam, L K; Xun, Luying

2011-11-01

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD(+) to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K ( d ) of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD(+) (e.g., K ( d ) of 87 μM for FurX-NAD(+)). The kinetic data suggest that the four enzymes are efficient "furfural reductases" with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°') for ethanol-dependent reduction of furfural was determined to be -1.1 kJ mol(-1). The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.

Molecular evolution of the nicotinic acid requirement within the Shigella/EIEC pathotype.

PubMed

Di Martino, Maria Letizia; Fioravanti, Rosa; Barbabella, Giada; Prosseda, Gianni; Colonna, Bianca; Casalino, Mariassunta

2013-12-01

Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor in several anabolic and catabolic reactions. NAD derives from quinolinic acid (QUIN) which in Escherichia coli is obtained through a pyridine salvage pathway or a de novo synthesis pathway. In the latter case, two enzymes, L-aspartate oxidase (NadB) and quinolinate synthase (NadA), are required for the synthesis of QUIN. In contrast to its E. coli ancestor, Shigella spp., the causative agent of bacillary dissentery, lacks the de novo pathway and strictly requires nicotinic acid for growth (Nic⁻ phenotype). This phenotype depends on the silencing of the nadB and nadA genes and its pathoadaptive nature is suggested by the observation that QUIN attenuates the Shigella invasive process. Shigella shares the pathogenicity mechanism with enteronvasive E. coli (EIEC), a group of pathogenic E. coli. On the basis of this similarity EIEC and Shigella have been grouped into a single E. coli pathotype. However EIEC strains do not constitute a homogeneous group and do not possess the complete set of characters that define Shigella strains. In this work we have analysed thirteen EIEC strains belonging to different serotypes and originating from different geographic areas. We show that, in contrast to Shigella, only some EIEC strains require nicotinic acid for growth in minimal medium. Moreover, by studying the emergence of the Nic⁻ phenotype in all serotypes of S. flexneri, as well as in S. sonnei and S. dysenteriae, we describe which molecular rearrangements occurred and which mutations are responsible for the inactivation of the nadA and nadB genes. Our data confirm that the genome of Shigella is extremely dynamic and support the hypothesis that EIEC might reflect an earlier stage of the pathoadaptation process undergone by Shigella. Copyright © 2013. Published by Elsevier GmbH.

A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices.

PubMed

Rösner, Jörg; Liotta, Agustin; Schmitz, Dietmar; Heinemann, Uwe; Kovács, Richard

2013-01-30

Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence. Copyright © 2012 Elsevier B.V. All rights reserved.

Short-term effects of different genetically modified maize varieties on arthropod food web properties: an experimental field assessment.

PubMed

Szénási, Ágnes; Pálinkás, Zoltán; Zalai, Mihály; Schmitz, Oswald J; Balog, Adalbert

2014-06-17

There is concern that genetically modified (GM) plants may have adverse affects on the arthropod biodiversity comprising agricultural landscapes. The present study report on a two year field experimental test of whether four different genotypic lines, some are novel with no previous field tests, of GM maize hybrids alter the structure of arthropod food webs that they harbour, relative to non-GM maize (control) that is widely used in agriculture. The different GM genotypes produced either Bt toxins, conferred glyphosate tolerance or a combination of the two traits. Quantitative food web analysis, based on short-term assessment assigning a total of 243,896 arthropod individuals collected from the treatments to their positions in food webs, revealed that complex and stable food webs persisted in each maize treatment. Moreover, food web structure remained relatively unchanged by the GM-genotype. The results suggest that at least in short-term period these particular GM maize genotypes will not have adverse effects on arthropod biota of agricultural landscapes.

A study of the metabolism of l-αγ-diaminobutyric acid in a Xanthomonas species

PubMed Central

Rao, D. Rajagopal; Hariharan, K.; Vijayalakshmi, K. R.

1969-01-01

1. l-αγ-Diaminobutyric acid is metabolized in Xanthomonas sp. to aspartic β-semialdehyde, aspartic acid and oxaloacetic acid. 2. Aspartic β-semialdehyde is formed from diaminobutyric acid by a pyruvate-dependent γ-transamination. 3. The transaminase has a pH optimum of 9 and exhibits a high degree of substrate specificity, as analogues of diaminobutyric acid and pyruvate are inert in the system. The transaminase is inhibited by carbonyl-binding agents such as hydroxylamine. 4. Aspartic acid is formed from aspartic β-semialdehyde by an NAD+-dependent dehydrogenation. 5. The dehydrogenase has a pH optimum of 8·5 and is a thiol enzyme. It is specific for aspartic β-semialdehyde but analogues of NAD+ such as 3-acetylpyridine–adenine dinucleotide and deamino-NAD are partly active in the system. 6. The significance of these reactions is discussed in relation to diaminobutyric acid metabolism in plants and mammalian systems. PMID:4390206

Recombinant NAD-dependent SIR-2 protein of Leishmania donovani: immunobiochemical characterization as a potential vaccine against visceral leishmaniasis.

PubMed

Baharia, Rajendra K; Tandon, Rati; Sharma, Tanuj; Suthar, Manish K; Das, Sanchita; Siddiqi, Mohammad Imran; Saxena, Jitendra Kumar; Sundar, Shaym; Sunder, Shyam; Dube, Anuradha

2015-03-01

The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach. The immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.

Transport of Boron by the tassel-less1 Aquaporin Is Critical for Vegetative and Reproductive Development in Maize[C][W][OPEN

PubMed Central

Durbak, Amanda R.; Phillips, Kimberly A.; Pike, Sharon; O’Neill, Malcolm A.; Mares, Jonathan; Gallavotti, Andrea; Malcomber, Simon T.; Gassmann, Walter; McSteen, Paula

2014-01-01

The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function. PMID:25035406

NAMPT-Mediated NAD Biosynthesis as the Internal Timing Mechanism: In NAD+ World, Time Is Running in Its Own Way.

PubMed

Poljsak, Borut

2017-09-08

The biological age of organisms differs from the chronological age and is determined by internal aging clock(s). How cells estimate time on a scale of 24 hours is relatively well studied; however, how biological time is measured by cells, tissues, organs, or organisms in longer time periods (years and decades) is largely unknown. What is clear and widely agreed upon is that the link to age and age-related diseases is not chronological, as it does not depend on a fixed passage of time. Rather, this link depends on the biological age of an individual cell, tissue, organ, or organism and not on time in a strictly chronological sense. Biological evolution does not invent new methods as often as improving upon already existing ones. It should be easier to evolve and remodel the existing (circadian) time clock mechanism to use it for measurement or regulation of longer time periods than to invent a new time mechanism/clock. Specifically, it will be demonstrated that the circadian clock can also be used to regulate circannual or even longer time periods. Nicotinamide phosphoribosyltransferase (NAMPT)-mediated nicotinamide adenine dinucleotide (NAD+) levels, being regulated by the circadian clock, might be the missing link between aging, cell cycle control, DNA damage repair, cellular metabolism and the aging clock, which is responsible for the biological age of an organism. The hypothesis that NAMPT/NAD+/SIRT1 might represent the time regulator that determines the organismal biological age will be presented. The biological age of tissues and organs might be regulated and synchronized through eNAMPT blood secretion. The "NAD World 2.0" concept will be upgraded with detailed insights into mechanisms that regulate NAD + -mediated aging clock ticking, the duration and amplitude of which are responsible for the aging rate of humans.

CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain.

PubMed Central

Ceni, Claire; Pochon, Nathalie; Brun, Virginie; Muller-Steffner, Hélène; Andrieux, Annie; Grunwald, Didier; Schuber, Francis; De Waard, Michel; Lund, Frances; Villaz, Michel; Moutin, Marie-Jo

2003-01-01

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue. PMID:12403647

Analysis of metabolisms and transports of xylitol using xylose- and xylitol-assimilating Saccharomyces cerevisiae.

PubMed

Tani, Tatsunori; Taguchi, Hisataka; Akamatsu, Takashi

2017-05-01

To clarify the relationship between NAD(P) + /NAD(P)H redox balances and the metabolisms of xylose or xylitol as carbon sources, we analyzed aerobic and anaerobic batch cultures of recombinant Saccharomyces cerevisiae in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C. The TDH3p-GAL2 or gal80Δ strain completely consumed the xylose within 24 h and aerobically consumed 92-100% of the xylitol within 96 h, but anaerobically consumed only 20% of the xylitol within 96 h. Cells of both strains grew well in aerobic culture. The addition of acetaldehyde (an effective oxidizer of NADH) increased the xylitol consumption by the anaerobically cultured strain. These results indicate that in anaerobic culture, NAD + generated in the NAD(P)H-dependent xylose reductase reaction was likely needed in the NAD + -dependent xylitol dehydrogenase reaction, whereas in aerobic culture, the NAD + generated by oxidation of NADH in the mitochondria is required in the xylitol dehydrogenase reaction. The role of Gal2 and Fps1 in importing xylitol into the cytosol and exporting it from the cells was analyzed by examining the xylitol consumption in aerobic culture and the export of xylitol metabolized from xylose in anaerobic culture, respectively. The xylitol consumptions of gal80Δ gal2Δ and gal80Δ gal2Δ fps1Δ strains were reduced by 81% and 88% respectively, relative to the gal80Δ strain. The maximum xylitol concentration accumulated by the gal80Δ, gal80Δ gal2Δ, and gal80Δ gal2Δ fps1Δ strains was 7.25 g/L, 5.30 g/L, and 4.27 g/L respectively, indicating that Gal2 and Fps1 transport xylitol both inward and outward. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Plants on constant alert: elevated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar resistant maize

USDA-ARS?s Scientific Manuscript database

Plant defense responses against insect herbivores frequently depend on the biosynthesis and action of jasmonic acid (JA) and its conjugates. To better understand JA signaling pathways in maize (Zea mays L.), we have examined two maize genotypes, Mp708 and Tx601. Mp708 is resistant to feeding by le...

Curcumin prevents cisplatin-induced renal alterations in mitochondrial bioenergetics and dynamic.

PubMed

Ortega-Domínguez, Bibiana; Aparicio-Trejo, Omar Emiliano; García-Arroyo, Fernando E; León-Contreras, Juan Carlos; Tapia, Edilia; Molina-Jijón, Eduardo; Hernández-Pando, Rogelio; Sánchez-Lozada, Laura Gabriela; Barrera-Oviedo, Diana; Pedraza-Chaverri, José

2017-09-01

Cisplatin is widely used as chemotherapeutic agent for treatment of diverse types of cancer, however, acute kidney injury (AKI) is an important side effect of this treatment. Diverse mechanisms have been involved in cisplatin-induced AKI, such as oxidative stress, apoptosis and mitochondrial damage. On the other hand, curcumin is a polyphenol extracted from the rhizome of Curcuma longa L. Previous studies have shown that curcumin protects against the cisplatin-induced AKI; however, it is unknown whether curcumin can reduce alterations in mitochondrial bioenergetics and dynamic in this model. It was found that curcumin prevents cisplatin-induced: (a) AKI and (b) alterations in the following mitochondrial parameters: bioenergetics, ultrastructure, hydrogen peroxide production and dynamic. In fact, curcumin prevented the increase of mitochondrial fission 1 protein (FIS1), the decrease of optic atrophy 1 protein (OPA1) and the decrease of NAD + -dependent deacetylase sirtuin-3 (SIRT3), a mitochondrial dynamic regulator as well as the increase in the mitophagy associated proteins parkin and phosphatase and tensin homologue (PTEN)-induced putative kinase protein 1 (PINK1). In conclusion, the protective effect of curcumin in cisplatin-induced AKI was associated with the prevention of the alterations in mitochondrial bioenergetics, ultrastructure, redox balance, dynamic, and SIRT3 levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

Kinetic competence of the cADP-ribose-CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Oppenheimer, N; Schuber, F

2001-01-01

CD38/NAD(+) glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD(+) into ADP-ribose (NAD(+) glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38-cADPR complex is kinetically competent in the conversion of NAD(+) into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD(+) glycohydrolase-catalysed hydrolysis of 2'-deoxy-2'-aminoribo-NAD(+) and ADP-cyclo[N1,C1']-2'-deoxy-2'-aminoribose (slowly hydrolysable analogues of NAD(+) and cADPR respectively) ruled out that the CD38-cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E.ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD(+) glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38-cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J. Biochem. 267, 3056-3064], we believe that the Michaelis CD38-NAD(+) complex could play such a role instead. PMID:11513738

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.

Transcriptome profiling of equine vitamin E deficient neuroaxonal dystrophy identifies upregulation of liver X receptor target genes

PubMed Central

Finno, Carrie J.; Bordbari, Matthew H.; Valberg, Stephanie J.; Lee, David; Herron, Josi; Hines, Kelly; Monsour, Tamer; Scott, Erica; Bannasch, Danika L.; Mickelson, James; Xu, Libin

2016-01-01

Specific spontaneous heritable neurodegenerative diseases have been associated with lower serum and cerebrospinal fluid α-tocopherol (α-TOH) concentrations. Equine neuroaxonal dystrophy (eNAD) has similar histologic lesions to human ataxia with vitamin E deficiency caused by mutations in the α-TOH transfer protein gene (TTPA). Mutations in TTPA are not present with eNAD and the molecular basis remains unknown. Given the neuropathologic phenotypic similarity of the conditions, we assessed the molecular basis of eNAD by global transcriptome sequencing of the cervical spinal cord. Differential gene expression analysis identified 157 significantly (FDR<0.05) dysregulated transcripts within the spinal cord of eNAD-affected horses. Statistical enrichment analysis identified significant downregulation of the ionotropic and metabotropic group III glutamate receptor, synaptic vesicle trafficking and cholesterol biosynthesis pathways. Gene co-expression analysis identified one module of upregulated genes significantly associated with the eNAD phenotype that included the liver X receptor (LXR) targets CYP7A1, APOE, PLTP and ABCA1. Validation of CYP7A1 and APOE dysregulation was performed in an independent biologic group and CYP7A1 was found to be additionally upregulated in the medulla oblongata of eNAD horses. Evidence of LXR activation supports a role for modulation of oxysterol-dependent LXR transcription factor activity by tocopherols. We hypothesize that the protective role of α-TOH in eNAD may reside in its ability to prevent oxysterol accumulation and subsequent activation of the LXR in order to decrease lipid peroxidation associated neurodegeneration. PMID:27751910

Alcohol alters hepatic FoxO1, p53, and mitochondrial SIRT5 deacetylation function

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

Lieber, Charles S.; Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029; Leo, Maria Anna

2008-08-22

Chronic alcohol consumption affects the gene expression of a NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-{gamma} coactivator1{alpha} (PGC-1{alpha}). Our aim was to verify that it also alters the forkhead (FoxO1) and p53 transcription factor proteins, critical in the hepatic response to oxidative stress and regulated by SIRT1 through its deacetylating capacity. Accordingly, rats were pair-fed the Lieber-DeCarli alcohol-containing liquid diets for 28 days. Alcohol increased hepatic mRNA expression of FoxO1 (p = 0.003) and p53 (p = 0.001) while corresponding protein levels remained unchanged. However phospho-FoxO1 and phospho-Akt (protein kinase) were both decreased by alcohol consumption (pmore » = 0.04 and p = 0.02, respectively) while hepatic p53 was found hyperacetylated (p = 0.017). Furthermore, mitochondrial SIRT5 was reduced (p = 0.0025), and PGC-1{alpha} hyperacetylated (p = 0.027), establishing their role in protein modification. Thus, alcohol consumption disrupts nuclear-mitochondrial interactions by post-translation protein modifications, which contribute to alteration of mitochondrial biogenesis through the newly discovered reduction of SIRT5.« less

Relationship of source and sink in determining kernel composition of maize

PubMed Central

Seebauer, Juliann R.; Singletary, George W.; Krumpelman, Paulette M.; Ruffo, Matías L.; Below, Frederick E.

2010-01-01

The relative role of the maternal source and the filial sink in controlling the composition of maize (Zea mays L.) kernels is unclear and may be influenced by the genotype and the N supply. The objective of this study was to determine the influence of assimilate supply from the vegetative source and utilization of assimilates by the grain sink on the final composition of maize kernels. Intermated B73×Mo17 recombinant inbred lines (IBM RILs) which displayed contrasting concentrations of endosperm starch were grown in the field with deficient or sufficient N, and the source supply altered by ear truncation (45% reduction) at 15 d after pollination (DAP). The assimilate supply into the kernels was determined at 19 DAP using the agar trap technique, and the final kernel composition was measured. The influence of N supply and kernel ear position on final kernel composition was also determined for a commercial hybrid. Concentrations of kernel protein and starch could be altered by genotype or the N supply, but remained fairly constant along the length of the ear. Ear truncation also produced a range of variation in endosperm starch and protein concentrations. The C/N ratio of the assimilate supply at 19 DAP was directly related to the final kernel composition, with an inverse relationship between the concentrations of starch and protein in the mature endosperm. The accumulation of kernel starch and protein in maize is uniform along the ear, yet adaptable within genotypic limits, suggesting that kernel composition is source limited in maize. PMID:19917600

Effects of AT1 receptor antagonism on kainate-induced seizures and concomitant changes in hippocampal extracellular noradrenaline, serotonin, and dopamine levels in Wistar-Kyoto and spontaneously hypertensive rats.

PubMed

Tchekalarova, Jana; Loyens, Ellen; Smolders, Ilse

2015-05-01

In the management of epilepsy, AT1 receptor antagonists have been suggested as an additional treatment strategy. A hyperactive brain angiotensin (Ang) II system and upregulated AT1 receptors are implicated in the cerebrovascular alterations in a genetic form of hypertension. Uncontrolled hypertension could also, in turn, be a risk factor for a seizure threshold decrease and development of epileptogenesis. The present study aimed to assess the effects of the selective AT1 receptor antagonist ZD7155 on kainic acid (KA)-induced status epilepticus (SE) development and accompanying changes in the hippocampal extracellular (EC) neurotransmitter levels of noradrenaline (NAD), serotonin (5-HT), and dopamine (DA) in spontaneously hypertensive rats (SHRs) and their parent strain Wistar-Kyoto (WKY) rats, since monoamines are well-known neurotransmitters involved in mechanisms of both epilepsy and hypertension. Status epilepticus was evoked in freely moving rats by a repetitive intraperitoneal (i.p.) administration of KA in subconvulsant doses. In the treatment group, ZD7155 (5mg/kg i.p.) was coadministered with the first KA injection. Spontaneously hypertensive rats exhibited higher susceptibility to SE than WKY rats, but the AT1 receptor antagonist did not alter the development of SE in SHRs or in WKY rats. In vivo microdialysis demonstrated significant KA-induced increases of the hippocampal NAD and DA levels in SHRs and of NAD, 5-HT, and DA in WKY rats. Although SHRs developed more severe seizures while receiving a lower dose of KA compared to WKY rats, AT1 receptor antagonism completely prevented all KA-induced increases of hippocampal monoamine levels in both rat strains without affecting seizure development per se. These results suggest a lack of direct relationship between KA-induced seizure susceptibility and adaptive changes of hippocampal NAD, 5-HT, and DA levels in the effects of ZD7155 in WKY rats and SHRs. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Nicotinamide Improves Aspects of Healthspan, but Not Lifespan, in Mice.

PubMed

Mitchell, Sarah J; Bernier, Michel; Aon, Miguel A; Cortassa, Sonia; Kim, Eun Young; Fang, Evandro F; Palacios, Hector H; Ali, Ahmed; Navas-Enamorado, Ignacio; Di Francesco, Andrea; Kaiser, Tamzin A; Waltz, Tyler B; Zhang, Ning; Ellis, James L; Elliott, Peter J; Frederick, David W; Bohr, Vilhelm A; Schmidt, Mark S; Brenner, Charles; Sinclair, David A; Sauve, Anthony A; Baur, Joseph A; de Cabo, Rafael

2018-03-06

The role in longevity and healthspan of nicotinamide (NAM), the physiological precursor of NAD + , is elusive. Here, we report that chronic NAM supplementation improves healthspan measures in mice without extending lifespan. Untargeted metabolite profiling of the liver and metabolic flux analysis of liver-derived cells revealed NAM-mediated improvement in glucose homeostasis in mice on a high-fat diet (HFD) that was associated with reduced hepatic steatosis and inflammation concomitant with increased glycogen deposition and flux through the pentose phosphate and glycolytic pathways. Targeted NAD metabolome analysis in liver revealed depressed expression of NAM salvage in NAM-treated mice, an effect counteracted by higher expression of de novo NAD biosynthetic enzymes. Although neither hepatic NAD + nor NADP + was boosted by NAM, acetylation of some SIRT1 targets was enhanced by NAM supplementation in a diet- and NAM dose-dependent manner. Collectively, our results show health improvement in NAM-supplemented HFD-fed mice in the absence of survival effects. Published by Elsevier Inc.

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+.

Targeting SIRT1 to improve metabolism: all you need is NAD+?

PubMed Central

Cantó, Carles; Auwerx, Johan

2013-01-01

SIRT1 is an evolutionary conserved NAD+-dependent deacetylase that is at the pinnacle of metabolic control, all the way from yeast to humans. SIRT1 senses changes in intracellular NAD+ levels, which reflect energy level, and uses this information to adapt the cellular energy output, such that the it matches cellular energy requirements. Generally, but not exclusively, the changes induced by SIRT1 activation are transcriptional in nature and are related to an increase in mitochondrial metabolism and antioxidant protection. These attractive features have validated SIRT1 as a therapeutic target in the management of metabolic disease and prompted an intensive search to identify pharmacological SIRT1 activators. In this review we will first give an overview of the SIRT1 biology with a particular focus on its role in metabolic control. We will then analyze the pros and cons of the current strategies used to activate SIRT1 and explore the emerging evidence indicating that modulation of NAD+ levels could provide an effective way to achieve such goals. PMID:22106091

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.

Hydrothermal treatment of maize: Changes in physical, chemical, and functional properties.

PubMed

Rocha-Villarreal, Verónica; Hoffmann, Jessica Fernanda; Vanier, Nathan Levien; Serna-Saldivar, Sergio O; García-Lara, Silverio

2018-10-15

The objective of this work was to assess the effects of a traditional parboiling treatment on physical, chemical and functional properties of yellow maize kernels. For this, maize kernels were subjected to the three main stages of a traditional parboiling process (soaking, steaming, and drying) at different moisture contents (15%, 25%, or 35%), and different pressure steaming times (0, 15, or 30 min). Kernels were evaluated for physical and chemical changes, while manually generated endosperm fractions were further evaluated for nutritional and functional changes. The parboiling process negatively altered the maize kernels properties by increasing the number of kernels with burst pericarp and decreasing the total carotenoid content in the endosperm by 42%. However, the most intense conditions (35% moisture and 30 min steam) lowered the number of broken kernels by 41%, and the number of stress cracks by 36%. Results also demonstrated that soaking enhanced the nutritional value of soaked yellow maize by increasing the thiamine content and the bound phenolic content in the endosperm fraction up to 102%. The proper implementation of this hydrothermal treatment could lead to significant enhancements in nutritional and functionality of maize products. Copyright © 2018 Elsevier Ltd. All rights reserved.

Resistant starch alters gut microbiome and metabolomics profiles concurrent with amelioration of chronic kidney disease in rats

USDA-ARS?s Scientific Manuscript database

Patients and animals with chronic kidney disease (CKD) exhibit profound alterations in the gut environment including shifts in microbial composition, increased fecal pH, and increased blood levels of gut microbe-derived metabolites (xeno-metabolites). The fermentable dietary fiber—high amylose maize...

A Mitochondrial Mutator System in Maize1[w

PubMed Central

Kuzmin, Evgeny V.; Duvick, Donald N.; Newton, Kathleen J.

2005-01-01

The P2 line of maize (Zea mays) is characterized by mitochondrial genome destabilization, initiated by recessive nuclear mutations. These alleles alter copy number control of mitochondrial subgenomes and disrupt normal transfer of mitochondrial genomic components to progeny, resulting in differences in mitochondrial DNA profiles among sibling plants and between parents and progeny. The mitochondrial DNA changes are often associated with variably defective phenotypes, reflecting depletion of essential mitochondrial genes. The P2 nuclear genotype can be considered a natural mutagenesis system for maize mitochondria. It dramatically accelerates mitochondrial genomic divergence by increasing low copy-number subgenomes, by rapidly amplifying aberrant recombination products, and by causing the random loss of normal components of the mitochondrial genomes. PMID:15681663

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications.

PubMed

Zárate-Romero, Andrés; Murillo-Melo, Darío S; Mújica-Jiménez, Carlos; Montiel, Carmina; Muñoz-Clares, Rosario A

2016-04-01

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL. © 2016 Authors; published by Portland Press Limited.

The NAD(P)H-dependent glutamate dehydrogenase activities of Prevotella ruminicola B(1)4 can be attributed to one enzyme (GdhA), and gdhA expression is regulated in response to the nitrogen source available for growth.

PubMed Central

Wen, Z; Morrison, M

1996-01-01

Prevotella ruminicola B(1)4 possesses both NADPH- and NADH-linked glutamate dehydrogenase (GDH) activities, with the greatest specific activity being measured from ammonia-limited cultures. Relative to cells grown in the presence of 1 mM ammonium chloride, the NADPH-dependent activity was decreased approximately 10-fold when peptides were provided as a nitrogen source. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was used to visualize the GDH protein(s) in cell extracts of P. ruminicola. For all growth conditions tested, only one GDH protein was detectable, and its relative abundance, as well as its reactivity with either NAD(P)+ or NAD(P)H, correlated well with the specific activities measured from whole-cell assays. Consistent with the findings from enzyme assays and PAGE activity gels, Northern (RNA) blot analysis revealed that expression of a gene encoding NAD(P)H-GDH activity was greatest in ammonia-grown cultures and that GDH activity is regulated in response to nitrogen source (ammonia versus peptides), probably at the level of transcription. A gene encoding the NAD(P)H-utilizing GDH activity (gdhA) was cloned, and its nucleotide sequence was determined and shown to contain an open reading frame of 1,332 bp which would encode a polypeptide of 48.8 kDa. The deduced amino acid sequence possesses three highly conserved motifs typical of family I GDHs, but several unique amino acid substitutions within these motifs were evident. These results are discussed within the context of ruminal nitrogen metabolism and the growth efficiency of succinate- and propionate-producing anaerobic bacteria. PMID:8837439

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation*

PubMed Central

Kalous, Kelsey S.; Wynia-Smith, Sarah L.; Olp, Michael D.

2016-01-01

The sirtuin family of proteins catalyze the NAD+-dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1–7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD+ and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn2+ release from the conserved sirtuin Zn2+-tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD+ and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn2+, consistent with S-nitrosation of the Zn2+-tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. PMID:27756843

Fate of Transgenic DNA from Orally Administered Bt MON810 Maize and Effects on Immune Response and Growth in Pigs

PubMed Central

Walsh, Maria C.; Buzoianu, Stefan G.; Gardiner, Gillian E.; Rea, Mary C.; Gelencsér, Eva; Jánosi, Anna; Epstein, Michelle M.; Ross, R. Paul; Lawlor, Peadar G.

2011-01-01

We assessed the effect of short-term feeding of genetically modified (GM: Bt MON810) maize on immune responses and growth in weanling pigs and determined the fate of the transgenic DNA and protein in-vivo. Pigs were fed a diet containing 38.9% GM or non-GM isogenic parent line maize for 31 days. We observed that IL-12 and IFNγ production from mitogenic stimulated peripheral blood mononuclear cells decreased (P<0.10) following 31 days of GM maize exposure. While Cry1Ab-specific IgG and IgA were not detected in the plasma of GM maize-fed pigs, the detection of the cry1Ab gene and protein was limited to the gastrointestinal digesta and was not found in the kidneys, liver, spleen, muscle, heart or blood. Feeding GM maize to weanling pigs had no effect on growth performance or body weight. IL-6 and IL-4 production from isolated splenocytes were increased (P<0.05) in response to feeding GM maize while the proportion of CD4+ T cells in the spleen decreased. In the ileum, the proportion of B cells and macrophages decreased while the proportion of CD4+ T cells increased in GM maize-fed pigs. IL-8 and IL-4 production from isolated intraepithelial and lamina propria lymphocytes were also increased (P<0.05) in response to feeding GM maize. In conclusion, there was no evidence of cry1Ab gene or protein translocation to the organs and blood of weaning pigs. The growth of pigs was not affected by feeding GM maize. Alterations in immune responses were detected; however, their biologic relevance is questionable. PMID:22132091

Ozone-Induced Alterations in the Accumulation of Newly Synthesized Proteins in Leaves of Maize.

PubMed Central

Pino, M. E.; Mudd, J. B.; Bailey-Serres, J.

1995-01-01

We examined the response of leaves of 3-week-old maize (Zea mays L.) to short-term (5 h) fumigation with O3-enriched air (0, 0.12, 0.24, or 0.36 [mu]L/L). Older leaves and leaf tissue developed more severe visible damage at higher external O3 concentrations. To investigate the immediate effect of O3 exposure on the accumulation of newly synthesized leaf proteins, leaves were labeled with [35S]methionine after 2 h and fumigated for an additional 3 h. O3-induced alterations of leaf proteins were observed in a concentration-dependent manner. There was a significant decrease in [35S]methionine incorporation into protein at the highest O3 concentration. Developmental differences in accumulation of de novo-synthesized leaf proteins were observed when the leaf tip, middle, and basal sections were labeled under 0 [mu]L/L O3, and additional changes were apparent upon exposure to increasing O3 concentrations. Changes in leaf protein synthesis were observed in the absence of visible leaf injury. Subcellular fractionation revealed O3-induced alterations in soluble and membrane-associated proteins. A number of thylakoid membrane-associated proteins showed specific increases in response to O3 fumigation. In contrast, the synthesis of a 32-kD polypeptide associated with thylakoid membranes was reduced in response to O3 fumigation in parallel with reduced incorporation of [35S]methionine into protein. Immunoprecipitation identified this polypeptide as the D1 protein of photosystem II. A reduction in the accumulation of newly synthesized D1 could have consequences for the efficiency of photosynthesis and other cellular processes. PMID:12228510

Shifts in microbial communities in soil, rhizosphere and roots of two major crop systems under elevated CO2 and O3.

PubMed

Wang, Peng; Marsh, Ellen L; Ainsworth, Elizabeth A; Leakey, Andrew D B; Sheflin, Amy M; Schachtman, Daniel P

2017-11-03

Rising atmospheric concentrations of CO 2 and O 3 are key features of global environmental change. To investigate changes in the belowground bacterial community composition in response to elevated CO 2 and O 3 (eCO 2 and eO 3 ) the endosphere, rhizosphere and soil were sampled from soybeans under eCO 2 and maize under eO 3 . The maize rhizosphere and endosphere α-diversity was higher than soybean, which may be due to a high relative abundance of Rhizobiales. Only the rhizosphere microbiome composition of the soybeans changed in response to eCO 2 , associated with an increased abundance of nitrogen fixing microbes. In maize, the microbiome composition was altered by the genotype and linked to differences in root exudate profiles. The eO 3 treatment did not change the microbial communities in the rhizosphere, but altered the soil communities where hybrid maize was grown. In contrast to previous studies that focused exclusively on the soil, this study provides new insights into the effects of plant root exudates on the composition of the belowground microbiome in response to changing atmospheric conditions. Our results demonstrate that plant species and plant genotype were key factors driving the changes in the belowground bacterial community composition in agroecosystems that experience rising levels of atmospheric CO 2 and O 3 .

The effect of altered dosage of a mutant allele of Teosinte branched 1 (tb1-ref) on the root system of modern maize.

PubMed

Gaudin, Amelie C M; McClymont, Sarah A; Soliman, Sameh S M; Raizada, Manish N

2014-02-14

There was ancient human selection on the wild progenitor of modern maize, Balsas teosinte, for decreased shoot branching (tillering), in order to allow more nutrients to be diverted to grain. Mechanistically, the decline in shoot tillering has been associated with selection for increased expression of the major domestication gene Teosinte Branched 1 (Tb1) in shoot primordia. Therefore, TB1 has been defined as a repressor of shoot branching. It is known that plants respond to changes in shoot size by compensatory changes in root growth and architecture. However, it has not been reported whether altered TB1 expression affects any plant traits below ground. Previously, changes in dosage of a well-studied mutant allele of Tb1 in modern maize, called tb1-ref, from one to two copies, was shown to increase tillering. As a result, plants with two copies of the tb1-ref allele have a larger shoot biomass than heterozygotes. Here we used aeroponics to phenotype the effects of tb1-ref copy number on maize roots at macro-, meso- and micro scales of development. An increase in the tb1-ref copy number from one to two copies resulted in: (1) an increase in crown root number due to the cumulative initiation of crown roots from successive tillers; (2) higher density of first and second order lateral roots; and (3) reduced average lateral root length. The resulting increase in root system biomass in homozygous tb1-ref mutants balanced the increase in shoot biomass caused by enhanced tillering. These changes caused homozygous tb1-ref mutants of modern maize to more closely resemble its ancestor Balsas teosinte below ground. We conclude that a decrease in TB1 function in maize results in a larger root system, due to an increase in the number of crown roots and lateral roots. Given that decreased TB1 expression results in a more highly branched and larger shoot, the impact of TB1 below ground may be direct or indirect. We discuss the potential implications of these findings for whole plant coordination of biomass accumulation and maize domestication.

Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases

NASA Technical Reports Server (NTRS)

Biermann, B.; Johnson, E. M.; Feldman, L. J.

1990-01-01

Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

Metabolic imaging of tumor for diagnosis and response for therapy

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

DNA repair deficiency sensitizes lung cancer cells to NAD+ biosynthesis blockade.

PubMed

Touat, Mehdi; Sourisseau, Tony; Dorvault, Nicolas; Chabanon, Roman M; Garrido, Marlène; Morel, Daphné; Krastev, Dragomir B; Bigot, Ludovic; Adam, Julien; Frankum, Jessica R; Durand, Sylvère; Pontoizeau, Clement; Souquère, Sylvie; Kuo, Mei-Shiue; Sauvaigo, Sylvie; Mardakheh, Faraz; Sarasin, Alain; Olaussen, Ken A; Friboulet, Luc; Bouillaud, Frédéric; Pierron, Gérard; Ashworth, Alan; Lombès, Anne; Lord, Christopher J; Soria, Jean-Charles; Postel-Vinay, Sophie

2018-04-02

Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non-small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house-generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro - ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells - and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.

Metformin and liraglutide ameliorate high glucose-induced oxidative stress via inhibition of PKC-NAD(P)H oxidase pathway in human aortic endothelial cells.

PubMed

Batchuluun, Battsetseg; Inoguchi, Toyoshi; Sonoda, Noriyuki; Sasaki, Shuji; Inoue, Tomoaki; Fujimura, Yoshinori; Miura, Daisuke; Takayanagi, Ryoichi

2014-01-01

Metformin and glucagon like peptide-1 (GLP-1) prevent diabetic cardiovascular complications and atherosclerosis. However, the direct effects on hyperglycemia-induced oxidative stress in endothelial cells are not fully understood. Thus, we aimed to evaluate the effects of metformin and a GLP-1 analog, liraglutide on high glucose-induced oxidative stress. Production of reactive oxygen species (ROS), activation of protein kinase C (PKC) and NAD(P)H oxidase, and changes in signaling molecules in response to high glucose exposure were evaluated in human aortic endothelial cells with and without treatment of metformin and liraglutide, alone or in combination. PKC-NAD(P)H oxidase pathway was assessed by translocation of GFP-fused PKCβ2 isoform and GFP-fused p47phox, a regulatory subunit of NAD(P)H oxidase, in addition to endogenous PKC phosphorylation and NAD(P)H oxidase activity. High glucose-induced ROS overproduction was blunted by metformin or liraglutide treatment, with a further decrease by a combination of these drugs. Exposure to high glucose caused PKCβ2 translocation and a time-dependent phosphorylation of endogenous PKC but failed to induce its translocation and phosphorylation in the cells treated with metformin and liraglutide. Furthermore, both drugs inhibited p47phox translocation and NAD(P)H oxidase activation, and prevented the high glucose-induced changes in intracellulalr diacylglycerol (DAG) level and phosphorylation of AMP-activated protein kinase (AMPK). A combination of these drugs further enhanced all of these effects. Metformin and liraglutide ameliorate high glucose-induced oxidative stress by inhibiting PKC-NAD(P)H oxidase pathway. A combination of these two drugs provides augmented protective effects, suggesting the clinical usefulness in prevention of diabetic vascular complications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2017-03-01

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

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

Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

PubMed

Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

2009-09-04

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

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

Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi

2009-09-04

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Infrared monitoring of dinitrotoluenes in sunflower and maize roots.

PubMed

Dokken, K M; Davis, L C

2011-01-01

Infrared microspectroscopy (IMS) is emerging as an important analytical tool for the structural analysis of biological tissue. This report describes the use of IMS coupled to a synchrotron source combined with principal components analysis (PCA) to monitor the fate and effect of dinitrotoluenes in the roots of maize and sunflower plants. Infrared imaging revealed that maize roots metabolized 2,4-dinitrotoluene (DNT) and 2,6-DNT. The DNTs and their derivative aromatic amines were predominantly associated with epidermis and xylem. Both isomers of DNT altered the structure and production of pectin and pectic polysaccharides in maize and sunflower plant roots. Infrared peaks diagnostic for aromatic amines were seen at the 5 mg L concentrations for both DNTs in maize and sunflower treated tissue. However, only infrared peaks for nitro groups, not aromatic amines, were present in the maize treated at 10 mg L For sunflower, the 10 mg L level was toxic and also produced very dark root systems making spectra difficult to obtain. Maize and sunflower seem unable to metabolize effectively at concentrations higher than about 5 mg L DNT in hydroponic solution. Based on the results of this study, IMS combined with PCA can be an effective means of determining the fate and metabolism of organic contaminants in plant tissue when isotopically labeled compounds are not available. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.

An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

PubMed

Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

2015-05-01

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity. Copyright © 2015 Elsevier Inc. All rights reserved.

Ligand Binding Phenomena that Pertain to the Metabolic Function of Renalase

PubMed Central

Beaupre, Brett A.; Roman, Joseph V.; Hoag, Matthew R.; Meneely, Kathleen M.; Silvaggi, Nicholas R.; Lamb, Audrey L.; Moran, Graham R.

2017-01-01

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P)+. This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucloetides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (kred/Kd) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, ADP for mononucloetide or AMP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site. PMID:27769837

Urinary fumonisin B1 and estimated fumonisin intake in women from high- and low-exposure communities in Guatemala.

PubMed

Torres, Olga; Matute, Jorge; Gelineau-van Waes, Janee; Maddox, Joyce R; Gregory, Simon G; Ashley-Koch, Allison E; Showker, Jency L; Zitomer, Nicholas C; Voss, Kenneth A; Riley, Ronald T

2014-05-01

Fumonisin (FB) intake can be high when maize is a dietary staple. We determined (i) urinary FB (UFB) in women consuming maize in high- and low-exposure communities in Guatemala, (ii) the FB levels in maize, (iii) the relationship between UFB and FB intake, and (iv) the relative excretion of UFB1 , UFB2 , and UFB3 . Urine and maize were analyzed for FB for 1 year in three departments. Maize consumption was estimated by an interview questionnaire. Fumonisin B1 , B2 , and B3 (FB1 , FB2 and FB3 ), were detected in 100% of maize samples. FB1 in maize and urine was significantly higher in Jutiapa compared to Chimaltenango or Escuintla. The FB intake paralleled UFB1 in a dose-dependent manner but UFB1 was present in much higher levels than UFB2 or UFB3 compared to maize. In Jutiapa, agroecological conditions favored FB production. UFB1 mirrored the estimated FB intake. UFB1 > 0.1 ng/mL resulted in a dose-dependent increase in the risk of exceeding FB intake of 2 μg/kg b.w./day compared to women with no detectable UFB1 . More than 50% exceeded 2 μg/kg b.w./day when UFB1 was >0.5 ng/mL. UFB2 and UFB3 were rarely detected confirming that FB1 is either absorbed better or preferentially excreted in urine. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Some thoughts on the factors that controlled prehistoric maize production in the American Southwest with application to southwestern Colorado

USGS Publications Warehouse

Benson, L.V.; Ramsey, D.K.; Stahle, D.W.; Petersen, K.L.

2013-01-01

In this paper, we present a model of prehistoric southwestern Colorado maize productivity. The model is based on a tree-ring reconstruction of water-year precipitation for Mesa Verde for the period A.D. 480 to 2011. Correlation of historic Mesa Verde precipitation with historic precipitation at 11 other weather stations enabled the construction of an elevation-dependent precipitation function. Prehistoric water-year precipitation values for Mesa Verde together with the elevation-dependent precipitation function allowed construction of the elevation of southwest Colorado precipitation contours for each year since A.D. 480, including the 30-cm contour, which represents the minimum amount of precipitation necessary for the production of maize and the 50-cm contour, which represents the optimum amount of precipitation necessary for the production of maize. In this paper, calculations of prehistoric maize productivity and field life for any specific elevation are also demonstrated. These calculations were performed using organic nitrogen measurements made on seven southwestern Colorado soil groups together with values of reconstructed water-year precipitation and estimations of the organic nitrogen mineralization rate.

The NQO1 bioactivatable drug, β-lapachone, alters the redox state of NQO1+ pancreatic cancer cells, causing perturbation in central carbon metabolism.

PubMed

Silvers, Molly A; Deja, Stanislaw; Singh, Naveen; Egnatchik, Robert A; Sudderth, Jessica; Luo, Xiuquan; Beg, Muhammad S; Burgess, Shawn C; DeBerardinis, Ralph J; Boothman, David A; Merritt, Matthew E

2017-11-03

Many cancer treatments, such as those for managing recalcitrant tumors like pancreatic ductal adenocarcinoma, cause off-target toxicities in normal, healthy tissue, highlighting the need for more tumor-selective chemotherapies. β-Lapachone is bioactivated by NAD(P)H:quinone oxidoreductase 1 (NQO1). This enzyme exhibits elevated expression in most solid cancers and therefore is a potential cancer-specific target. β-Lapachone's therapeutic efficacy partially stems from the drug's induction of a futile NQO1-mediated redox cycle that causes high levels of superoxide and then peroxide formation, which damages DNA and causes hyperactivation of poly(ADP-ribose) polymerase, resulting in extensive NAD + /ATP depletion. However, the effects of this drug on energy metabolism due to NAD + depletion were never described. The futile redox cycle rapidly consumes O 2 , rendering standard assays of Krebs cycle turnover unusable. In this study, a multimodal analysis, including metabolic imaging using hyperpolarized pyruvate, points to reduced oxidative flux due to NAD + depletion after β-lapachone treatment of NQO1+ human pancreatic cancer cells. NAD + -sensitive pathways, such as glycolysis, flux through lactate dehydrogenase, and the citric acid cycle (as inferred by flux through pyruvate dehydrogenase), were down-regulated by β-lapachone treatment. Changes in flux through these pathways should generate biomarkers useful for in vivo dose responses of β-lapachone treatment in humans, avoiding toxic side effects. Targeting the enzymes in these pathways for therapeutic treatment may have the potential to synergize with β-lapachone treatment, creating unique NQO1-selective combinatorial therapies for specific cancers. These findings warrant future studies of intermediary metabolism in patients treated with β-lapachone. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Temperature-dependent IR spectroscopic and structural study of 18-crown-6 chelating ligand in the complexation with sodium surfactant salts and potassium picrate.

PubMed

Mihelj, Tea; Tomašić, Vlasta; Biliškov, Nikola; Liu, Feng

2014-04-24

18-crown-6 ether (18C6) complexes with the following anionic surfactants: sodium n-dodecylsulfate (18C6-NaDS), sodium 4-(1-pentylheptyl)benzenesulfonate (18C6-NaDBS); and potassium picrate (18C6-KP) were synthesized and studied in terms of their thermal and structural properties. Physico-chemical properties of new solid 1:1 coordination complexes were characterized by infrared (IR) spectroscopy, thermogravimetry and differential thermal analysis, differential scanning calorimetry, X-ray diffraction and microscopic observations. The strength of coordination between Na(+) and oxygen atoms of 18C6 ligand does not depend on anionic part of the surfactant, as established by thermodynamical parameters obtained by temperature-dependent IR spectroscopy. Each of these complexes exhibit different kinds of endothermic transitions in heating scan. Diffraction maxima obtained by SAXS and WAXS, refer the behavior of the compounds 18C6-NaDS and 18C6-NaDBS as smectic liquid crystalline. Distortion of 18C6-NaDS and 18C6-KP complexes occurs in two steps. Temperature of the decomplexation of solid crystal complex 18C6-KP is considerably higher than of mesophase complexes, 18C6-NaDS, and 18C6-NaDBS. The structural and liquid crystalline properties of novel 18-crown-ether complexes are function of anionic molecule geometry, type of chosen cation (Na(+), K(+)), as well as architecture of self-organized aggregates. A good combination of crown ether unit and amphiphile may provide a possibility for preparing new functionalized materials, opening the research field of ion complexation and of host-guest type behavior. Copyright © 2013 Elsevier B.V. All rights reserved.

Catalases Are NAD(P)H-Dependent Tellurite Reductases

PubMed Central

Calderón, Iván L.; Arenas, Felipe A.; Pérez, José Manuel; Fuentes, Derie E.; Araya, Manuel A.; Saavedra, Claudia P.; Tantaleán, Juan C.; Pichuantes, Sergio E.; Youderian, Philip A.; Vásquez, Claudio C.

2006-01-01

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO3 2−) to the less toxic, insoluble metal, tellurium (Te°), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical. PMID:17183702

Catalases are NAD(P)H-dependent tellurite reductases.

PubMed

Calderón, Iván L; Arenas, Felipe A; Pérez, José Manuel; Fuentes, Derie E; Araya, Manuel A; Saavedra, Claudia P; Tantaleán, Juan C; Pichuantes, Sergio E; Youderian, Philip A; Vásquez, Claudio C

2006-12-20

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

PubMed

Deutch, Charles E

2013-11-01

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

Impact of anatomical traits of maize (Zea mays L.) leaf as affected by nitrogen supply and leaf age on bundle sheath conductance.

PubMed

Retta, Moges; Yin, Xinyou; van der Putten, Peter E L; Cantre, Denis; Berghuijs, Herman N C; Ho, Quang Tri; Verboven, Pieter; Struik, Paul C; Nicolaï, Bart M

2016-11-01

The mechanism of photosynthesis in C 4 crops depends on the archetypal Kranz-anatomy. To examine how the leaf anatomy, as altered by nitrogen supply and leaf age, affects the bundle sheath conductance (g bs ), maize (Zea mays L.) plants were grown under three contrasting nitrogen levels. Combined gas exchange and chlorophyll fluorescence measurements were done on fully grown leaves at two leaf ages. The measured data were analysed using a biochemical model of C 4 photosynthesis to estimate g bs . The leaf microstructure and ultrastructure were quantified using images obtained from micro-computed tomography and microscopy. There was a strong positive correlation between g bs and leaf nitrogen content (LNC) while old leaves had lower g bs than young leaves. Leaf thickness, bundle sheath cell wall thickness and surface area of bundle sheath cells per unit leaf area (S b ) correlated well with g bs although they were not significantly affected by LNC. As a result, the increase of g bs with LNC was little explained by the alteration of leaf anatomy. In contrast, the combined effect of LNC and leaf age on S b was responsible for differences in g bs between young leaves and old leaves. Future investigations should consider changes at the level of plasmodesmata and membranes along the CO 2 leakage pathway to unravel LNC and age effects further. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effect of cotton bollworm (Helicoverpa armigera Hübner) caused injury on maize grain content, especially regarding to the protein alteration.

PubMed

Keszthelyi, S; Pál-Fám, F; Kerepesi, I

2011-03-01

The cotton bollworm (Helicoverpa armigera Hübner), which migrated in the Carpathian-basin from Mediterraneum in the last decades, is becoming an increasingly serious problem for maize producers in Hungary. In several regions the damage it causes has reached the threshold of economic loss, especially in the case of the sweet maize cultivation. The aim of the research was to determine the changing of ears weights and in-kernel accumulation and alteration in grain as a function of cotton bollworm mastication.Our investigation confirmed that there is an in-kernel and protein pattern change of maize grain by cotton bollworm. Our results proved the significant damaging of each part of ears by cotton bollworm masticating (the average weight loss of ears: 13.99%; the average weight loss of grains: 14.03%; the average weight loss of cobs: 13.74%), with the exception of the increasing of the grain-cob ratio. Our examinations did not prove the water loss - that is the "forced maturing" - caused by the damage. Decreasing of raw fat (control: 2.8%; part-damaged: 2.6%; damaged: 2.4%) and starch content (control: 53.1%; part-damaged: 46.6%; damaged: 44.7%) were registered as a function of injury. In contrast, the raw protein content was increased (control: 4.7%; part-damaged: 5.3%; damaged: 7.4%) by maize ear masticating. The most conspicuous effect on protein composition changing was proved by comparison of damaged grain samples by SDS PAGE. Increased amounts of 114, 50, 46 and 35 kDa molecular mass proteins were detected which explained the more than 50% elevation of raw protein content. The statistical analysis of molecular weights proved the protein realignment as a function of the pest injuries, too.

Aggressiveness of Cephalosporium maydis causing late wilt of maize in Spain.

PubMed

García-Carneros, A B; Girón, I; Molinero-Ruiz, L

2012-01-01

Late wilt of maize, caused by the vascular and soilborne pathogen Cephalosporium maydis, was identified in the Iberian Peninsula in 2008. During the last years the incidence and economical impact of the disease has importantly increased both in Portugal and Spain. Varieties of maize displaying tolerance to the pathogen are available, but the effectiveness can be dependent on the virulence of the fungus (i.e. ability to cause disease on a specific genotype). On the other hand, strains of crop pathogens from different geographic origins can differ with regard to the degree of disease caused on a specific genotype (i.e. aggressiveness). Our working hypothesis was that isolates of C. maydis from different maize growing areas may differ in aggressiveness towards maize plants. Seven fungal strains were isolated in 2009 from diseased plants collected in the most important maize growing regions of Spain and used to inoculate two susceptible maize varieties grown in shadehouse from March to July 2010. The experimental unit consisted of two 4-day-old seedlings planted in an 8-liter pot filled with sand/silt previously infested with 200 g of wheat grains colonized by the fungi. Non colonized wheat grains were used for the control treatments. Six replications (pots) were established for each variety/isolate combination according to a complete randomized 2 x 8 factorial design. The percentage of necrotic and dry aboveground tissues was recorded 14 weeks after inoculation and thereafter weekly until physiological senescence of the control plants. At the end of the experiment, weights of roots and aboveground parts of the plants were recorded. Initial occurrence of symptoms in the plants was significantly dependent on the isolate of C. maydis and on the maize variety. However, final severity of aboveground symptoms (leaf necroses and drying up) was only dependent on the fungal isolate. All the isolates significantly reduced the root weight of both varieties of maize. The highest root weight reductions were also associated to a significant low weight of above-ground parts. Considering all the symptoms analysed and their progression in the maize plants, our results reveal that a diversity of aggressiveness exists among isolates of C. maydis. The need for a characterization of maize genotypes by their reaction against highly aggressive isolates of the fungus in the Iberian Peninsula is suggested. This study is a first step towards a recommendation of crop varieties that are tolerant to C. maydis in different areas of the Iberian Peninsula. Future research aims at studying the relationship between aggressiveness levels, molecular characteristics and geographical origin whithin C. maydis.

Maize, tropical (Zea mays L.).

PubMed

Assem, Shireen K

2015-01-01

Maize (Zea mays L.) is the third most important food crop globally after wheat and rice. In sub-Saharan Africa, tropical maize has traditionally been the main staple of the diet; 95 % of the maize grown is consumed directly as human food and as an important source of income for the resource-poor rural population. The biotechnological approach to engineer biotic and abiotic traits implies the availability of an efficient plant transformation method. The production of genetically transformed plants depends both on the ability to integrate foreign genes into target cells and the efficiency with which plants are regenerated. Maize transformation and regeneration through immature embryo culture is the most efficient system to regenerate normal transgenic plants. However, this system is highly genotype dependent. Genotypes adapted to tropic areas are difficult to regenerate. Therefore, transformation methods used with model genotypes adapted to temperate areas are not necessarily efficient with tropical lines. Agrobacterium-mediated transformation is the method of choice since it has been first achieved in 1996. In this report, we describe a transformation method used successfully with several tropical maize lines. All the steps of transformation and regeneration are described in details. This protocol can be used with a wide variety of tropical lines. However, some modifications may be needed with recalcitrant lines.

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p

PubMed Central

Helliwell, Stephen B.; Karkare, Shantanu; Bergdoll, Marc; Rahier, Alain; Leighton-Davis, Juliet R.; Fioretto, Celine; Aust, Thomas; Filipuzzi, Ireos; Frederiksen, Mathias; Gounarides, John; Hoepfner, Dominic; Hofmann, Andreas; Imbert, Pierre-Eloi; Jeker, Rolf; Knochenmuss, Richard; Krastel, Philipp; Margerit, Anais; Memmert, Klaus; Miault, Charlotte V.; Rao Movva, N.; Muller, Alban; Naegeli, Hans-Ulrich; Oberer, Lukas; Prindle, Vivian; Riedl, Ralph; Schuierer, Sven; Sexton, Jessica A.; Tao, Jianshi; Wagner, Trixie; Yin, Hong; Zhang, Juan; Roggo, Silvio; Reinker, Stefan; Parker, Christian N.

2015-01-01

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae—Erg26p, Homo sapiens—NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound. PMID:26456460

Characteristics of the Mg2+-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor 1

PubMed Central

Cohen, William S.

1989-01-01

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg2+-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na2SO3-dependent Mg2+-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg2+-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg2+-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg2+-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one. PMID:16667119

Evolutionary divergence of phytochrome protein function in Zea mays PIF3 signaling.

PubMed

Kumar, Indrajit; Swaminathan, Kankshita; Hudson, Karen; Hudson, Matthew E

2016-07-01

Two maize phytochrome-interacting factor (PIF) basic helix-loop-helix (bHLH) family members, ZmPIF3.1 and ZmPIF3.2, were identified, cloned and expressed in vitro to investigate light-signaling interactions. A phylogenetic analysis of sequences of the maize bHLH transcription factor gene family revealed the extent of the PIF family, and a total of seven predicted PIF-encoding genes were identified from genes encoding bHLH family VIIa/b proteins in the maize genome. To investigate the role of maize PIFs in phytochrome signaling, full-length cDNAs for phytochromes PhyA2, PhyB1, PhyB2 and PhyC1 from maize were cloned and expressed in vitro as chromophorylated holophytochromes. We showed that ZmPIF3.1 and ZmPIF3.2 interact specifically with the Pfr form of maize holophytochrome B1 (ZmphyB1), showing no detectable affinity for the Pr form. Maize holophytochrome B2 (ZmphyB2) showed no detectable binding affinity for PIFs in either Pr or Pfr forms, but phyB Pfr from Arabidopsis interacted with ZmPIF3.1 similarly to ZmphyB1 Pfr. We conclude that subfunctionalization at the protein-protein interaction level has altered the role of phyB2 relative to that of phyB1 in maize. Since the phyB2 mutant shows photomorphogenic defects, we conclude that maize phyB2 is an active photoreceptor, without the binding of PIF3 seen in other phyB family proteins. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease.

PubMed

Gariani, Karim; Ryu, Dongryeol; Menzies, Keir J; Yi, Hyon-Seung; Stein, Sokrates; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Katsyuba, Elena; Jha, Pooja; Vijgen, Sandrine; Rubbia-Brandt, Laura; Kim, Yong Kyung; Kim, Jung Tae; Kim, Koon Soon; Shong, Minho; Schoonjans, Kristina; Auwerx, Johan

2017-01-01

To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD + ), we hypothesized that overactivation of PARPs drives NAD + depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD + and activate NAD + -dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD + levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD + , increasing mitochondrial biogenesis and β-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1 hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse models of NAFLD confer a protection against its development. PARP inhibitors may therefore represent a novel and practical pharmacological approach for treating NAFLD. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Climate change and maize yield in Iowa

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

Xu, Hong; Twine, Tracy E.; Girvetz, Evan

Climate is changing across the world, including the major maize-growing state of Iowa in the USA. To maintain crop yields, farmers will need a suite of adaptation strategies, and choice of strategy will depend on how the local to regional climate is expected to change. Here we predict how maize yield might change through the 21 st century as compared with late 20 th century yields across Iowa, USA, a region representing ideal climate and soils for maize production that contributes substantially to the global maize economy. To account for climate model uncertainty, we drive a dynamic ecosystem model withmore » output from six climate models and two future climate forcing scenarios. Despite a wide range in the predicted amount of warming and change to summer precipitation, all simulations predict a decrease in maize yields from late 20 th century to middle and late 21 st century ranging from 15% to 50%. Linear regression of all models predicts a 6% state-averaged yield decrease for every 1°C increase in warm season average air temperature. When the influence of moisture stress on crop growth is removed from the model, yield decreases either remain the same or are reduced, depending on predicted changes in warm season precipitation. Lastly, our results suggest that even if maize were to receive all the water it needed, under the strongest climate forcing scenario yields will decline by 10-20% by the end of the 21 st century.« less

Climate change and maize yield in Iowa

DOE PAGES

Xu, Hong; Twine, Tracy E.; Girvetz, Evan

2016-05-24

Climate is changing across the world, including the major maize-growing state of Iowa in the USA. To maintain crop yields, farmers will need a suite of adaptation strategies, and choice of strategy will depend on how the local to regional climate is expected to change. Here we predict how maize yield might change through the 21 st century as compared with late 20 th century yields across Iowa, USA, a region representing ideal climate and soils for maize production that contributes substantially to the global maize economy. To account for climate model uncertainty, we drive a dynamic ecosystem model withmore » output from six climate models and two future climate forcing scenarios. Despite a wide range in the predicted amount of warming and change to summer precipitation, all simulations predict a decrease in maize yields from late 20 th century to middle and late 21 st century ranging from 15% to 50%. Linear regression of all models predicts a 6% state-averaged yield decrease for every 1°C increase in warm season average air temperature. When the influence of moisture stress on crop growth is removed from the model, yield decreases either remain the same or are reduced, depending on predicted changes in warm season precipitation. Lastly, our results suggest that even if maize were to receive all the water it needed, under the strongest climate forcing scenario yields will decline by 10-20% by the end of the 21 st century.« less

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors

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

Wilsbacher, Julie L.; Cheng, Min; Cheng, Dong

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell deathmore » when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236–45.« less

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

Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong

NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvatemore » to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.« less

Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

PubMed

Bremer, Daniel; Leben, Ruth; Mothes, Ronja; Radbruch, Helena; Niesner, Raluca

2017-04-03

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Downregulation of transcription factor aflR in Aspergillus flavus confers reduction to aflatoxin accumulation in transgenic maize with alteration of host plant architecture.

PubMed

Masanga, Joel Okoyo; Matheka, Jonathan Mutie; Omer, Rasha Adam; Ommeh, Sheila Cecily; Monda, Ethel Oranga; Alakonya, Amos Emitati

2015-08-01

We report success of host-induced gene silencing in downregulation of aflatoxin biosynthesis in Aspergillus flavus infecting maize transformed with a hairpin construct targeting transcription factor aflR. Infestation of crops by aflatoxin-producing fungi results in economic losses as well as negative human and animal health effects. Currently, the control strategies against aflatoxin accumulation are not effective to the small holder farming systems in Africa and this has led to widespread aflatoxin exposure especially in rural populations of sub-Saharan Africa that rely on maize as a staple food crop. A recent strategy called host-induced gene silencing holds great potential for developing aflatoxin-resistant plant germplasm for the African context where farmers are unable to make further investments other than access to the germplasm. We transformed maize with a hairpin construct targeting the aflatoxin biosynthesis transcription factor aflR. The developed transgenic maize were challenged with an aflatoxigenic Aspergillus flavus strain from Eastern Kenya, a region endemic to aflatoxin outbreaks. Our results indicated that aflR was downregulated in A. flavus colonizing transgenic maize. Further, maize kernels from transgenic plants accumulated significantly lower levels of aflatoxins (14-fold) than those from wild type plants. Interestingly, we observed that our silencing cassette caused stunting and reduced kernel placement in the transgenic maize. This could have been due to "off-target" silencing of unintended genes in transformed plants by aflR siRNAs. Overall, this work indicates that host-induced gene silencing has potential in developing aflatoxin-resistant germplasm.

C acid decarboxylases required for C photosynthesis are active in the mid-vein of the C species Arabidopsis thaliana, and are important in sugar and amino acid metabolism.

PubMed

Brown, Naomi J; Palmer, Ben G; Stanley, Susan; Hajaji, Hana; Janacek, Sophie H; Astley, Holly M; Parsley, Kate; Kajala, Kaisa; Quick, W Paul; Trenkamp, Sandra; Fernie, Alisdair R; Maurino, Veronica G; Hibberd, Julian M

2010-01-01

Cells associated with veins of petioles of C(3) tobacco possess high activities of the decarboxylase enzymes required in C(4) photosynthesis. It is not clear whether this is the case in other C(3) species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C(4) acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.

The NAMPT/E2F2/SIRT1 axis promotes proliferation and inhibits p53-dependent apoptosis in human melanoma cells.

PubMed

Zhao, Hailong; Tang, Weiwei; Chen, Xiaowen; Wang, Siyu; Wang, Xianyan; Xu, Haiyan; Li, Lijuan

2017-11-04

Melanoma is the most common primary malignant neoplasm in adults, causing more deaths than any other skin cancer, necessitating the development of new target-based approaches. Current evidence suggests SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD + biosynthetic enzyme, together comprise a novel systemic regulatory network to play a pivotal role in cell proliferation and apoptosis. Nevertheless, how the regulation of this cofactor interfaces with signal transduction network remains poorly understood in melanoma. Here, we report NAMPT is highly expressed in melanomaassociated with poor overall survival in patients. Pharmacological and genetic inhibition of NAMPT decreased NAD + levels and melanoma cell proliferation capacity, and NAMPT knockdown induced apoptosis through the activity of the tumor suppressor p53. Next, we demonstrate NAMPT regulates the transcription factor E2F family member 2 (E2F2) in the apoptosis process. Downstream, E2F2 control the mRNA and protein levels of SIRT1. Finally, we find NAMPT mediates the apoptosis resistance of melanoma cells through NAMPT-E2F2-SIRT1 axis, more than NAD + -driven transcriptional program. Accordingly, our results demonstrated that NAMPT is a prognostic marker in melanoma, and the identificationofNAMPT-E2F2-SIRT1 pathway establishes another link between NAMPT and apoptosis events in melanoma, with therapeutic implications for this deadly cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

PubMed

Kalous, Kelsey S; Wynia-Smith, Sarah L; Olp, Michael D; Smith, Brian C

2016-12-02

The sirtuin family of proteins catalyze the NAD + -dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD + and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn 2+ release from the conserved sirtuin Zn 2+ -tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn 2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD + and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn 2+ , consistent with S-nitrosation of the Zn 2+ -tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic Factors Involved in Fumonisin Accumulation in Maize Kernels and Their Implications in Maize Agronomic Management and Breeding

PubMed Central

Santiago, Rogelio; Cao, Ana; Butrón, Ana

2015-01-01

Contamination of maize with fumonisins depends on the environmental conditions; the maize resistance to contamination and the interaction between both factors. Although the effect of environmental factors is a determinant for establishing the risk of kernel contamination in a region, there is sufficient genetic variability among maize to develop resistance to fumonisin contamination and to breed varieties with contamination at safe levels. In addition, ascertaining which environmental factors are the most important in a region will allow the implementation of risk monitoring programs and suitable cultural practices to reduce the impact of such environmental variables. The current paper reviews all works done to address the influence of environmental variables on fumonisin accumulation, the genetics of maize resistance to fumonisin accumulation, and the search for the biochemical and/or structural mechanisms of the maize plant that could be involved in resistance to fumonisin contamination. We also explore the outcomes of breeding programs and risk monitoring of undertaken projects. PMID:26308050

Genetic Factors Involved in Fumonisin Accumulation in Maize Kernels and Their Implications in Maize Agronomic Management and Breeding.

PubMed

Santiago, Rogelio; Cao, Ana; Butrón, Ana

2015-08-20

Contamination of maize with fumonisins depends on the environmental conditions; the maize resistance to contamination and the interaction between both factors. Although the effect of environmental factors is a determinant for establishing the risk of kernel contamination in a region, there is sufficient genetic variability among maize to develop resistance to fumonisin contamination and to breed varieties with contamination at safe levels. In addition, ascertaining which environmental factors are the most important in a region will allow the implementation of risk monitoring programs and suitable cultural practices to reduce the impact of such environmental variables. The current paper reviews all works done to address the influence of environmental variables on fumonisin accumulation, the genetics of maize resistance to fumonisin accumulation, and the search for the biochemical and/or structural mechanisms of the maize plant that could be involved in resistance to fumonisin contamination. We also explore the outcomes of breeding programs and risk monitoring of undertaken projects.

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.

Use of Drought Index and Crop Modelling for Drought Impacts Analysis on Maize (Zea mays L.) Yield Loss in Bandung District

NASA Astrophysics Data System (ADS)

Kurniasih, E.; Impron; Perdinan

2017-03-01

Drought impacts on crop yield loss depend on drought magnitude and duration and on plant genotype at every plant growth stages when droughts occur. This research aims to assess the difference calculation results of 2 drought index methods and to study the maize yield loss variability impacted by drought magnitude and duration during maize growth stages in Bandung district, province of West Java, Indonesia. Droughts were quantified by the Standardized Precipitation Index (SPI) and the Standardized Precipitation Evapotranspiration Index (SPEI) at 1- to 3-month lags for the January1986-December 2015 period data. Maize yield responses to droughts were simulated by AquaCrop for the January 1986-May 2016 period of growing season. The analysis showed that the SPI and SPEI methods provided similar results in quantifying drought event. Droughts during maize reproductive stages caused the highest maize yield loss.

Cloning and Characterization of GLOSSY1, a Maize Gene Involved in Cuticle Membrane and Wax Production1[w

PubMed Central

Sturaro, Monica; Hartings, Hans; Schmelzer, Elmon; Velasco, Riccardo; Salamini, Francesco; Motto, Mario

2005-01-01

The cuticle covering the aerial organs of land plants plays a protective role against several biotic and abiotic stresses and, in addition, participates in a variety of plant-insect interactions. Here, we describe the molecular cloning and characterization of the maize (Zea mays) GLOSSY1 (GL1) gene, a component of the pathway leading to cuticular wax biosynthesis in seedling leaves. The genomic and cDNA sequences we isolated differ significantly in length and in most of the coding region from those previously identified. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes. GL1 expression is not restricted to the juvenile developmental stage of the maize plant, pointing to a broader function of the gene product than anticipated on the basis of the mutant phenotype. Indeed, in addition to affecting cuticular wax biosynthesis, gl1 mutations have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure. Of the many wax biosynthetic genes identified so far, only a few from Arabidopsis (Arabidopsis thaliana) were found to be essential for normal cutin formation. Among these is WAX2, which shares 62% identity with GL1 at the protein level. In wax2-defective plants, cutin alterations induce postgenital organ fusion. This trait is not displayed by gl1 mutants, suggesting a different role of the maize and Arabidopsis cuticle in plant development. PMID:15849306

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

PubMed Central

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

2011-01-01

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

Soil Selenium (Se) Biofortification Changes the Physiological, Biochemical and Epigenetic Responses to Water Stress in Zea mays L. by Inducing a Higher Drought Tolerance.

PubMed

Bocchini, Marika; D'Amato, Roberto; Ciancaleoni, Simona; Fontanella, Maria C; Palmerini, Carlo A; Beone, Gian M; Onofri, Andrea; Negri, Valeria; Marconi, Gianpiero; Albertini, Emidio; Businelli, Daniela

2018-01-01

Requiring water and minerals to grow and to develop its organs, Maize ( Zea mays L.) production and distribution is highly rainfall-dependent. Current global climatic changes reveal irregular rainfall patterns and this could represent for maize a stressing condition resulting in yield and productivity loss around the world. It is well known that low water availability leads the plant to adopt a number of metabolic alterations to overcome stress or reduce its effects. In this regard, selenium (Se), a trace element, can help reduce water damage caused by the overproduction of reactive oxygen species (ROS). Here we report the effects of exogenous Se supply on physiological and biochemical processes that may influence yield and quality of maize under drought stress conditions. Plants were grown in soil fertilized by adding 150 mg of Se (sodium selenite). We verified the effects of drought stress and Se treatment. Selenium biofortification proved more beneficial for maize plants when supplied at higher Se concentrations. The increase in proline, K concentrations and nitrogen metabolism in aerial parts of plants grown in Se-rich substrates, seems to prove that Se-biofortification increased plant resistance to water shortage conditions. Moreover, the increase of SeMeSeCys and SeCys2 forms in roots and aerial parts of Se-treated plants suggest resistance strategies to Se similar to those existing in Se-hyperaccumulator species. In addition, epigenetic changes in DNA methylation due to water stress and Se treatment were also investigated using methylation sensitive amplified polymorphism (MSAP). Results suggest that Se may be an activator of particular classes of genes that are involved in tolerance to abiotic stresses. In particular, PSY (phytoene synthase) gene, essential for maintaining leaf carotenoid contents, SDH (sorbitol dehydrogenase), whose activity regulates the level of important osmolytes during drought stress and ADH (alcohol dehydrogenase), whose activity plays a central role in biochemical adaptation to environmental stress. In conclusion, Se-biofortification could help maize plants to cope with drought stress conditions, by inducing a higher drought tolerance.

Soil Selenium (Se) Biofortification Changes the Physiological, Biochemical and Epigenetic Responses to Water Stress in Zea mays L. by Inducing a Higher Drought Tolerance

PubMed Central

Bocchini, Marika; D’Amato, Roberto; Ciancaleoni, Simona; Fontanella, Maria C.; Palmerini, Carlo A.; Beone, Gian M.; Onofri, Andrea; Negri, Valeria; Marconi, Gianpiero; Albertini, Emidio; Businelli, Daniela

2018-01-01

Requiring water and minerals to grow and to develop its organs, Maize (Zea mays L.) production and distribution is highly rainfall-dependent. Current global climatic changes reveal irregular rainfall patterns and this could represent for maize a stressing condition resulting in yield and productivity loss around the world. It is well known that low water availability leads the plant to adopt a number of metabolic alterations to overcome stress or reduce its effects. In this regard, selenium (Se), a trace element, can help reduce water damage caused by the overproduction of reactive oxygen species (ROS). Here we report the effects of exogenous Se supply on physiological and biochemical processes that may influence yield and quality of maize under drought stress conditions. Plants were grown in soil fertilized by adding 150 mg of Se (sodium selenite). We verified the effects of drought stress and Se treatment. Selenium biofortification proved more beneficial for maize plants when supplied at higher Se concentrations. The increase in proline, K concentrations and nitrogen metabolism in aerial parts of plants grown in Se-rich substrates, seems to prove that Se-biofortification increased plant resistance to water shortage conditions. Moreover, the increase of SeMeSeCys and SeCys2 forms in roots and aerial parts of Se-treated plants suggest resistance strategies to Se similar to those existing in Se-hyperaccumulator species. In addition, epigenetic changes in DNA methylation due to water stress and Se treatment were also investigated using methylation sensitive amplified polymorphism (MSAP). Results suggest that Se may be an activator of particular classes of genes that are involved in tolerance to abiotic stresses. In particular, PSY (phytoene synthase) gene, essential for maintaining leaf carotenoid contents, SDH (sorbitol dehydrogenase), whose activity regulates the level of important osmolytes during drought stress and ADH (alcohol dehydrogenase), whose activity plays a central role in biochemical adaptation to environmental stress. In conclusion, Se-biofortification could help maize plants to cope with drought stress conditions, by inducing a higher drought tolerance. PMID:29636765

Chemically modified maize cobs waste with enhanced adsorption properties upon methyl orange and arsenic.

PubMed

Elizalde-González, María P; Mattusch, J; Wennrich, R

2008-07-01

The surface chemistry of maize naturasorbent was altered in this work by the modifying agents: phosphoric acid and different amines (triethanolamine, diethylenetriamine and 1,4-diaminobutane). Removal of methyl orange (25 mg l(-1)) was <50% by maize corn cobs modified by phosphorylation and higher by the quaternized samples: 68% with the 1,4-diaminobutane and 73% with the diethylenetriamine modificators. Adsorption of arsenite by the samples modified with phosphoric acid/ammonia was 11 microg g(-1), which corresponds to 98% removal from a 550 microg As l(-1) solution for an adsorbent dose of 50 mg ml(-1). The samples modified by phosphoric acid/urea removed 0.4 microg g(-1) arsenate from a 300 mug As l(-1) solution. Adsorption of methyl orange, arsenite and arsenate was superior by the chemically modified maize cobs judged against the initial naturasorbent. For comparison, removal by the commercial anion exchanger was 100% for methyl orange, 45% (5 microg g(-1)) for arsenite and 99% (5 microg g(-1)) for arsenate.

Metabolic changes in transgenic maize mature seeds over-expressing the Aspergillus niger phyA2.

PubMed

Rao, Jun; Yang, Litao; Guo, Jinchao; Quan, Sheng; Chen, Guihua; Zhao, Xiangxiang; Zhang, Dabing; Shi, Jianxin

2016-02-01

Non-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2. Genetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC-MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

Genome-wide identification, expression analysis of auxin-responsive GH3 family genes in maize (Zea mays L.) under abiotic stresses.

PubMed

Feng, Shangguo; Yue, Runqing; Tao, Sun; Yang, Yanjun; Zhang, Lei; Xu, Mingfeng; Wang, Huizhong; Shen, Chenjia

2015-09-01

Auxin is involved in different aspects of plant growth and development by regulating the expression of auxin-responsive family genes. As one of the three major auxin-responsive families, GH3 (Gretchen Hagen3) genes participate in auxin homeostasis by catalyzing auxin conjugation and bounding free indole-3-acetic acid (IAA) to amino acids. However, how GH3 genes function in responses to abiotic stresses and various hormones in maize is largely unknown. Here, the latest updated maize (Zea mays L.) reference genome sequence was used to characterize and analyze the ZmGH3 family genes from maize. The results showed that 13 ZmGH3 genes were mapped on five maize chromosomes (total 10 chromosomes). Highly diversified gene structures and tissue-specific expression patterns suggested the possibility of function diversification for these genes in response to environmental stresses and hormone stimuli. The expression patterns of ZmGH3 genes are responsive to several abiotic stresses (salt, drought and cadmium) and major stress-related hormones (abscisic acid, salicylic acid and jasmonic acid). Various environmental factors suppress auxin free IAA contents in maize roots suggesting that these abiotic stresses and hormones might alter GH3-mediated auxin levels. The responsiveness of ZmGH3 genes to a wide range of abiotic stresses and stress-related hormones suggested that ZmGH3s are involved in maize tolerance to environmental stresses. © 2014 Institute of Botany, Chinese Academy of Sciences.

The Bacterial Community Structure and Dynamics of Carbon and Nitrogen when Maize (Zea mays L.) and Its Neutral Detergent Fibre Were Added to Soil from Zimbabwe with Contrasting Management Practices.

PubMed

De la Cruz-Barrón, Magali; Cruz-Mendoza, Alejandra; Navarro-Noya, Yendi E; Ruiz-Valdiviezo, Victor M; Ortíz-Gutiérrez, Daniel; Ramírez-Villanueva, Daniel A; Luna-Guido, Marco; Thierfelder, Cristian; Wall, Patrick C; Verhulst, Nele; Govaerts, Bram; Dendooven, Luc

2017-01-01

Water infiltration, soil carbon content, aggregate stability and yields increased in conservation agriculture practices compared to conventionally ploughed control treatments at the Henderson research station near Mazowe (Zimbabwe). How these changes in soil characteristics affect the bacterial community structure and the bacteria involved in the degradation of applied organic material remains unanswered. Soil was sampled from three agricultural systems at Henderson, i.e. (1) conventional mouldboard ploughing with continuous maize (conventional tillage), (2) direct seeding with a Fitarelli jab planter and continuous maize (direct seeding with continuous maize) and (3) direct seeding with a Fitarelli jab planter with rotation of maize sunn hemp (direct seeding with crop rotation). Soil was amended with young maize plants or their neutral detergent fibre (NDF) and incubated aerobically for 56 days, while C and N mineralization and the bacterial community structure were monitored. Bacillus (Bacillales), Micrococcaceae (Actinomycetales) and phylotypes belonging to the Pseudomonadales were first degraders of the applied maize plants. At day 3, Streptomyces (Actinomycetales), Chitinophagaceae ([Saprospirales]) and Dyella (Xanthomonadales) participated in the degradation of the applied maize and at day 7 Oxalobacteraceae (Burkholderiales). Phylotypes belonging to Halomonas (Oceanospirillales) were the first degraders of NDF and were replaced by Phenylobacterium (Caulobacterales) and phylotypes belonging to Pseudomonadales at day 3. Afterwards, similar bacterial groups were favoured by application of NDF as they were by the application of maize plants, but there were also clear differences. Phylotypes belonging to the Micrococcaceae and Bacillus did not participate in the degradation of NDF or its metabolic products, while phylotypes belonging to the Acidobacteriaceae participated in the degradation of NDF but not in that of maize plants. It was found that agricultural practices had a limited effect on the bacterial community structure, but application of organic material altered it substantially.

Regulation of 11 beta-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol.

PubMed

Gomez-Sanchez, Elise P; Ganjam, Venkataseshu; Chen, Yuan Jian; Liu, Ying; Zhou, Ming Yi; Toroslu, Cigdem; Romero, Damian G; Hughson, Michael D; de Rodriguez, Angela; Gomez-Sanchez, Celso E

2003-08-01

The 11beta-hydroxysteroid dehydrogenase (11betaHSD) type 1 (11betaHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11betaHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11betaHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11betaHSD1 and -2 enzymes are described herein. Renal 11betaHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11betaHSD2 mRNA and protein expressions were significantly increased by E2 treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11betaHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11betaHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.

Adipose tissue NAD+-homeostasis, sirtuins and poly(ADP-ribose) polymerases -important players in mitochondrial metabolism and metabolic health.

PubMed

Jokinen, Riikka; Pirnes-Karhu, Sini; Pietiläinen, Kirsi H; Pirinen, Eija

2017-08-01

Obesity, a chronic state of energy overload, is characterized by adipose tissue dysfunction that is considered to be the major driver for obesity associated metabolic complications. The reasons for adipose tissue dysfunction are incompletely understood, but one potential contributing factor is adipose tissue mitochondrial dysfunction. Derangements of adipose tissue mitochondrial biogenesis and pathways associate with obesity and metabolic diseases. Mitochondria are central organelles in energy metabolism through their role in energy derivation through catabolic oxidative reactions. The mitochondrial processes are dependent on the proper NAD + /NADH redox balance and NAD + is essential for reactions catalyzed by the key regulators of mitochondrial metabolism, sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs). Notably, obesity is associated with disturbed adipose tissue NAD + homeostasis and the balance of SIRT and PARP activities. In this review we aim to summarize existing literature on the maintenance of intracellular NAD + pools and the function of SIRTs and PARPs in adipose tissue during normal and obese conditions, with the purpose of comprehending their potential role in mitochondrial derangements and obesity associated metabolic complications. Understanding the molecular mechanisms that are the root cause of the adipose tissue mitochondrial derangements is crucial for developing new effective strategies to reverse obesity associated metabolic complications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Ligand binding phenomena that pertain to the metabolic function of renalase.

PubMed

Beaupre, Brett A; Roman, Joseph V; Hoag, Matthew R; Meneely, Kathleen M; Silvaggi, Nicholas R; Lamb, Audrey L; Moran, Graham R

2016-12-15

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P) + . This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucleotides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (k red /K d ) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, AMP for mononucleotide or ADP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site. Copyright © 2016 Elsevier Inc. All rights reserved.

Ethylene-dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize.

PubMed

Yamauchi, Takaki; Tanaka, Akihiro; Mori, Hitoshi; Takamure, Itsuro; Kato, Kiyoaki; Nakazono, Mikio

2016-10-01

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen-deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene-dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1-aminocyclopropane-1-carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen-deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis. © 2016 John Wiley & Sons Ltd.

Rapid transcriptome responses of maize (Zea mays) to UV-B in irradiated and shielded tissues

PubMed Central

Casati, Paula; Walbot, Virginia

2004-01-01

Background Depletion of stratospheric ozone has raised terrestrial levels of ultraviolet-B radiation (UV-B), an environmental change linked to an increased risk of skin cancer and with potentially deleterious consequences for plants. To better understand the processes of UV-B acclimation that result in altered plant morphology and physiology, we investigated gene expression in different organs of maize at several UV-B fluence rates and exposure times. Results Microarray hybridization was used to assess UV-B responses in directly exposed maize organs and organs shielded by a plastic that absorbs UV-B. After 8 hours of high UV-B, the abundance of 347 transcripts was altered: 285 were increased significantly in at least one organ and 80 were downregulated. More transcript changes occurred in directly exposed than in shielded organs, and the levels of more transcripts were changed in adult compared to seedling tissues. The time course of transcript abundance changes indicated that the response kinetics to UV-B is very rapid, as some transcript levels were altered within 1 hour of exposure. Conclusions Most of the UV-B regulated genes are organ-specific. Because shielded tissues, including roots, immature ears, and leaves, displayed altered transcriptome profiles after exposure of the plant to UV-B, some signal(s) must be transmitted from irradiated to shielded tissues. These results indicate that there are integrated responses to UV-B radiation above normal levels. As the same total UV-B irradiation dose applied at three intensities elicited different transcript profiles, the transcriptome changes exhibit threshold effects rather than a reciprocal dose-effect response. Transcriptome profiling highlights possible signaling pathways and molecules for future research. PMID:15003119

Effects of sulfur dioxide on expansion of lesions caused by Corynebacterium nebraskense in maize and by Xanthomonas phaseoli var. sojensis in soybean

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

Laurence, J.A.; Aluisio, A.L.

1981-01-01

In order to assess the effects of air pollution on plant disease development, the authors investigated the effects of SO/sub 2/ on lesion development by two bacterial pathogens. Maize or soybean plants were exposed to sulfur dioxide (SO/sub 2/) at 524 ..mu..g m/sup -3/ or 262 ..mu..g m/sup -3/ before, after or before and after inoculation with Corynebacterium nebraskense or Xanthomonas phaseoli var. sojensis, respectively. Lesion development was inhibited in both cases, regardless of when the exposures occurred. The time of exposure, however, altered the subsequent effect on lesion size. Dry weight and sulfur content of host tissue were notmore » altered by the joint effects of the pollutant and the pathogens.« less

Root foraging elicits niche complementarity-dependent yield advantage in the ancient ‘three sisters’ (maize/bean/squash) polyculture

PubMed Central

Zhang, Chaochun; Postma, Johannes A.; York, Larry M.; Lynch, Jonathan P.

2014-01-01

Background and Aims Since ancient times in the Americas, maize, bean and squash have been grown together in a polyculture known as the ‘three sisters’. This polyculture and its maize/bean variant have greater yield than component monocultures on a land-equivalent basis. This study shows that below-ground niche complementarity may contribute to this yield advantage. Methods Monocultures and polycultures of maize, bean and squash were grown in two seasons in field plots differing in nitrogen (N) and phosphorus (P) availability. Root growth patterns of individual crops and entire polycultures were determined using a modified DNA-based technique to discriminate roots of different species. Key Results The maize/bean/squash and maize/bean polycultures had greater yield and biomass production on a land-equivalent basis than the monocultures. Increased biomass production was largely caused by a complementarity effect rather than a selection effect. The differences in root crown architecture and vertical root distribution among the components of the ‘three sisters’ suggest that these species have different, possibly complementary, nutrient foraging strategies. Maize foraged relatively shallower, common bean explored the vertical soil profile more equally, while the root placement of squash depended on P availability. The density of lateral root branching was significantly greater for all species in the polycultures than in the monocultures. Conclusions It is concluded that species differences in root foraging strategies increase total soil exploration, with consequent positive effects on the growth and yield of these ancient polycultures. PMID:25274551

Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation.

PubMed

Spiekermann, Stephan; Landmesser, Ulf; Dikalov, Sergey; Bredt, Martin; Gamez, Graciela; Tatge, Helma; Reepschläger, Nina; Hornig, Burkhard; Drexler, Helmut; Harrison, David G

2003-03-18

Increased inactivation of nitric oxide by superoxide (O2*-) contributes to endothelial dysfunction in patients with coronary disease (CAD). We therefore characterized the vascular activities of xanthine oxidase and NAD(P)H oxidase, 2 major O2*--producing enzyme systems, and their relationship with flow-dependent, endothelium-mediated vasodilation (FDD) in patients with CAD. Xanthine- and NAD(P)H-mediated O*.- formation was determined in coronary arteries from 10 patients with CAD and 10 controls by using electron spin resonance spectroscopy. Furthermore, activity of endothelium-bound xanthine oxidase in vivo and FDD of the radial artery were determined in 21 patients with CAD and 10 controls. FDD was measured before and after infusion of the antioxidant vitamin C (25 mg/min i.a.) to determine the portion of FDD inhibited by radicals. In coronary arteries from patients with CAD, xanthine- and NAD(P)H-mediated O2*- formation was increased compared with controls (xanthine: 12+/-2 versus 7+/-1 nmol O2*-/ microg protein; NADH: 11+/-1 versus 7+/-1 nmol O2*-/ microg protein; and NADPH: 12+/-2 versus 9+/-1 nmol O2*-/ microg protein; each P<0.05). Endothelium-bound xanthine oxidase activity was increased by >200% in patients with CAD (25+/-4 versus 9+/-1 nmol O2*-/ microL plasma per min; P<0.05) and correlated inversely with FDD (r=-0.55; P<0.05) and positively with the effect of vitamin C on FDD (r=0.54; P<0.05). The present study represents the first electron spin resonance measurements of xanthine and NAD(P)H oxidase activity in human coronary arteries and supports the concept that increased activities of both enzymes contribute to increased vascular oxidant stress in patients with CAD. Furthermore, the present study suggests that increased xanthine oxidase activity contributes to endothelial dysfunction in patients with CAD and may thereby promote the atherosclerotic process.

The effect of altered dosage of a mutant allele of Teosinte branched 1 (tb1-ref) on the root system of modern maize

PubMed Central

2014-01-01

Background There was ancient human selection on the wild progenitor of modern maize, Balsas teosinte, for decreased shoot branching (tillering), in order to allow more nutrients to be diverted to grain. Mechanistically, the decline in shoot tillering has been associated with selection for increased expression of the major domestication gene Teosinte Branched 1 (Tb1) in shoot primordia. Therefore, TB1 has been defined as a repressor of shoot branching. It is known that plants respond to changes in shoot size by compensatory changes in root growth and architecture. However, it has not been reported whether altered TB1 expression affects any plant traits below ground. Previously, changes in dosage of a well-studied mutant allele of Tb1 in modern maize, called tb1-ref, from one to two copies, was shown to increase tillering. As a result, plants with two copies of the tb1-ref allele have a larger shoot biomass than heterozygotes. Here we used aeroponics to phenotype the effects of tb1-ref copy number on maize roots at macro-, meso- and micro scales of development. Results An increase in the tb1-ref copy number from one to two copies resulted in: (1) an increase in crown root number due to the cumulative initiation of crown roots from successive tillers; (2) higher density of first and second order lateral roots; and (3) reduced average lateral root length. The resulting increase in root system biomass in homozygous tb1-ref mutants balanced the increase in shoot biomass caused by enhanced tillering. These changes caused homozygous tb1-ref mutants of modern maize to more closely resemble its ancestor Balsas teosinte below ground. Conclusion We conclude that a decrease in TB1 function in maize results in a larger root system, due to an increase in the number of crown roots and lateral roots. Given that decreased TB1 expression results in a more highly branched and larger shoot, the impact of TB1 below ground may be direct or indirect. We discuss the potential implications of these findings for whole plant coordination of biomass accumulation and maize domestication. PMID:24524734

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

PubMed

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

2018-01-01

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

Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

PubMed

Herbst, Eric A F; George, Mitchell A J; Brebner, Karen; Holloway, Graham P; Kane, Daniel A

2018-05-01

The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD + , suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD + -dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD + -dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

Quantum gas in the fast forward scheme of adiabatically expanding cavities: Force and equation of state

NASA Astrophysics Data System (ADS)

Babajanova, Gulmira; Matrasulov, Jasur; Nakamura, Katsuhiro

2018-04-01

With use of the scheme of fast forward which realizes quasistatic or adiabatic dynamics in shortened timescale, we investigate a thermally isolated ideal quantum gas confined in a rapidly dilating one-dimensional (1D) cavity with the time-dependent size L =L (t ) . In the fast-forward variants of equation of states, i.e., Bernoulli's formula and Poisson's adiabatic equation, the force or 1D analog of pressure can be expressed as a function of the velocity (L ˙) and acceleration (L ̈) of L besides rapidly changing state variables like effective temperature (T ) and L itself. The force is now a sum of nonadiabatic (NAD) and adiabatic contributions with the former caused by particles moving synchronously with kinetics of L and the latter by ideal bulk particles insensitive to such a kinetics. The ratio of NAD and adiabatic contributions does not depend on the particle number (N ) in the case of the soft-wall confinement, whereas such a ratio is controllable in the case of hard-wall confinement. We also reveal the condition when the NAD contribution overwhelms the adiabatic one and thoroughly changes the standard form of the equilibrium equation of states.

[Immunologic indexes, enzyme status of lymphocytes and functional activity of blood neutrophils in children with infectious mononucleosis caused by Epstein-Barr virus].

PubMed

Kurtasova, L M; Tolstikova, A E; Savchenko, A A

2013-01-01

Explore the immunological parameters, levels of activity of NAD(P)-dependent dehydrogenases lymphocytes, interferon status parameters, phagocytic activity and chemiluminescence response of neutrophils in the blood of children in the acute phase of infectious mononucleosis caused by the Epstein-Barr virus. 65 children at the age of 4-6 years old with infectious mononucleosis caused by EBV in acute phase were observed. Such indexes as cell-mediated, humoral and interferon immunity, NAD(P)-depended dehydrogenases activity in blood lymphocyte, phagocytes activity, levels of spontaneous and induced chemiluminescence ofperipheral blood neutrophils were studied. Children with EVB-infection have immunophenotype spectrum changes and changes of enzymes status of blood lymphocytes against the increasing in leucocytes and the useful increasing in lymphocytes. The useful increasing in IgA, IgM, IgG contenting in serum blood were found. The decreasing of spontaneous production of IFN alpha and the decreasing of induced production of IFNalpha, IFNgamma were determined. The breach of phagocytes activity and chemiluminescent response of blood neutrophils were found. The children in the acute phase of infectious mononucleosis caused by the Epstein-Barr virus, there are changes in the immune status, changes the activity of NAD(P)-dependent dehydrogenases in blood lymphocytes, marked changes in functional and metabolic state of peripheral blood neutrophils.

Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity.

PubMed

Lim, J H; Choi, J; Kim, W; Ahn, B Y; Han, Y S

2001-04-15

We constructed nine deletion mutants of NAD+-dependent DNA ligase from Aquifex pyrophilus to characterize the functional domains. All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD+-dependent self-adenylation, DNA binding, and nick-closing activity. Although the mutant lsub1 (91-362) included the active site lysine (KxDG), self-adenylation was not shown. However, the mutants lsub6 (1-362), lsub7 (1-516), and lsub9 (1-635) showed the same adenylation activity as that of wild type. The lsub5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity. These results suggest that the presence of N-terminal 90 residues is essential for the formation of an enzyme-AMP complex, while C-terminal domain (487-719) appears to play a minimal role in adenylation. It was found that the presence of C-terminal domain (487-719) is indispensable for DNA binding activity of lsub5 (91-719). The mutant lsub9 (1-635) showed reduced DNA binding activity compared to that of wild type, suggesting the contribution of the domain (636-719) for the DNA binding activity. Thus, we concluded that the N-terminal 90 residues and C-terminal domain (487-719) of NAD+-dependent DNA ligase from A. pyrophilus are mutually indispensable for binding of DNA substrate.

Analysis of Maize versus Ethanol Production in Nebraska, United States and International Agricultural Droughts: Lessons for Global Food Security

NASA Astrophysics Data System (ADS)

Boken, V.; Tenkorang, F.

2012-04-01

Nebraska is one of the eight main corn (maize) belt states of the United States. Maize is the major crop of Nebraska with an average annual production of about 38 million tons (about 12% of U.S. production), which contributes billions of dollars to the state's economy. The yield of maize has increased significantly over the past century - from 1.6 t/ha in 1900 to 10.4 t/ha in 2010. While the majority of maize (about 40%) is currently used for animal feed and ethanol production, only about six percent is exported. It is estimated that about one billion people accounting for about 15% population of the world live in chronic hunger because of low agricultural productivity and drought. Most of these people depend on the U.S. for grains including maize. If a greater quantity of maize is diverted to ethanol production, considerably less quantity of maize would be available for export to developing countries where it could be used for human consumption and to mitigate hunger and improve food security. This paper presents analysis of maize production in Nebraska for the past three decades and examines how its commercialization for ethanol production has affected its exports in the face of drought at an international level.

Mice fed a high-fat diet supplemented with resistant starch display marked shifts in the liver metabolome concurrent with altered gut bacteria

USDA-ARS?s Scientific Manuscript database

High-amylose maize resistant starch type 2 (HAMRS2) is a fermentable dietary fiber known to alter the gut milieu, including the gut microbiota, which may explain reported effects of resistant starch to ameliorate obesity-associated metabolic dysfunction. Our working hypothesis is that HAMRS2-induced...

Comparative Proteomic Analyses Provide New Insights into Low Phosphorus Stress Responses in Maize Leaves

PubMed Central

Zhang, Kewei; Liu, Hanhan; Tao, Peilin; Chen, Huan

2014-01-01

Phosphorus deficiency limits plant growth and development. To better understand the mechanisms behind how maize responds to phosphate stress, we compared the proteome analysis results of two groups of maize leaves that were treated separately with 1,000 µM (control, +P) and 5 µM of KH2PO4 (intervention group, −P) for 25 days. In total, 1,342 protein spots were detected on 2-DE maps and 15.43% had changed (P<0.05; ≥1.5-fold) significantly in quantity between the +P and −P groups. These proteins are involved in several major metabolic pathways, including photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, protein synthesis, cell rescue and cell defense and virulence. The results showed that the reduction in photosynthesis under low phosphorus treatment was due to the down-regulation of the proteins involved in CO2 enrichment, the Calvin cycle and the electron transport system. Electron transport and photosynthesis restrictions resulted in a large accumulation of peroxides. Maize has developed many different reactive oxygen species (ROS) scavenging mechanisms to cope with low phosphorus stress, including up-regulating its antioxidant content and antioxidase activity. After being subjected to phosphorus stress over a long period, maize may increase its internal phosphorus utilization efficiency by altering photorespiration, starch synthesis and lipid composition. These results provide important information about how maize responds to low phosphorus stress. PMID:24858307

A few sequence polymorphisms among isolates of Maize bushy stunt phytoplasma associate with organ proliferation symptoms of infected maize plants

PubMed Central

Orlovskis, Zigmunds; Canale, Maria Cristina; Haryono, Mindia; Lopes, João Roberto Spotti

2017-01-01

Background and Aims Maize bushy stunt phytoplasma (MBSP) is a bacterial pathogen of maize (Zea mays L.) across Latin America. MBSP belongs to the 16SrI-B sub-group within the genus ‘Candidatus Phytoplasma’. MBSP and its insect vector Dalbulus maidis (Hemiptera: Cicadellidae) are restricted to maize; both are thought to have coevolved with maize during its domestication from a teosinte-like ancestor. MBSP-infected maize plants show a diversity of symptoms. and it is likely that MBSP is under strong selection for increased virulence and insect transmission on maize hybrids that are widely grown in Brazil. In this study it was investigated whether the differences in genome sequences of MBSP isolates from two maize-growing regions in South-east Brazil explain variations in symptom severity of the MBSP isolates on various maize genotypes. Methods MBSP isolates were collected from maize production fields in Guaíra and Piracicaba in South-east Brazil for infection assays. One representative isolate was chosen for de novo whole-genome assembly and for the alignment of sequence reads from the genomes of other phytoplasma isolates to detect polymorphisms. Statistical methods were applied to investigate the correlation between variations in disease symptoms of infected maize plants and MBSP sequence polymorphisms. Key Results MBSP isolates contributed consistently to organ proliferation symptoms and maize genotype to leaf necrosis, reddening and yellowing of infected maize plants. The symptom differences are associated with polymorphisms in a phase-variable lipoprotein, which is a candidate effector, and an ATP-dependent lipoprotein ABC export protein, whereas no polymorphisms were observed in other candidate effector genes. Lipoproteins and ABC export proteins activate host defence responses, regulate pathogen attachment to host cells and activate effector secretion systems in other pathogens. Conclusions Polymorphisms in two putative virulence genes among MBSP isolates from maize-growing regions in South-east Brazil are associated with variations in organ proliferation symptoms of MBSP-infected maize plants. PMID:28069632

Talc

MedlinePlus

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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.

Impacts of Fertilization Regimes on Arbuscular Mycorrhizal Fungal (AMF) Community Composition Were Correlated with Organic Matter Composition in Maize Rhizosphere Soil

PubMed Central

Zhu, Chen; Ling, Ning; Guo, Junjie; Wang, Min; Guo, Shiwei; Shen, Qirong

2016-01-01

The understanding of the response of arbuscular mycorrhizal fungi (AMF) community composition to fertilization is of great significance in sustainable agriculture. However, how fertilization influences AMF diversity and composition is not well-established yet. A field experiment located in northeast China in typical black soil (Chernozem) was conducted and high-throughput sequencing approach was used to investigate the effects of different fertilizations on the variation of AMF community in the rhizosphere soil of maize crop. The results showed that AMF diversity in the maize rhizosphere was significantly altered by different fertilization regimes. As revealed by redundancy analysis, the application of organic manure was the most important factor impacting AMF community composition between samples with and without organic manure, followed by N fertilizer and P fertilizer inputs. Moreover, the organic matter composition in the rhizosphere, determined by GC–MS, was significantly altered by the organic manure amendment. Many of the chemical components displayed significant relationships with the AMF community composition according to the Mantel test, among those, 2-ethylnaphthalene explained the highest percentage (54.2%) of the variation. The relative contents of 2-ethylnaphthalene and 2, 6, 10-trimethyltetradecane had a negative correlation with Glomus relative abundance, while the relative content of 3-methylbiphenyl displayed a positive correlation with Rhizophagus. The co-occurrence patterns in treatments with and without organic manure amendment were analyzed, and more hubs were detected in the network of soils with organic manure amendment. Additionally, three operational taxonomic units (OTUs) belonging to Glomerales were identified as hubs in all treatments, indicating these OTUs likely occupied broad ecological niches and were always active for mediating AMF species interaction in the maize rhizosphere. Taken together, impacts of fertilization regimes on AMF community composition were correlated with organic matter composition in maize rhizosphere soil and the application of manure could activate more AMF species to interact with other species in the maize rhizosphere. This knowledge can be valuable in regulating the symbiotic system of plants and AMF, maintaining the health and high yields of crops and providing a primary basis for rational fertilization. PMID:27899920

Evolutionary response of landraces to climate change in centers of crop diversity

PubMed Central

Mercer, Kristin L; Perales, Hugo R

2010-01-01

Landraces cultivated in centers of crop diversity result from past and contemporary patterns of natural and farmer-mediated evolutionary forces. Successful in situ conservation of crop genetic resources depends on continuity of these evolutionary processes. Climate change is projected to affect agricultural production, yet analyses of impacts on in situ conservation of crop genetic diversity and farmers who conserve it have been absent. How will crop landraces respond to alterations in climate? We review the roles that phenotypic plasticity, evolution, and gene flow might play in sustaining production, although we might expect erosion of genetic diversity if landrace populations or entire races lose productivity. For example, highland maize landraces in southern Mexico do not express the plasticity necessary to sustain productivity under climate change, but may evolve in response to altered conditions. The outcome for any given crop in a given region will depend on the distribution of genetic variation that affects fitness and patterns of climate change. Understanding patterns of neutral and adaptive diversity from the population to the landscape scale is essential to clarify how landraces conserved in situ will continue to evolve and how to minimize genetic erosion of this essential natural resource. PMID:25567941

Evolutionary response of landraces to climate change in centers of crop diversity.

PubMed

Mercer, Kristin L; Perales, Hugo R

2010-09-01

Landraces cultivated in centers of crop diversity result from past and contemporary patterns of natural and farmer-mediated evolutionary forces. Successful in situ conservation of crop genetic resources depends on continuity of these evolutionary processes. Climate change is projected to affect agricultural production, yet analyses of impacts on in situ conservation of crop genetic diversity and farmers who conserve it have been absent. How will crop landraces respond to alterations in climate? We review the roles that phenotypic plasticity, evolution, and gene flow might play in sustaining production, although we might expect erosion of genetic diversity if landrace populations or entire races lose productivity. For example, highland maize landraces in southern Mexico do not express the plasticity necessary to sustain productivity under climate change, but may evolve in response to altered conditions. The outcome for any given crop in a given region will depend on the distribution of genetic variation that affects fitness and patterns of climate change. Understanding patterns of neutral and adaptive diversity from the population to the landscape scale is essential to clarify how landraces conserved in situ will continue to evolve and how to minimize genetic erosion of this essential natural resource.

Bt-maize (MON810) and Non-GM Soybean Meal in Diets for Atlantic Salmon (Salmo salar L.) Juveniles – Impact on Survival, Growth Performance, Development, Digestive Function, and Transcriptional Expression of Intestinal Immune and Stress Responses

PubMed Central

Gu, Jinni; Bakke, Anne Marie; Valen, Elin C.; Lein, Ingrid; Krogdahl, Åshild

2014-01-01

Responses in Atlantic salmon (Salmo salar L.) juveniles (fry) fed diets containing genetically modified maize (Bt-maize, MON810) expressing Cry1Ab protein from first-feeding were investigated during a 99-day feeding trial. Four experimental diets were made; each diet contained ∼20% maize, either Bt-maize or its near-isogenic maternal line (non-GM maize). One pair was fishmeal-based while the other pair included standard (extracted) soybean meal (SBM; 16.7% inclusion level), with the intention of investigating responses to the maize varieties in healthy fish as well as in immunologically challenged fish with SBM-induced distal intestinal inflammation, respectively. Three replicate tanks of fry (0.17±0.01 g; initial mean weight ± SEM) were fed one of the four diets and samples were taken on days 15, 36, 48 and 99. Survival, growth performance, whole body composition, digestive function, morphology of intestine, liver and skeleton, and mRNA expression of some immune and stress response parameters in the distal intestine were evaluated. After 99 days of feeding, survival was enhanced and the intended SBM-induced inflammatory response in the distal intestine of the two groups of SBM-fed fish was absent, indicating that the juvenile salmon were tolerant to SBM. Mortality, growth performance and body composition were similar in fish fed the two maize varieties. The Bt-maize fed fish, however, displayed minor but significantly decreased digestive enzyme activities of leucine aminopeptidase and maltase, as well as decreased concentration of gut bile salts, but significantly increased amylase activity at some sampling points. Histomorphological, radiographic and mRNA expression evaluations did not reveal any biologically relevant effects of Bt-maize in the gastrointestinal tract, liver or skeleton. The results suggest that the Cry1Ab protein or other compositional differences in GM Bt-maize may cause minor alterations in intestinal responses in juvenile salmon, but without affecting overall survival, growth performance, development or health. PMID:24923786

Bt-maize (MON810) and non-GM soybean meal in diets for Atlantic salmon (Salmo salar L.) juveniles--impact on survival, growth performance, development, digestive function, and transcriptional expression of intestinal immune and stress responses.

PubMed

Gu, Jinni; Bakke, Anne Marie; Valen, Elin C; Lein, Ingrid; Krogdahl, Åshild

2014-01-01

Responses in Atlantic salmon (Salmo salar L.) juveniles (fry) fed diets containing genetically modified maize (Bt-maize, MON810) expressing Cry1Ab protein from first-feeding were investigated during a 99-day feeding trial. Four experimental diets were made; each diet contained ∼20% maize, either Bt-maize or its near-isogenic maternal line (non-GM maize). One pair was fishmeal-based while the other pair included standard (extracted) soybean meal (SBM; 16.7% inclusion level), with the intention of investigating responses to the maize varieties in healthy fish as well as in immunologically challenged fish with SBM-induced distal intestinal inflammation, respectively. Three replicate tanks of fry (0.17±0.01 g; initial mean weight ± SEM) were fed one of the four diets and samples were taken on days 15, 36, 48 and 99. Survival, growth performance, whole body composition, digestive function, morphology of intestine, liver and skeleton, and mRNA expression of some immune and stress response parameters in the distal intestine were evaluated. After 99 days of feeding, survival was enhanced and the intended SBM-induced inflammatory response in the distal intestine of the two groups of SBM-fed fish was absent, indicating that the juvenile salmon were tolerant to SBM. Mortality, growth performance and body composition were similar in fish fed the two maize varieties. The Bt-maize fed fish, however, displayed minor but significantly decreased digestive enzyme activities of leucine aminopeptidase and maltase, as well as decreased concentration of gut bile salts, but significantly increased amylase activity at some sampling points. Histomorphological, radiographic and mRNA expression evaluations did not reveal any biologically relevant effects of Bt-maize in the gastrointestinal tract, liver or skeleton. The results suggest that the Cry1Ab protein or other compositional differences in GM Bt-maize may cause minor alterations in intestinal responses in juvenile salmon, but without affecting overall survival, growth performance, development or health.

Agents for replacement of NAD+/NADH system in enzymatic reactions

DOEpatents

Fish, Richard H.; Kerr, John B.; Lo, Christine H.

2004-04-06

Novel agents acting as co-factors for replacement of NAD(P).sup.+ /NAD(P)H co-enzyme systems in enzymatic oxido-reductive reactions. Agents mimicking the action of NAD(P).sup.+ /NAD(P)H system in enzymatic oxidation/reduction of substrates into reduced or oxidized products. A method for selection and preparation of the mimicking agents for replacement of NAD(P).sup.+ /NAD(P)H system and a device comprising co-factors for replacement of NAD(P).sup.+ /NAD(P)H system.

Mycorrhizal phosphate uptake pathway in maize: vital for growth and cob development on nutrient poor agricultural and greenhouse soils

PubMed Central

Willmann, Martin; Gerlach, Nina; Buer, Benjamin; Polatajko, Aleksandra; Nagy, Réka; Koebke, Eva; Jansa, Jan; Flisch, René; Bucher, Marcel

2013-01-01

Arbuscular mycorrhizal fungi (AMF) form a mutually beneficial symbiosis with plant roots providing predominantly phosphorus in the form of orthophosphate (Pi) in exchange for plant carbohydrates on low P soils. The goal of this work was to generate molecular-genetic evidence in support of a major impact of the mycorrhizal Pi uptake (MPU) pathway on the productivity of the major crop plant maize under field and controlled conditions. Here we show, that a loss-of-function mutation in the mycorrhiza-specific Pi transporter gene Pht1;6 correlates with a dramatic reduction of above-ground biomass and cob production in agro-ecosystems with low P soils. In parallel mutant pht1;6 plants exhibited an altered fingerprint of chemical elements in shoots dependent on soil P availability. In controlled environments mycorrhiza development was impaired in mutant plants when grown alone. The presence of neighboring mycorrhizal nurse plants enhanced the reduced mycorrhiza formation in pht1;6 roots. Uptake of 33P-labeled orthophosphate via the MPU pathway was strongly impaired in colonized mutant plants. Moreover, repression of the MPU pathway resulted in a redirection of Pi to neighboring plants. In line with previous results, our data highlight the relevance of the MPU pathway in Pi allocation within plant communities and in particular the role of Pht1;6 for the establishment of symbiotic Pi uptake and for maize productivity and nutritional value in low-input agricultural systems. In a first attempt to identify cellular pathways which are affected by Pht1;6 activity, gene expression profiling via RNA-Seq was performed and revealed a set of maize genes involved in cellular signaling which exhibited differential regulation in mycorrhizal pht1;6 and control plants. The RNA data provided support for the hypothesis that fungal supply of Pi and/or Pi transport across Pht1;6 affects cell wall biosynthesis and hormone metabolism in colonized root cells. PMID:24409191

A de novo NADPH generation pathway for improving lysine production of Corynebacterium glutamicum by rational design of the coenzyme specificity of glyceraldehyde 3-phosphate dehydrogenase.

PubMed

Bommareddy, Rajesh Reddy; Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

2014-09-01

Engineering the cofactor availability is a common strategy of metabolic engineering to improve the production of many industrially important compounds. In this work, a de novo NADPH generation pathway is proposed by altering the coenzyme specificity of a native NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to NADP, which consequently has the potential to produce additional NADPH in the glycolytic pathway. Specifically, the coenzyme specificity of GAPDH of Corynebacterium glutamicum is systematically manipulated by rational protein design and the effect of the manipulation for cellular metabolism and lysine production is evaluated. By a combinatorial modification of four key residues within the coenzyme binding sites, different GAPDH mutants with varied coenzyme specificity were constructed. While increasing the catalytic efficiency of GAPDH towards NADP enhanced lysine production in all of the tested mutants, the most significant improvement of lysine production (~60%) was achieved with the mutant showing similar preference towards both NAD and NADP. Metabolic flux analysis with (13)C isotope studies confirmed that there was no significant change of flux towards the pentose phosphate pathway and the increased lysine yield was mainly attributed to the NADPH generated by the mutated GAPDH. The present study highlights the importance of protein engineering as a key strategy in de novo pathway design and overproduction of desired products. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

PubMed

Papapetridis, Ioannis; van Dijk, Marlous; Dobbe, Arthur P A; Metz, Benjamin; Pronk, Jack T; van Maris, Antonius J A

2016-04-26

Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption. Replacement of the native NADP(+)-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD(+)-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15% increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP(+)-dependent acetaldehyde dehydrogenase, led to a 39% increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44% increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13% increase in the ethanol yield on glucose. The combination of NAD(+)-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

PubMed

Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

2014-08-25

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs

PubMed Central

Reinhardt, Klaus; Breunig, Hans Georg; Uchugonova, Aisada; König, Karsten

2015-01-01

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype. PMID:26333813

Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2.

PubMed

Preissler, Janina; Wahlefeld, Stefan; Lorent, Christian; Teutloff, Christian; Horch, Marius; Lauterbach, Lars; Cramer, Stephen P; Zebger, Ingo; Lenz, Oliver

2018-01-01

Biocatalysts that mediate the H 2 -dependent reduction of NAD + to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD + -reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O 2 , which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD + -reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1 T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H 2 -oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H 2 -mediated NAD + reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O 2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD + -reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H 2 -driven cofactor recycling under oxic conditions at elevated temperatures. Copyright © 2017 Elsevier B.V. All rights reserved.

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)

Initial Genomics of the Human Nucleolus

PubMed Central

Németh, Attila; Conesa, Ana; Santoyo-Lopez, Javier; Medina, Ignacio; Montaner, David; Péterfia, Bálint; Solovei, Irina; Cremer, Thomas; Dopazo, Joaquin; Längst, Gernot

2010-01-01

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD–localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD–specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture. PMID:20361057

Pleiotropic effects of ZmLAZY1 on the auxin-mediated responses to gravity and light in maize shoot and inflorescences.

PubMed

Dong, Zhaobin; Jin, Weiwei

2013-01-01

Auxin has been found to control both gravitropism and inflorescence development in plant. Auxin transport has also been demonstrated to be responsible for tropism. Maize, a key agricultural crop, has distinct male (tassel) and female (ear) inflorescence, and this morphogenesis depends on auxin maximum and gradient. The classic maize mutant lazy plant1 (la1) has defective gravitropic response. The mechanism underlining maize gravitropism remains unclear. Recently, we isolated the ZmLA1 gene by map-based cloning, and our findings suggest that ZmLA1 might mediate the crosstalk between shoot gravitropism and inflorescence development by regulating auxin transport, auxin signaling, and auxin-mediated light response in maize. Here, we propose a model describing the ZmLA1-mediated complex interactions among auxin, gravity, light, and inflorescent development.

NAD+-Carrying Mesoporous Silica Nanoparticles Can Prevent Oxidative Stress-Induced Energy Failures of Both Rodent Astrocytes and PC12 Cells

PubMed Central

Chen, Heyu; Wang, Yao; Zhang, Jixi; Ma, Yingxin; Wang, Caixia; Zhou, Ying; Gu, Hongchen; Ying, Weihai

2013-01-01

Aim To test the hypothesis that NAD+-carrying mesoporous silica nanoparticles (M-MSNs@NAD+) can effectively deliver NAD+ into cells to produce cytoprotective effects. Methods & Materials NAD+ was incorporated into M-MSNs. Primary rat astrocyte cultures and PC12 cells were treated with H2O2, followed by post-treatment with M-MSNs@NAD+. After various durations of the post-treatment, intracellular NAD+ levels, intracellular ATP levels and lactate dehydrogenase (LDH) release were determined. Results & Discussion M-MSNs can be effectively loaded with NAD+. The M-MSNs@NAD+ can significantly attenuate H2O2-induced NAD+ and ATP decreases in both astrocyte cultures and PC12 cells. M-MSNs@NAD+ can also partially prevent the H2O2-induced LDH release from both astrocyte cultures and PC12 cells. In contrast, the NAD+ that is spontaneously released from the M-MSNs@NAD+ is insufficient to prevent the H2O2-induced damage. Conclusions Our study has suggested the first approach that can effectively deliver NAD+ into cells, which provides an important basis both for elucidating the roles of intracellular NAD+ in biological functions and for therapeutic applications of NAD+. Our study has also provided the first direct evidence demonstrating a key role of NAD+ depletion in oxidative stress-induced ATP decreases. PMID:24040179

Ameliorative Effects of Neutral Electrolyzed Water on Growth Performance, Biochemical Constituents, and Histopathological Changes in Turkey Poults during Aflatoxicosis

PubMed Central

Gómez-Espinosa, Denise; Cervantes-Aguilar, Francisco Javier; Del Río-García, Juan Carlos; Villarreal-Barajas, Tania; Vázquez-Durán, Alma; Méndez-Albores, Abraham

2017-01-01

Different in vitro and in silico approaches from our research group have demonstrated that neutral electrolyzed water (NEW) can be used to detoxify aflatoxins. The objective of this investigation was to evaluate the ability of NEW to detoxify B-aflatoxins (AFB1 and AFB2) in contaminated maize and to confirm detoxification in an in vivo experimental model. Batches of aflatoxin-contaminated maize were detoxified with NEW and mixed in commercial feed. A total of 240 6-day-old female large white Nicholas-700 turkey poults were randomly divided into four treatments of six replicates each (10 turkeys per replicate), which were fed ad libitum for two weeks with the following dietary treatments: (1) control feed containing aflatoxin-free maize (CONTROL); (2) feed containing the aflatoxin-contaminated maize (AF); (3) feed containing the aflatoxin-contaminated maize detoxified with NEW (AF + NEW); and (4) control feed containing aflatoxin-free maize treated with NEW (NEW). Compared to the control groups, turkey poults of the AF group significantly reduced body weight gain and increased feed conversion ratio and mortality rate; whereas turkey poults of the AF + NEW group did not present significant differences on productive parameters. In addition, alterations in serum biochemical constituents, enzyme activities, relative organ weight, gross morphological changes and histopathological studies were significantly mitigated by the aflatoxin-detoxification procedure. From these results, it is concluded that the treatment of aflatoxin-contaminated maize with NEW provided reasonable protection against the effects caused by aflatoxins in young turkey poults. PMID:28335412

Estimating plant distance in maize using Unmanned Aerial Vehicle (UAV).

PubMed

Zhang, Jinshui; Basso, Bruno; Price, Richard F; Putman, Gregory; Shuai, Guanyuan

2018-01-01

Distance between rows and plants are essential parameters that affect the final grain yield in row crops. This paper presents the results of research intended to develop a novel method to quantify the distance between maize plants at field scale using an Unmanned Aerial Vehicle (UAV). Using this method, we can recognize maize plants as objects and calculate the distance between plants. We initially developed our method by training an algorithm in an indoor facility with plastic corn plants. Then, the method was scaled up and tested in a farmer's field with maize plant spacing that exhibited natural variation. The results of this study demonstrate that it is possible to precisely quantify the distance between maize plants. We found that accuracy of the measurement of the distance between maize plants depended on the height above ground level at which UAV imagery was taken. This study provides an innovative approach to quantify plant-to-plant variability and, thereby final crop yield estimates.

Construction and functional analysis of Trichoderma harzianum mutants that modulate maize resistance to the pathogen Curvularia lunata.

PubMed

Fan, Lili; Fu, Kehe; Yu, Chuanjin; Ma, Jia; Li, Yaqian; Chen, Jie

2014-01-01

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the mycelial fungus Trichoderma harzianum. From a total of 450 mutants, six mutants that showed significant influence on maize resistance to C. lunata were analyzed in detail. Maize coated with these mutants was more susceptible to C. lunata compared with those coated with a wild-type (WT) strain. Similar to other fungal ATMT libraries, all six mutants were single copy integrations, which occurred preferentially in noncoding regions (except two mutants) and were frequently accompanied by the loss of border sequences. Two mutants (T66 and T312) that were linked to resistance were characterized further. Maize seeds coated with T66 and T312 were more susceptible to C. lunata than those treated with WT. Moreover, the mutants affected the resistance of maize to C. lunata by enhancing jasmonate-responsive gene expression. T66 and T312 induced maize resistance to C. lunata infection through a jasmonic acid-dependent pathway.

Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport

USDA-ARS?s Scientific Manuscript database

Carbohydrate import into seeds directly determines seed size and must have been increased through domestication. However, evidence for domestication of sugar translocation and the identity of seed filling transporters remained elusive. Maize ZmSWEET4c, as opposed to its sucrose-transporting homologs...

Near-infrared hyperspectral imaging for detecting Aflatoxin B1 of maize kernels

USDA-ARS?s Scientific Manuscript database

The feasibility of detecting the Aflatoxin B1 in maize kernels inoculated with Aspergillus flavus conidia in the field was assessed using near-infrared hyperspectral imaging technique. After pixel-level calibration, wavelength dependent offset, the masking method was adopted to reduce the noise and ...

The genetic basis of flecking and its relationship to disease resistance in the IBM maize mapping population

USDA-ARS?s Scientific Manuscript database

Flecking is defined as a mild, often environmentally-dependent lesion phenotype observed on the leaves of several commonly used maize inbred lines. Anecdotal evidence suggests a link between flecking and enhanced broad-spectrum disease resistance. Neither the genetic basis underlying flecking nor ...

A maize inbred exhibits resistance against western corn root worm, Diabrotica vergifera vergifera.

USDA-ARS?s Scientific Manuscript database

Plants respond to insect infestations with a suite of natural defenses that vary depending on their genetic and phenotypic traits. Insect resistance traits against root herbivores like western corn rootworm (WCR, Diabrotica virgifera) are not well understood in non-transgenic maize. Using biomechani...

Metabolome analysis of food-chain between plants and insects

USDA-ARS?s Scientific Manuscript database

Evolution has shown the co-dependency of host plants-predators (insects), especially inevitable dependency of predators on plant biomass for securing their energy sources. In this respect, it had been believed that NAD+ source used for major energy producing pathway in insects is a glycerol-3-phosph...

Chemical and structural biology of protein lysine deacetylases

PubMed Central

YOSHIDA, Minoru; KUDO, Norio; KOSONO, Saori; ITO, Akihiro

2017-01-01

Histone acetylation is a reversible posttranslational modification that plays a fundamental role in regulating eukaryotic gene expression and chromatin structure/function. Key enzymes for removing acetyl groups from histones are metal (zinc)-dependent and NAD+-dependent histone deacetylases (HDACs). The molecular function of HDACs have been extensively characterized by various approaches including chemical, molecular, and structural biology, which demonstrated that HDACs regulate cell proliferation, differentiation, and metabolic homeostasis, and that their alterations are deeply involved in various human disorders including cancer. Notably, drug discovery efforts have achieved success in developing HDAC-targeting therapeutics for treatment of several cancers. However, recent advancements in proteomics technology have revealed much broader aspects of HDACs beyond gene expression control. Not only histones but also a large number of cellular proteins are subject to acetylation by histone acetyltransferases (HATs) and deacetylation by HDACs. Furthermore, some of their structures can flexibly accept and hydrolyze other acyl groups on protein lysine residues. This review mainly focuses on structural aspects of HDAC enzymatic activity regulated by interaction with substrates, co-factors, small molecule inhibitors, and activators. PMID:28496053

SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington’s Disease Mice

PubMed Central

Tulino, Raffaella; Benjamin, Agnesska C.; Jolinon, Nelly; Smith, Donna L.; Chini, Eduardo N.; Carnemolla, Alisia; Bates, Gillian P.

2016-01-01

Huntington’s disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies. PMID:26815359

Trans-Homolog Interactions Facilitating Paramutation in Maize

PubMed Central

2015-01-01

Paramutations represent locus-specific trans-homolog interactions affecting the heritable silencing properties of endogenous alleles. Although examples of paramutation are well studied in maize (Zea mays), the responsible mechanisms remain unclear. Genetic analyses indicate roles for plant-specific DNA-dependent RNA polymerases that generate small RNAs, and current working models hypothesize that these small RNAs direct heritable changes at sequences often acting as transcriptional enhancers. Several studies have defined specific sequences that mediate paramutation behaviors, and recent results identify a diversity of DNA-dependent RNA polymerase complexes operating in maize. Other reports ascribe broader roles for some of these complexes in normal genome function. This review highlights recent research to understand the molecular mechanisms of paramutation and examines evidence relevant to small RNA-based modes of transgenerational epigenetic inheritance. PMID:26149572

Proliferation of the biocontrol agent Fusarium oxysporum f. sp. strigae and its impact on indigenous rhizosphere fungal communities in maize under different agro-ecologies.

PubMed

Zimmermann, Judith; Musyoki, Mary K; Cadisch, Georg; Rasche, Frank

2016-06-01

Our objectives were to (1) monitor the proliferation of the biocontrol agent (BCA) Fusarium oxysporum f. sp. strigae strain "Foxy-2", an effective soil-borne BCA against the parasitic weed Striga hermonthica , in the rhizosphere of maize under different agro-ecologies, and (2) investigate its impact on indigenous rhizosphere fungal community abundance and composition. Field experiments were conducted in Busia and Homa Bay districts in western Kenya during two cropping seasons to account for effects of soil type, climate, growth stage and seasonality. Maize seeds were coated with or without "Foxy-2" and soils were artificially infested with S. hermonthica seeds. One treatment with nitrogen rich organic residues ( Tithonia diversifolia ) was established to compensate hypothesized resource competition between "Foxy-2" and the indigenous fungal community. Rhizosphere soil samples collected at three growth stages (i.e., EC30, EC60, EC90) of maize were subjected to abundance measurement of "Foxy-2" and total indigenous fungi using quantitative polymerase chain reaction (qPCR) analysis. Terminal restriction fragment length polymorphism (TRFLP) analysis was used to assess potential alterations in the fungal community composition in response to "Foxy-2" presence. "Foxy-2" proliferated stronger in the soils with a sandy clay texture (Busia) than in those with a loamy sand texture (Homa Bay) and revealed slightly higher abundance in the second season. "Foxy-2" had, however, only a transient suppressive effect on total indigenous fungal abundance which ceased in the second season and was further markedly compensated after addition of T. diversifolia residues. Likewise, community structure of the indigenous fungal community was mainly altered by maize growth stages, but not by "Foxy-2". In conclusion, no adverse effects of "Foxy-2" inoculation on indigenous fungal rhizosphere communities were observed corroborating the safety of this BCA under the given agro-ecologies.

Altering the axial light gradient affects photomorphogenesis in emerging seedlings of Zea mays L

NASA Technical Reports Server (NTRS)

Parks, B. M.; Poff, K. L.

1986-01-01

The axial (longitudinal) red light gradient (632 nanometers) of 4 day old dark-grown maize seedlings is increased by staining the peripheral cells of the coleoptile. The magnitude of increase in the light gradient is dependent solely on the light-absorbing qualities of the stain used. Metanil yellow has no effect on the axial red-light gradient, while methylene blue causes a large increase in this light gradient. These stains did not affect growth in darkness or the sensitivity of mesocotyl elongation to red light. However, mesocotyl elongation was altered for the dark-grown seedlings stained with methylene blue when these seedlings were transplanted, covered with soil, and permitted to emerge under natural lighting conditions. These observations are consistent with the idea that there is a single perceptive site below the coleoptilar node, and suggest that this perceptive site gives the actinic light which has traveled downward through the length of the shoot from an entry point in the plant tip region.

Introducing non-flooded crops in rice-dominated landscapes: Impact on carbon, nitrogen and water budgets

NASA Astrophysics Data System (ADS)

Jauker, Frank; Wassmann, Reiner; Amelung, Wulf; Breuer, Lutz; Butterbach-Bahl, Klaus; Conrad, Ralf; Ekschmitt, Klemens; Goldbach, Heiner; He, Yao; John, Katharina; Kiese, Ralf; Kraus, David; Reinhold-Hurek, Barbara; Siemens, Jan; Weller, Sebastian; Wolters, Volkmar

2013-04-01

Rice production consumes about 30% of all freshwater used worldwide and 45% in Asia. Turning away from permanently flooded rice cropping systems for mitigating future water scarcity and reducing methane emissions, however, will alter a variety of ecosystem services with potential adverse effects to both the environment and agricultural production. Moreover, implementing systems that alternate between flooded and non-flooded crops increases the risk of disruptive effects. The multi-disciplinary DFG research unit ICON aims at exploring and quantifying the ecological consequences of altered water regimes (flooded vs. non-flooded), crop diversification (irrigated rice vs. aerobic rice vs. maize), and different fertilization strategies (conventional, site-specific, and zero N fertilization). ICON particularly focuses on the biogeochemical cycling of carbon and nitrogen, green-house gas (GHG) emissions, water balance, soil biotic processes and other important ecosystem services. The overarching goal is to provide the basic process understanding that is necessary for balancing the revenues and environmental impacts of high-yield rice cropping systems while maintaining their vital ecosystem services. To this aim, a large-scale field experiment has been established at the experimental farm of the International Rice Research Institute (IRRI, Philippines). Ultimately, the experimental results are analyzed in the context of management scenarios by an integrated modeling of crop development (ORYZA), carbon and nitrogen cycling (MoBiLE-DNDC), and water fluxes (CMF), providing the basis for developing pathways to a conversion of rice-based systems towards higher yield potentials under minimized environmental impacts. In our presentation, we demonstrate the set-up of the controlled large-scale field experiment for simultaneous assessment of carbon and nitrogen fluxes and water budgets. We show and discuss first results for: - Quantification and assessment of the net-fluxes of CH4, N2O and CO2 from rice-rice and rice-maize rotations. The conversion of flooded to non-flooded cropping systems resulted in pollution swapping of greenhouse gas emissions, shifting from CH4 under wet conditions to N2O under dry conditions. - Quantification and assessment of water budgets and nutrient loss in rice-rice and rice-maize rotations. Switching from rice-rice dominated growing systems to upland rice or maize-rice cropping systems resulted in reduced water use efficiency and increased nitrogen loss. - Quantification and assessment of soil functions affected by soil fauna community structure in flooded and non-flooded cropping rotations. In contrast to temperate soils, earthworms reduced the peaks of microbial C and N decomposition depending on soil water content.

Aerobic denitration of 2,4,6-trinitrotoluene in the presence of phenazine compounds and reduced pyridine nucleotides.

PubMed

Stenuit, Ben; Lamblin, Guillaume; Cornelis, Pierre; Agathos, Spiros N

2012-10-02

Phenazine-containing spent culture supernatants of Pseudomonas aeruginosa concentrated with a C18 solid-phase extraction cartridge initiate NAD(P)H-dependent denitration of 2,4,6-trinitrotoluene (TNT). In this study, TNT denitration was investigated under aerobic conditions using two phenazine secondary metabolites excreted by P. aeruginosa, pyocyanin (Py) and its precursor phenazine-1- carboxylic acid (PCA), and two chemically synthesized pyocyanin analogs, phenazine methosulfate (PMS+) and phenazine ethosulfate (PES+). The biomimetic Py/NAD(P)H/O2 system was characterized and found to extensively denitrate TNT in unbuffered aqueous solution with minor production of toxic amino aromatic derivatives. To a much lesser extent, TNT denitration was also observed with PMS+ and PES+ in the presence of NAD(P)H. No TNT denitration was detected with the biomimetic PCA/NAD(P)H/O2 system. Electron paramagnetic resonance (EPR) spectroscopy analysis of the biomimetic Py/NAD(P)H/O2 system revealed the generation of superoxide radical anions (O2 •−). In vitro TNT degradation experiments in the presence of specific inhibitors of reactive oxygen species suggest a nucleophilic attack of superoxide radical anion followed by TNT denitration through an as yet unknown mechanism. The results of this research confirm the high functional versatility of the redox-active metabolite pyocyanin and the susceptibility of aromatic compounds bearing electron withdrawing substituents, such as nitro groups, to superoxide-driven nucleophilic attack.

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.

Consequences of C4 differentiation for chloroplast membrane proteomes in maize mesophyll and bundle sheath cells.

PubMed

Majeran, Wojciech; Zybailov, Boris; Ytterberg, A Jimmy; Dunsmore, Jason; Sun, Qi; van Wijk, Klaas J

2008-09-01

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

PubMed Central

Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

2000-01-01

The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide. PMID:10727402

When Genomes Collide: Aberrant Seed Development Following Maize Interploidy Crosses

PubMed Central

Pennington, Paul D.; Costa, Liliana M.; Gutierrez-Marcos, Jose F.; Greenland, Andy J.; Dickinson, Hugh G.

2008-01-01

Background and Aims The results of wide- or interploidy crosses in angiosperms are unpredictable and often lead to seed abortion. The consequences of reciprocal interploidy crosses have been explored in maize in detail, focusing on alterations to tissue domains in the maize endosperm, and changes in endosperm-specific gene expression. Methods Following reciprocal interploidy crosses between diploid and tetraploid maize lines, development of endosperm domains was studied using GUS reporter lines, and gene expression in resulting kernels was investigated using semi-quantitative RT-PCR on endosperms isolated at different stages of development. Key Results Reciprocal interploidy crosses result in very small, largely infertile seeds with defective endosperms. Seeds with maternal genomic excess are smaller than those with paternal genomic excess, their endosperms cellularize earlier and they accumulate significant quantities of starch. Endosperms from the reciprocal cross undergo an extended period of cell proliferation, and accumulate little starch. Analysis of reporter lines and gene expression studies confirm that functional domains of the endosperm are severely disrupted, and are modified differently according to the direction of the interploidy cross. Conclusions Interploidy crosses affect factors which regulate the balance between cell proliferation and cell differentiation within the endosperm. In particular, unbalanced crosses in maize affect transfer cell differentiation, and lead to the temporal deregulation of the ontogenic programme of endosperm development. PMID:18276791

Spore associated bacteria regulates maize root K+/Na+ ion homeostasis to promote salinity tolerance during arbuscular mycorrhizal symbiosis.

PubMed

Selvakumar, Gopal; Shagol, Charlotte C; Kim, Kiyoon; Han, Seunggab; Sa, Tongmin

2018-06-05

The interaction between arbuscular mycorrhizal fungi (AMF) and AMF spore associated bacteria (SAB) were previously found to improve mycorrhizal symbiotic efficiency under saline stress, however, the information about the molecular basis of this interaction remain unknown. Therefore, the present study aimed to investigate the response of maize plants to co-inoculation of AMF and SAB under salinity stress. The co-inoculation of AMF and SAB significantly improved plant dry weight, nutrient content of shoot and root tissues under 25 or 50 mM NaCl. Importantly, co-inoculation significantly reduced the accumulation of proline in shoots and Na + in roots. Co-inoculated maize plants also exhibited high K + /Na + ratios in roots at 25 mM NaCl concentration. Mycorrhizal colonization significantly positively altered the expression of ZmAKT2, ZmSOS1, and ZmSKOR genes, to maintain K + and Na + ion homeostasis. Confocal laser scanning microscope (CLSM) view showed that SAB were able to move and localize into inter- and intracellular spaces of maize roots and were closely associated with the spore outer hyaline layer. These new findings indicate that co-inoculation of AMF and SAB effectively alleviates the detrimental effects of salinity through regulation of SOS pathway gene expression and K + /Na + homeostasis to improve maize plant growth.

Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization.

PubMed

Balsanelli, Eduardo; Serrato, Rodrigo V; de Baura, Valter A; Sassaki, Guilherme; Yates, Marshall G; Rigo, Liu Un; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A

2010-08-01

In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Maize root culture as a model system for studying azoxystrobin biotransformation in plants.

PubMed

Gautam, Maheswor; Elhiti, Mohamed; Fomsgaard, Inge S

2018-03-01

Hairy roots induced by Agrobacterium rhizogenes are well established models to study the metabolism of xenobiotics in plants for phytoremediation purposes. However, the model requires special skills and resources for growing and is a time-consuming process. The roots induction process alters the genetic construct of a plant and is known to express genes that are normally absent from the non-transgenic plants. In this study, we propose and establish a non-transgenic maize root model to study xenobiotic metabolism in plants for phytoremediation purpose using azoxystrobin as a xenobiotic compound. Maize roots were grown aseptically in Murashige and Skoog medium for two weeks and were incubated in 100 μM azoxystrobin solution. Azoxystrobin was taken up by the roots to the highest concentration within 15 min of treatment and its phase I metabolites were also detected at the same time. Conjugated metabolites of azoxystrobin were detected and their identities were confirmed by enzymatic and mass spectrometric methods. Further, azoxystrobin metabolites identified in maize root culture were compared against azoxystrobin metabolites in azoxystrobin sprayed lettuce grown in green house. A very close similarity between metabolites identified in maize root culture and lettuce plant was obtained. The results from this study establish that non-transgenic maize roots can be used for xenobiotic metabolism studies instead of genetically transformed hairy roots due to the ease of growing and handling. Copyright © 2017 Elsevier Ltd. All rights reserved.

Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments.

PubMed

Boily, Monique; Sarrasin, Benoit; Deblois, Christian; Aras, Philippe; Chagnon, Madeleine

2013-08-01

In Québec, as observed globally, abnormally high honey bee mortality rates have been reported recently. Several potential contributing factors have been identified, and exposure to pesticides is of increasing concern. In maize fields, foraging bees are exposed to residual concentrations of insecticides such as neonicotinoids used for seed coating. Highly toxic to bees, neonicotinoids are also reported to increase AChE activity in other invertebrates exposed to sub-lethal doses. The purpose of this study was therefore to test if the honey bee's AChE activity could be altered by neonicotinoid compounds and to explore possible effects of other common products used in maize fields: atrazine and glyphosate. One week prior to pollen shedding, beehives were placed near three different field types: certified organically grown maize, conventionally grown maize or non-cultivated. At the same time, caged bees were exposed to increasing sub-lethal doses of neonicotinoid insecticides (imidacloprid and clothianidin) and herbicides (atrazine and glyphosate) under controlled conditions. While increased AChE activity was found in all fields after 2 weeks of exposure, bees close to conventional maize crops showed values higher than those in both organic maize fields and non-cultivated areas. In caged bees, AChE activity increased in response to neonicotinoids, and a slight decrease was observed by glyphosate. These results are discussed with regard to AChE activity as a potential biomarker of exposure for neonicotinoids.

High level of oxygen treatment causes cardiotoxicity with arrhythmias and redox modulation

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

Chapalamadugu, Kalyan C.; Panguluri, Siva K.; Bennett, Eric S.

2015-01-01

Hyperoxia exposure in mice leads to cardiac hypertrophy and voltage-gated potassium (Kv) channel remodeling. Because redox balance of pyridine nucleotides affects Kv function and hyperoxia alters cellular redox potential, we hypothesized that hyperoxia exposure leads to cardiac ion channel disturbances and redox changes resulting in arrhythmias. In the present study, we investigated the electrical changes and redox abnormalities caused by 72 h hyperoxia treatment in mice. Cardiac repolarization changes were assessed by acquiring electrocardiogram (ECG) and cardiac action potentials (AP). Biochemical assays were employed to identify the pyridine nucleotide changes, Kv1.5 expression and myocardial injury. Hyperoxia treatment caused marked bradycardia,more » arrhythmia and significantly prolonged (ms) the, RR (186.2 ± 10.7 vs. 146.4 ± 6.2), PR (46.8 ± 3.1 vs. 39.3 ± 1.6), QRS (10.8 ± 0.6 vs. 8.5 ± 0.2), QTc (57.1 ± 3.5 vs. 40 ± 1.4) and JT (13.4 ± 2.1 vs. 7.0 ± 0.5) intervals, when compared with normoxia group. Hyperoxia treatment also induced significant increase in cardiac action potential duration (APD) (ex-APD{sub 90}; 73.8 ± 9.5 vs. 50.9 ± 3.1 ms) and elevated levels of serum markers of myocardial injury; cardiac troponin I (TnI) and lactate dehydrogenase (LDH). Hyperoxia exposure altered cardiac levels of mRNA/protein expression of; Kv1.5, Kvβ subunits and SiRT1, and increased ratios of reduced pyridine nucleotides (NADH/NAD and NADPH/NADP). Inhibition of SiRT1 in H9C2 cells using Splitomicin resulted in decreased SiRT1 and Kv1.5 expression, suggesting that SiRT1 may mediate Kv1.5 downregulation. In conclusion, the cardiotoxic effects of hyperoxia exposure involve ion channel disturbances and redox changes resulting in arrhythmias. - Highlights: • Hyperoxia treatment leads to arrhythmia with prolonged QTc and action potential duration. • Hyperoxia treatment alters cardiac pyridine nucleotide [NAD(P)H/NAD(P)] levels. • SiRT1 and Kv1.5 are co-regulated in hyperoxic heart injury. • Hyperoxia may lead to cardiotoxicity.« less

Increased Rate of NAD Metabolism Shortens Plant Longevity by Accelerating Developmental Senescence in Arabidopsis.

PubMed

Hashida, Shin-Nosuke; Itami, Taketo; Takahara, Kentaro; Hirabayashi, Takayuki; Uchimiya, Hirofumi; Kawai-Yamada, Maki

2016-11-01

NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD + , NADH, NADP + or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Hosts of stolbur phytoplasmas in maize redness affected fields

USDA-ARS?s Scientific Manuscript database

The plant host range of a phytoplasma is strongly dependent on the host range of its insect vector. Maize redness in Serbia is caused by stolbur phytoplasma (subgroup 16SrXII-A) and is transmitted by the cixiid planthoper, Reptalus panzeri (Löw). R. panzeri was the only potential vector found to be ...

Gibberella ear rot of maize (Zea mays) in Nepal: distribution of the mycotoxins nivalenol and deoxynivalenol in naturally and experimentally infected maize.

PubMed

Desjardins, Anne E; Busman, Mark; Manandhar, Gyanu; Jarosz, Andrew M; Manandhar, Hira K; Proctor, Robert H

2008-07-09

The fungus Fusarium graminearum (sexual stage Gibberella zeae) causes ear rot of maize (Zea mays) and contamination with the 8-ketotrichothecenes nivalenol (1) or 4-deoxynivalenol (2), depending on diversity of the fungal population for the 4-oxygenase gene (TRI13). To determine the importance of 1 and 2 in maize ear rot, a survey of naturally contaminated maize in Nepal was combined with experiments in the field and in a plant growth room. In the survey, 1 contamination was 4-fold more frequent than 2 contamination and 1-producers (TRI13) were isolated more than twice as frequently as 2-producers (Psi TRI13). In maize ear rot experiments, genetically diverse 1-producers and 2-producers caused ear rot and trichothecene contamination. Among strains with the same genetic background, however, 1-producers caused less ear rot and trichothecene contamination than did 2-producers. The high frequency of 1 contamination and the high virulence of many 1-producers are of concern because maize is a staple food of rural populations in Nepal and because 1 has proven to be more toxic than 2 to animals.

Retrogradation of Maize Starch after High Hydrostatic Pressure Gelation: Effect of Amylose Content and Depressurization Rate.

PubMed

Yang, Zhi; Swedlund, Peter; Gu, Qinfen; Hemar, Yacine; Chaieb, Sahraoui

2016-01-01

High hydrostatic pressure (HHP) has been employed to gelatinize or physically modify starch dispersions. In this study, waxy maize starch, normal maize starch, and two high amylose content starch were processed by a HHP of the order of 600 MPa, at 25°C for 15min. The effect of HHP processing on the crystallization of maize starches with various amylose content during storage at 4°C was investigated. Crystallization kinetics of HHP treated starch gels were investigated using rheology and FTIR. The effect of crystallization on the mechanical properties of starch gel network were evaluated in terms of dynamic complex modulus (G*). The crystallization induced increase of short-range helices structures were investigated using FTIR. The pressure releasing rate does not affect the starch retrogradation behaviour. The rate and extent of retrogradation depends on the amylose content of amylose starch. The least retrogradation was observed in HHP treated waxy maize starch. The rate of retrogradation is higher for HHP treated high amylose maize starch than that of normal maize starch. A linear relationship between the extent of retrogradation (phase distribution) measured by FTIR and G* is proposed.

Retrogradation of Maize Starch after High Hydrostatic Pressure Gelation: Effect of Amylose Content and Depressurization Rate

PubMed Central

Yang, Zhi; Swedlund, Peter; Gu, Qinfen; Hemar, Yacine; Chaieb, Sahraoui

2016-01-01

High hydrostatic pressure (HHP) has been employed to gelatinize or physically modify starch dispersions. In this study, waxy maize starch, normal maize starch, and two high amylose content starch were processed by a HHP of the order of 600 MPa, at 25°C for 15min. The effect of HHP processing on the crystallization of maize starches with various amylose content during storage at 4°C was investigated. Crystallization kinetics of HHP treated starch gels were investigated using rheology and FTIR. The effect of crystallization on the mechanical properties of starch gel network were evaluated in terms of dynamic complex modulus (G*). The crystallization induced increase of short-range helices structures were investigated using FTIR. The pressure releasing rate does not affect the starch retrogradation behaviour. The rate and extent of retrogradation depends on the amylose content of amylose starch. The least retrogradation was observed in HHP treated waxy maize starch. The rate of retrogradation is higher for HHP treated high amylose maize starch than that of normal maize starch. A linear relationship between the extent of retrogradation (phase distribution) measured by FTIR and G* is proposed. PMID:27219066

Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

PubMed

Salusjärvi, Laura; Kaunisto, Sanna; Holmström, Sami; Vehkomäki, Maija-Leena; Koivuranta, Kari; Pitkänen, Juha-Pekka; Ruohonen, Laura

2013-12-01

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

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

Sirtuin 3 mediates neuroprotection of ketones against ischemic stroke

PubMed Central

Yin, Junxiang; Han, Pengcheng; Tang, Zhiwei; Liu, Qingwei; Shi, Jiong

2015-01-01

Stroke is one of the leading causes of death. Growing evidence indicates that ketone bodies have beneficial effects in treating stroke, but their underlying mechanism remains unclear. Our previous study showed ketone bodies reduced reactive oxygen species by using NADH as an electron donor, thus increasing the NAD+/NADH ratio. In this study, we investigated whether mitochondrial NAD+-dependent Sirtuin 3 (SIRT3) could mediate the neuroprotective effects of ketone bodies after ischemic stroke. We injected mice with either normal saline or ketones (beta-hydroxybutyrate and acetoacetate) at 30 minutes after ischemia induced by transient middle cerebral artery (MCA) occlusion. We found that ketone treatment enhanced mitochondria function, reduced oxidative stress, and therefore reduced infarct volume. This led to improved neurologic function after ischemia, including the neurologic score and the performance in Rotarod and open field tests. We further showed that ketones' effects were achieved by upregulating NAD+-dependent SIRT3 and its downstream substrates forkhead box O3a (FoxO3a) and superoxide dismutase 2 (SOD2) in the penumbra region since knocking down SIRT3 in vitro diminished ketones' beneficial effects. These results provide us a foundation to develop novel therapeutics targeting this SIRT3-FoxO3a-SOD2 pathway. PMID:26058697

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 biochemical pathways. In the next few years, the refinement of personalized therapy for the use of NAD + precursors and improved detection methodologies allowing the administration of specific NAD + precursors in the context of patients' NAD + levels will lead to a better understanding of the therapeutic role of NAD + precursors in human diseases. Antioxid. Redox Signal. 00, 000-000.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion

NASA Technical Reports Server (NTRS)

Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P.; McNally, J. Scott; Holland, Steven M.; Dikalov, Sergei; Giddens, Don P.; Griendling, Kathy K.; Harrison, David G.;

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).

PubMed

Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

2008-06-01

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.

Expression of OsMYB55 in maize activates stress-responsive genes and enhances heat and drought tolerance.

PubMed

Casaretto, José A; El-Kereamy, Ashraf; Zeng, Bin; Stiegelmeyer, Suzy M; Chen, Xi; Bi, Yong-Mei; Rothstein, Steven J

2016-04-29

Plant response mechanisms to heat and drought stresses have been considered in strategies for generating stress tolerant genotypes, but with limited success. Here, we analyzed the transcriptome and improved tolerance to heat stress and drought of maize plants over-expressing the OsMYB55 gene. Over-expression of OsMYB55 in maize decreased the negative effects of high temperature and drought resulting in improved plant growth and performance under these conditions. This was evidenced by the higher plant biomass and reduced leaf damage exhibited by the transgenic lines compared to wild type when plants were subjected to individual or combined stresses and during or after recovery from stress. A global transcriptomic analysis using RNA sequencing revealed that several genes induced by heat stress in wild type plants are constitutively up-regulated in OsMYB55 transgenic maize. In addition, a significant number of genes up-regulated in OsMYB55 transgenic maize under control or heat treatments have been associated with responses to abiotic stresses including high temperature, dehydration and oxidative stress. The latter is a common and major consequence of imposed heat and drought conditions, suggesting that this altered gene expression may be associated with the improved stress tolerance in these transgenic lines. Functional annotation and enrichment analysis of the transcriptome also pinpoint the relevance of specific biological processes for stress responses. Our results show that expression of OsMYB55 can improve tolerance to heat stress and drought in maize plants. Enhanced expression of stress-associated genes may be involved in OsMYB55-mediated stress tolerance. Possible implications for the improved tolerance to heat stress and drought of OsMYB55 transgenic maize are discussed.

ZCN8 encodes a potential orthologue of Arabidopsis FT florigen that integrates both endogenous and photoperiod flowering signals in maize

PubMed Central

Lazakis, Chloë M.; Coneva, Viktoriya; Colasanti, Joseph

2011-01-01

Higher plants use multiple perceptive measures to coordinate flowering time with environmental and endogenous cues. Physiological studies show that florigen is a mobile factor that transmits floral inductive signals from the leaf to the shoot apex. Arabidopsis FT protein is widely regarded as the archetype florigen found in diverse plant species, particularly in plants that use inductive photoperiods to flower. Recently, a large family of FT homologues in maize, the Zea CENTRORADIALIS (ZCN) genes, was described, suggesting that maize also contains FT-related proteins that act as a florigen. The product of one member of this large family, ZCN8, has several attributes that make it a good candidate as a maize florigen. Mechanisms underlying the floral transition in maize are less well understood than those of other species, partly because flowering in temperate maize is dependent largely on endogenous signals. The maize indeterminate1 (id1) gene is an important regulator of maize autonomous flowering that acts in leaves to mediate the transmission or production of florigenic signals. This study finds that id1 acts upstream of ZCN8 to control its expression, suggesting a possible new link to flowering in day-neutral maize. Moreover, in teosinte, a tropical progenitor of maize that requires short-day photoperiods to induce flowering, ZCN8 is highly up-regulated in leaves under inductive photoperiods. Finally, vascular-specific expression of ZCN8 in Arabidopsis complements the ft-1 mutation, demonstrating that leaf-specific expression of ZCN8 can induce flowering. These results suggest that ZCN8 may encode a florigen that integrates both endogenous and environmental signals in maize. PMID:21730358

Effects of elevated [CO2] on maize defence against mycotoxigenic Fusarium verticillioides

PubMed Central

Vaughan, Martha M; Huffaker, Alisa; Schmelz, Eric A; Dafoe, Nicole J; Christensen, Shawn; Sims, James; Martins, Vitor F; Swerbilow, Jay; Romero, Maritza; Alborn, Hans T; Allen, Leon HARTWELL; Teal, Peter EA

2014-01-01

Maize is by quantity the most important C4 cereal crop; however, future climate changes are expected to increase maize susceptibility to mycotoxigenic fungal pathogens and reduce productivity. While rising atmospheric [CO2] is a driving force behind the warmer temperatures and drought, which aggravate fungal disease and mycotoxin accumulation, our understanding of how elevated [CO2] will effect maize defences against such pathogens is limited. Here we report that elevated [CO2] increases maize susceptibility to Fusarium verticillioides proliferation, while mycotoxin levels are unaltered. Fumonisin production is not proportional to the increase in F. verticillioides biomass, and the amount of fumonisin produced per unit pathogen is reduced at elevated [CO2]. Following F. verticillioides stalk inoculation, the accumulation of sugars, free fatty acids, lipoxygenase (LOX) transcripts, phytohormones and downstream phytoalexins is dampened in maize grown at elevated [CO2]. The attenuation of maize 13-LOXs and jasmonic acid production correlates with reduced terpenoid phytoalexins and increased susceptibility. Furthermore, the attenuated induction of 9-LOXs, which have been suggested to stimulate mycotoxin biosynthesis, is consistent with reduced fumonisin per unit fungal biomass at elevated [CO2]. Our findings suggest that elevated [CO2] will compromise maize LOX-dependent signalling, which will influence the interactions between maize and mycotoxigenic fungi. Elevated [CO2] increases maize susceptibility to Fusarium verticillioides proliferation but mycotoxin levels are unaltered. The attenuation of maize 13-LOXs and JA production correlates with reduced terpenoid phytoalexins and increased susceptibility. Furthermore, the attenuated induction of 9-LOXs, which have been suggested to stimulate mycotoxin biosynthesis, is consistent with reduced fumonisin per unit fungal biomass at elevated [CO2]. PMID:24689748

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

PubMed Central

VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

2015-01-01

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

Transposition of the maize transposable element Ac in barley (Hordeum vulgare L.).

PubMed

Scholz, S; Lörz, H; Lütticke, S

2001-01-01

Transposition of the maize autonomous element Ac (Activator) was investigated in barley (Hordeum vulgare L.) with the aim of developing a transposon tagging system for the latter. The Ac element was introduced into meristematic tissue of barley by microprojectile bombardment. Transposon activity was then examined in the resulting transgenic plants. Multiple excision events were detected in leaf tissue of all plant lines. The mobile elements generated empty donor sites with small DNA sequence alterations, similar to those found in maize. Reintegration of Ac at independent genomic loci in somatic tissue was demonstrated by isolation of new element-flanking regions by AIMS-PCR (amplification of insertion-mutagenized sites). In addition, transmission of transposed Ac elements to progeny plants was confirmed. The results indicate that the introduced Ac element is able to transpose in barley. This is a first step towards the establishment of a transposon tagging system in this economically important crop.

The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

PubMed

Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

2016-06-01

Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

Role of redox signaling in the autonomous proliferative response of endothelial cells to hypoxia.

PubMed

Schäfer, M; Schäfer, C; Ewald, N; Piper, H M; Noll, Th

2003-05-16

Endothelial cells exhibit an autonomous proliferative response to hypoxia, independent of paracrine effectors. In cultured endothelial cells of porcine aorta, we analyzed the signaling of this response, with a focus on the roles of redox signaling and the MEK/ERK pathway. Transient hypoxia (1 hour) stimulated proliferation by 61+/-4% (n=16; P<0.05 versus control), quantified after 24 hours normoxic postincubation. Hypoxia induced an activation of ERK2 and of NAD(P)H oxidase and a burst of reactive oxygen species (ROS), determined by DCF fluorescence. To inhibit the MEK/ERK pathway, we used PD 98059 (PD, 20 micromol/L); to downregulate NAD(P)H oxidase, we applied p22phox antisense oligonucleotides; and to inhibit mitochondrial ROS generation, we used the ubiquinone derivate mitoQ (MQ, 10 micromol/L). All three inhibitions suppressed the proliferative response: PD inhibited NAD(P)H oxidase activation; p22phox antisense transfection did not inhibit ERK2 activation, but suppressed ROS production; and MQ inhibited ERK2 activation and ROS production. The autonomous proliferative response depends on the MEK/ERK pathway and redox signaling steps upstream and downstream of ERK. Located upstream is ROS generation by mitochondria, downstream is NAD(P)H oxidase.

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

PubMed Central

Schulz, Margot; Filary, Barbara; Kühn, Sabine; Colby, Thomas; Harzen, Anne; Schmidt, Jürgen; Sicker, Dieter; Hennig, Lothar; Hofmann, Diana; Disko, Ulrich; Anders, Nico

2016-01-01

ABSTRACT The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe2+ ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis. PMID:26645909

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

PubMed

Schulz, Margot; Filary, Barbara; Kühn, Sabine; Colby, Thomas; Harzen, Anne; Schmidt, Jürgen; Sicker, Dieter; Hennig, Lothar; Hofmann, Diana; Disko, Ulrich; Anders, Nico

2016-01-01

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

Republished study: long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize.

PubMed

Séralini, Gilles-Eric; Clair, Emilie; Mesnage, Robin; Gress, Steeve; Defarge, Nicolas; Malatesta, Manuela; Hennequin, Didier; de Vendômois, Joël Spiroux

2014-01-01

The health effects of a Roundup-tolerant NK603 genetically modified (GM) maize (from 11% in the diet), cultivated with or without Roundup application and Roundup alone (from 0.1 ppb of the full pesticide containing glyphosate and adjuvants) in drinking water, were evaluated for 2 years in rats. This study constitutes a follow-up investigation of a 90-day feeding study conducted by Monsanto in order to obtain commercial release of this GMO, employing the same rat strain and analyzing biochemical parameters on the same number of animals per group as our investigation. Our research represents the first chronic study on these substances, in which all observations including tumors are reported chronologically. Thus, it was not designed as a carcinogenicity study. We report the major findings with 34 organs observed and 56 parameters analyzed at 11 time points for most organs. Biochemical analyses confirmed very significant chronic kidney deficiencies, for all treatments and both sexes; 76% of the altered parameters were kidney-related. In treated males, liver congestions and necrosis were 2.5 to 5.5 times higher. Marked and severe nephropathies were also generally 1.3 to 2.3 times greater. In females, all treatment groups showed a two- to threefold increase in mortality, and deaths were earlier. This difference was also evident in three male groups fed with GM maize. All results were hormone- and sex-dependent, and the pathological profiles were comparable. Females developed large mammary tumors more frequently and before controls; the pituitary was the second most disabled organ; the sex hormonal balance was modified by consumption of GM maize and Roundup treatments. Males presented up to four times more large palpable tumors starting 600 days earlier than in the control group, in which only one tumor was noted. These results may be explained by not only the non-linear endocrine-disrupting effects of Roundup but also by the overexpression of the EPSPS transgene or other mutational effects in the GM maize and their metabolic consequences. Our findings imply that long-term (2 year) feeding trials need to be conducted to thoroughly evaluate the safety of GM foods and pesticides in their full commercial formulations.

Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

PubMed

Rurek, Michał; Nuc, Katarzyna; Raczyńska, Katarzyna Dorota; Augustyniak, Halina

2003-10-02

The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.

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.

Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

PubMed Central

Burdette, D; Zeikus, J G

1994-01-01

The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

Mutagenic effects of heavy ion radiation in plants

NASA Astrophysics Data System (ADS)

Mei, M.; Deng, H.; Lu, Y.; Zhuang, C.; Liu, Z.; Qiu, Q.; Qiu, Y.; Yang, T. C.

1994-10-01

Genetic and developmental effects of heavy ions in maize and rice were investigated. Heavy particles with various charges and energies were accelerated at the BEVALAC. The frequency of occurence of white-yellow stripes on leaves of plants developed from irradiated maize seeds increased linearly with dose, and high-LET heavy charged particles, e.g., neon, argon, and iron, were 2-12 times as effective as gamma rays in inducing this type of mutation. The effectiveness of high-LET heavy ion in (1) inhibiting rice seedling growth, (2) reducing plant fertility, (3) inducing chromosome aberration and micronuclei in root tip cells and pollen mother cells of the first generation plants developed from exposed seeds, and (4) inducing mutation in the second generation, were greater than that of low-LET gamma rays. All effects observed were dose-dependent; however, there appeared to be an optimal range of doses for inducing certain types of mutation, for example, for argon ions (400 MeV/u) at 90-100 Gy, several valuable mutant lines with favorable characters, such as semidwarf, early maturity and high yield ability, were obtained. Experimental results suggest that the potential application of heavy ions in crop improvement is promising. RFLP analysis of two semidwarf mutants induced by argon particles revealed that large DNA alterations might be involved in these mutants.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

PubMed

VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

2015-11-13

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorus mining efficiency declines with decreasing soil P concentration and varies across crop species.

PubMed

Schelfhout, Stephanie; De Schrijver, An; Verheyen, Kris; De Beelde, Robbe; Haesaert, Geert; Mertens, Jan

2018-07-29

High soil P concentrations hinder ecological restoration of biological communities typical for nutrient-poor soils. Phosphorus mining, i.e., growing crops with fertilization other than P, might reduce soil P concentrations. However, crop species have different P-uptake rates and can affect subsequent P removal in crop rotation, both of which may also vary with soil P concentration. In a pot experiment with three soil-P-levels (High-P: 125-155 mg P Olsen /kg; Mid-P: 51-70 mg P Olsen /kg; Low-P: 6-21 mg P Olsen /kg), we measured how much P was removed by five crop species (buckwheat, maize, sunflower, flax, and triticale). Total P removal decreased with soil-P-level and depended upon crop identity. Buckwheat and maize removed most P from High-P and Mid-P soils and triticale removed less P than buckwheat, maize, and sunflower at every soil-P-level. The difference in P removal between crops was, however, almost absent in Low-P soils. Absolute and relative P removal with seeds depended upon crop species and, for maize and triticale, also upon soil-P-level. None of the previously grown crop species significantly affected P removal by the follow-up crop (perennial ryegrass). We can conclude that for maximizing P removal, buckwheat or maize could be grown.

Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair

PubMed Central

Goellner, Eva M.; Grimme, Bradford; Brown, Ashley R.; Lin, Ying-Chih; Wang, Xiao-Hong; Sugrue, Kelsey F.; Mitchell, Leah; Trivedi, Ram N.; Tang, Jiang-bo; Sobol, Robert W.

2011-01-01

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O6-methylguanine (O6-MeG) due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. Based on our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of NAD+, we have hypothesized that combined BER and NAD+ biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD+ biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD+ biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM. PMID:21406402

H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

PubMed

Furukawa, Ayako; Tada-Oikawa, Saeko; Kawanishi, Shosuke; Oikawa, Shinji

2007-01-01

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

A Maize Cystatin Suppresses Host Immunity by Inhibiting Apoplastic Cysteine Proteases[C][W

PubMed Central

van der Linde, Karina; Hemetsberger, Christoph; Kastner, Christine; Kaschani, Farnusch; van der Hoorn, Renier A.L.; Kumlehn, Jochen; Doehlemann, Gunther

2012-01-01

Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Δpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)–induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis. PMID:22454455

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

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

Coupling of NAD+ Biosynthesis and Nicotinamide Ribosyl Transport: Characterization of NadR Ribonucleotide Kinase Mutants of Haemophilus influenzae

PubMed Central

Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

2005-01-01

Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate. PMID:15968050

Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae.

PubMed

Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

2005-07-01

Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.

Concurrent targeting of nitrosative stress-PARP pathway corrects functional, behavioral and biochemical deficits in experimental diabetic neuropathy

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

Negi, Geeta; Kumar, Ashutosh; Sharma, Shyam S., E-mail: sssharma@niper.ac.in

2010-01-01

Peroxynitrite mediated nitrosative stress, an indisputable initiator of DNA damage and overactivation of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated after sensing DNA damage, are two crucial pathogenetic mechanisms in diabetic neuropathy. The intent of the present study was to investigate the effect of combination of a peroxynitrite decomposition catalyst (PDC), FeTMPyP and a PARP inhibitor, 4-ANI against diabetic peripheral neuropathy. The end points of evaluation of the study included motor nerve conduction velocity (MNCV) and nerve blood flow (NBF) for evaluating nerve functions; thermal hyperalgesia and mechanical allodynia for assessing nociceptive alterations, malondialdehyde and peroxynitrite levels to detect oxidativemore » stress-nitrosative stress; NAD concentration in sciatic nerve to assess overactivation of PARP. Additionally immunohistochemical studies for nitrotyrosine and Poly(ADP-ribose) (PAR) was also performed. Treatment with the combination of FeTMPyP and 4-ANI led to significant improvement in nerve functions and pain parameters and also attenuated the oxidative-nitrosative stress markers. Further, the combination also reduced the overactivation of PARP as evident from increased NAD levels and decreased PAR immunopositivity in sciatic nerve microsections. Thus, it can be concluded that treatment with the combination of a PDC and PARP inhibitor attenuates alteration in peripheral nerves in diabetic neuropathy (DN).« less

The Aux/IAA gene rum1 involved in seminal and lateral root formation controls vascular patterning in maize (Zea mays L.) primary roots.

PubMed

Zhang, Yanxiang; Paschold, Anja; Marcon, Caroline; Liu, Sanzhen; Tai, Huanhuan; Nestler, Josefine; Yeh, Cheng-Ting; Opitz, Nina; Lanz, Christa; Schnable, Patrick S; Hochholdinger, Frank

2014-09-01

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEMS 1) controls seminal and lateral root initiation. To identify RUM1-dependent gene expression patterns, RNA-Seq of the differentiation zone of primary roots of rum1 mutants and the wild type was performed in four biological replicates. In total, 2 801 high-confidence maize genes displayed differential gene expression with Fc ≥2 and FDR ≤1%. The auxin signalling-related genes rum1, like-auxin1 (lax1), lax2, (nam ataf cuc 1 nac1), the plethora genes plt1 (plethora 1), bbm1 (baby boom 1), and hscf1 (heat shock complementing factor 1) and the auxin response factors arf8 and arf37 were down-regulated in the mutant rum1. All of these genes except nac1 were auxin-inducible. The maize arf8 and arf37 genes are orthologues of Arabidopsis MP/ARF5 (MONOPTEROS/ARF5), which controls the differentiation of vascular cells. Histological analyses of mutant rum1 roots revealed defects in xylem organization and the differentiation of pith cells around the xylem. Moreover, histochemical staining of enlarged pith cells surrounding late metaxylem elements demonstrated that their thickened cell walls displayed excessive lignin deposition. In line with this phenotype, rum1-dependent mis-expression of several lignin biosynthesis genes was observed. In summary, RNA-Seq of RUM1-dependent gene expression in maize primary roots, in combination with histological and histochemical analyses, revealed the specific regulation of auxin signal transduction components by RUM1 and novel functions of RUM1 in vascular development. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease.

PubMed

Penberthy, W Todd

2007-04-01

Cells at the maternal-fetal interface express indoleamine 2,3 dioxygenase (IDO) to consume all local tryptophan for the express purpose of starving adjacent maternal T cells of this most limiting and essential amino acid. This stops local T cell proliferation to ultimately result in the most dramatic example of immune tolerance, acceptance of the fetus. By contrast, inhibition of IDO using 1-methyl-tryptophan causes a sudden catastrophic rejection of the mammalian fetus. Immunomodulatory factors including IFNgamma, TNFalpha, IL-1, and LPS use IDO induction in responsive antigen presenting cells (APCs) also to transmit tolerogenic signals to T cells. Thus it makes sense to consider IDO induction towards tolerance for autoimmune diseases in general. Approaches to cell specific therapeutic IDO induction with NAD precursor supplementation to prevent the collateral non-T cell pathogenesis due to chronic TNFalpha-IDO activated tryptophan depletion in autoimmune diseases are reviewed. Tryptophan is an essential amino acid most immediately because it is the only precursor for the endogenous biosynthesis of nicotinamide adenine dinucleotide (NAD). Both autoimmune disease and the NAD deficiency disease pellagra occur in women at greater than twice the frequency of occurrence in men. The importance of IDO dysregulation manifest as autoimmune pellagric dementia is genetically illustrated for Nasu-Hakola Disease (or PLOSL), which is caused by a mutation in the IDO antagonizing genes TYROBP/DAP12 or TREM2. Loss of function leads to psychotic symptoms rapidly progressing to presenile dementia likely due to unchecked increases in microglial IDO expression, which depletes neurons of tryptophan causing neurodegeneration. Administration of NAD precursors rescued entire mental hospitals of dementia patients literally overnight in the 1930's and NAD precursors should help Nasu-Hakola patients as well. NAD depletion mediated by peroxynitrate PARP1 activation is one of the few established mechanisms of necrosis. Chronic elevation of TNFalpha leading to necrotic events by NAD depletion in autoimmune disease likely occurs via combination of persistent IDO activation and iNOS-peroxynitrate activation of PARP1 both of which deplete NAD. Pharmacological doses of NAD precursors repeatedly provide dramatic therapeutic benefit for rheumatoid arthritis, type 1 diabetes, multiple sclerosis, colitis, other autoimmune diseases, and schizophrenia in either the clinic or animal models. Collectively these observations support the idea that autoimmune disease may in part be considered as localized pellagra manifesting symptoms particular to the inflamed target tissues. Thus pharmacological doses of NAD precursors (nicotinic acid/niacin, nicotinamide/niacinamide, or nicotinamide riboside) should be considered as potentially essential to the therapeutic success of any IDO-inducing regimen for treating autoimmune diseases. Distinct among the NAD precursors, nicotinic acid specifically activates the g-protein coupled receptor (GPCR) GPR109a to produce the IDO-inducing tolerogenic prostaglandins PGE(2) and PGD(2). Next, PGD(2) is converted to the anti-inflammatory prostaglandin, 15d-PGJ(2). These prostaglandins exert potent anti-inflammatory activities through endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma respectively. Nicotinamide prevents type 1 diabetes and ameliorates multiple sclerosis in animal models, while nothing is known about the therapeutic potential of nicotinamide riboside. Alternatively the direct targeting of the non-redox NAD-dependent proteins using resveratrol to activate SIRT1 or PJ34 in order to inhibit PARP1 and prevent autoimmune pathogenesis are also given consideration.

The quality of metabolic pathway resources depends on initial enzymatic function assignments: a case for maize

DOE PAGES

Walsh, Jesse R.; Schaeffer, Mary L.; Zhang, Peifen; ...

2016-11-29

As metabolic pathway resources become more commonly available, researchers have unprecedented access to information about their organism of interest. Despite efforts to ensure consistency between various resources, information content and quality can vary widely. Two maize metabolic pathway resources for the B73 inbred line, CornCyc 4.0 and MaizeCyc 2.2, are based on the same gene model set and were developed using Pathway Tools software. These resources differ in their initial enzymatic function assignments and in the extent of manual curation. Here, we present an in-depth comparison between CornCyc and MaizeCyc to demonstrate the effect of initial computational enzymatic function assignmentsmore » on the quality and content of metabolic pathway resources.« less

The quality of metabolic pathway resources depends on initial enzymatic function assignments: a case for maize

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

Walsh, Jesse R.; Schaeffer, Mary L.; Zhang, Peifen

As metabolic pathway resources become more commonly available, researchers have unprecedented access to information about their organism of interest. Despite efforts to ensure consistency between various resources, information content and quality can vary widely. Two maize metabolic pathway resources for the B73 inbred line, CornCyc 4.0 and MaizeCyc 2.2, are based on the same gene model set and were developed using Pathway Tools software. These resources differ in their initial enzymatic function assignments and in the extent of manual curation. Here, we present an in-depth comparison between CornCyc and MaizeCyc to demonstrate the effect of initial computational enzymatic function assignmentsmore » on the quality and content of metabolic pathway resources.« less

Economics, energy, and environmental assessment of diversified crop rotations in sub-Himalayas of India.

PubMed

Singh, Raman Jeet; Meena, Roshan Lal; Sharma, N K; Kumar, Suresh; Kumar, Kuldeep; Kumar, Dileep

2016-02-01

Reducing the carbon footprint and increasing energy use efficiency of crop rotations are the two most important sustainability issues of the modern agriculture. Present study was undertaken to assess economics, energy, and environmental parameters of common diversified crop rotations (maize-tomato, and maize-toria-wheat) vis-a-vis traditional crop rotations like maize-wheat, maize + ginger and rice-wheat of the north-western Himalayan region of India. Results revealed that maize-tomato and maize + ginger crop rotations being on par with each other produced significantly higher system productivity in terms of maize equivalent yield (30.2-36.2 t/ha) than other crop rotations (5.04-7.68 t/ha). But interestingly in terms of energy efficiencies, traditional maize-wheat system (energy efficiency 7.9, human energy profitability of 177.8 and energy profitability of 6.9 MJ/ha) was significantly superior over other systems. Maize + ginger rotation showed greater competitive advantage over other rotations because of less consumption of non-renewable energy resources. Similarly, maize-tomato rotation had ability of the production process to exploit natural resources due to 14-38% less use of commercial or purchased energy sources over other crop rotations. Vegetable-based crop rotations (maize + ginger and maize-tomato) maintained significantly the least carbon footprint (0.008 and 0.019 kg CO2 eq./kg grain, respectively) and the highest profitability (154,322 and 274,161 Rs./ha net return, respectively) over other crop rotations. As the greatest inputs of energy and carbon across the five crop rotations were nitrogen fertilizer (15-29% and 17-28%, respectively), diesel (14-24% and 8-19%, respectively) and irrigation (10-27% and 11-44%, respectively), therefore, alternative sources like organic farming, conservation agriculture practices, soil and water conservation measures, rain water harvesting etc. should be encouraged to reduce dependency of direct energy and external carbon inputs particularly in sub-Himalayas of India.

Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants.

PubMed

Planchamp, Chantal; Glauser, Gaetan; Mauch-Mani, Brigitte

2014-01-01

Pseudomonas putida KT2440 (KT2440) rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots) and systemic (leaves) early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots 3 days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR) against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal growth in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as resistance inducers.

Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants

PubMed Central

Planchamp, Chantal; Glauser, Gaetan; Mauch-Mani, Brigitte

2014-01-01

Pseudomonas putida KT2440 (KT2440) rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots) and systemic (leaves) early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots 3 days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR) against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal growth in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as resistance inducers. PMID:25628626

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

PubMed Central

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

2015-01-01

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

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 Association, Inc.

The maize lilliputian1 (lil1) gene, encoding a brassinosteroid cytochrome P450 C-6 oxidase, is involved in plant growth and drought response.

PubMed

Castorina, Giulia; Persico, Martina; Zilio, Massimo; Sangiorgio, Stefano; Carabelli, Laura; Consonni, Gabriella

2018-05-16

Brassinosteroids (BRs) are plant hormones involved in many developmental processes as well as in plant-environment interactions. Their role was investigated in this study through the analysis of lilliputian1-1 (lil1-1), a dwarf mutant impaired in BR biosynthesis in maize (Zea mays). We isolated lil1-1 through transposon tagging in maize. The action of lil1 was investigated through morphological and genetic analysis. Moreover, by comparing lil1-1 mutant and wild-type individuals grown under drought stress, the effect of BR reduction on the response to drought stress was examined. lil1-1 is a novel allele of the brassinosteroid-deficient dwarf1 (brd1) gene, encoding a brassinosteroid C-6 oxidase. We show in this study that lil1 is epistatic to nana plant1 (na1), a BR gene involved in earlier steps of the pathway. The lill-1 mutation causes alteration in the root gravitropic response, leaf epidermal cell density, epicuticular wax deposition and seedling adaptation to water scarcity conditions. Lack of active BR molecules in maize causes a pleiotropic effect on plant development and improves seedling tolerance of drought. BR-deficient maize mutants can thus be instrumental in unravelling novel mechanisms on which plant adaptations to abiotic stress are based.

Elevated ozone reduces photosynthetic carbon gain by accelerating leaf senescence of inbred and hybrid maize in a genotype-specific manner.

PubMed

Yendrek, Craig R; Erice, Gorka; Montes, Christopher M; Tomaz, Tiago; Sorgini, Crystal A; Brown, Patrick J; McIntyre, Lauren M; Leakey, Andrew D B; Ainsworth, Elizabeth A

2017-12-01

Exposure to elevated tropospheric ozone concentration ([O 3 ]) accelerates leaf senescence in many C 3 crops. However, the effects of elevated [O 3 ] on C 4 crops including maize (Zea mays L.) are poorly understood in terms of physiological mechanism and genetic variation in sensitivity. Using free air gas concentration enrichment, we investigated the photosynthetic response of 18 diverse maize inbred and hybrid lines to season-long exposure to elevated [O 3 ] (~100 nl L -1 ) in the field. Gas exchange was measured on the leaf subtending the ear throughout the grain filling period. On average over the lifetime of the leaf, elevated [O 3 ] led to reductions in photosynthetic CO 2 assimilation of both inbred (-22%) and hybrid (-33%) genotypes. There was significant variation among both inbred and hybrid lines in the sensitivity of photosynthesis to elevated [O 3 ], with some lines showing no change in photosynthesis at elevated [O 3 ]. Based on analysis of inbred line B73, the reduced CO 2 assimilation at elevated [O 3 ] was associated with accelerated senescence decreasing photosynthetic capacity and not altered stomatal limitation. These findings across diverse maize genotypes could advance the development of more O 3 tolerant maize and provide experimental data for parameterization and validation of studies modeling how O 3 impacts crop performance. © 2017 John Wiley & Sons Ltd.

Spinosad- and Deltamethrin-Induced Impact on Mating and Reproductive Output of the Maize Weevil Sitophilus zeamais.

PubMed

Vélez, Mayra; Botina, Lorena L; Turchen, Leonardo M; Barbosa, Wagner F; Guedes, Raul Narciso C

2018-04-02

Assessments of acute insecticide toxicity frequently focus on the lethal effects on individual arthropod pest species and populations neglecting the impacts and consequences of sublethal exposure. However, the sublethal effects of insecticides may lead to harmful, neutral, or even beneficial responses that may affect (or not) the behavior and sexual fitness of the exposed insects. Intriguingly, little is known about such effects on stored product insect pests in general and the maize weevil in particular. Thus, we assessed the sublethal effects of spinosad and deltamethrin on female mate-searching, mating behavior, progeny emergence, and grain consumption by maize weevils. Insecticide exposure did not affect the resting time, number of stops, and duration of mate-searching by female weevils, but their walking velocity was compromised. Maize weevil couples sublethally exposed to deltamethrin and spinosad exhibited altered reproductive behavior (walking, interacting, mounting, and copulating), but deltamethrin caused greater impairment. Curiously, higher grain consumption and increased progeny emergence were observed in deltamethrin-exposed insects, suggesting that this pyrethroid insecticide elicits hormesis in maize weevils that may compromise control efficacy by this compound. Although spinosad has less of an impact on weevil reproductive behavior than deltamethrin, this bioinsecticide also benefited weevil progeny emergence, but did not affect grain consumption. Therefore, our findings suggest caution using either compound, and particularly deltamethrin, for controlling the maize weevil, as they may actually favor this species population growth when in sublethal exposure requiring further assessments. The same concern may be valid for other insecticides as well, what deserves future attention.

Genetic resources for maize cell wall biology.

PubMed

Penning, Bryan W; Hunter, Charles T; Tayengwa, Reuben; Eveland, Andrea L; Dugard, Christopher K; Olek, Anna T; Vermerris, Wilfred; Koch, Karen E; McCarty, Donald R; Davis, Mark F; Thomas, Steven R; McCann, Maureen C; Carpita, Nicholas C

2009-12-01

Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions.

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...

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.

Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside.

PubMed

Tran, Ai; Yokose, Ryota; Cen, Yana

2018-05-15

As a cofactor for numerous reactions, NAD+ is found widely dispersed across many maps of cellular metabolism. This core redox role alone makes the biosynthesis of NAD+ of great interest. Recent studies have revealed new biological roles for NAD+ as a substrate for diverse enzymes that regulate a broad spectrum of key cellular tasks. These NAD+-consuming enzymes further highlight the importance of understanding NAD+ biosynthetic pathways. In this study, we developed a chemo-enzymatic synthesis of isotopically labeled NAD+ precursor, nicotinamide riboside (NR). The synthesis of NR isotopomers allowed us to unambiguously determine that NR is efficiently converted to NAD+ in the cellular environment independent of degradation to nicotinamide, and it is incorporated into NAD+ in its intact form. The versatile synthetic method along with the isotopically labeled NRs will provide powerful tools to further decipher the important yet complicated NAD+ metabolism.

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.

Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.

PubMed

Frederick, David W; Loro, Emanuele; Liu, Ling; Davila, Antonio; Chellappa, Karthikeyani; Silverman, Ian M; Quinn, William J; Gosai, Sager J; Tichy, Elisia D; Davis, James G; Mourkioti, Foteini; Gregory, Brian D; Dellinger, Ryan W; Redpath, Philip; Migaud, Marie E; Nakamaru-Ogiso, Eiko; Rabinowitz, Joshua D; Khurana, Tejvir S; Baur, Joseph A

2016-08-09

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function. Copyright © 2016 Elsevier Inc. All rights reserved.

Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

PubMed Central

Irani, Niloufer G; Grotewold, Erich

2005-01-01

Background Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS) cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. Results BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. Conclusion By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI)-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin. PMID:15907203

Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

PubMed Central

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L.; Evans, Charles R.; Brenner, Charles

2011-01-01

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins. PMID:21589930

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

PubMed

Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

2011-05-11

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

Genetically modified myths and realities.

PubMed

Parrott, Wayne

2010-11-30

Myths abound when it comes to GE crops. At their worst, myths play an active role in discouraging the use of GE to solve problems that afflict humankind, such as malnutrition and birth defects. Of all the various myths, two have been particularly important in preventing the use of GE maize in its areas of origin. The first is that transgenic maize will contaminate and destroy land races, thus destroying biodiversity and its associated cultural traditions. This myth totally ignores the fact that the gene flow that has taken place between maize and its progenitor, between the land races, and between land races and modern hybrids, has not led to any dire consequences. The second myth is that crops are natural and have not been modified by humans, or if they have, that plant breeding does not alter DNA. This myth ignores the fact that for the most part, it is impossible to alter the appearance of crops without changing the DNA. In fact, DNA movement within the crop genome is normal and its movement leads to double-strand DNA repair, with results like those found around transgene insertion sites. In addition, plants have ways to create novel genes. These changes help plants adapt to evolution and to human selection. The net result is that changes similar to what happens during the production of engineered plants takes place anyway in plant genomes. Copyright © 2010 Elsevier B.V. All rights reserved.

Effect of Cadmium Stress on Non-enzymatic Antioxidant and Nitric Oxide Levels in Two Varieties of Maize (Zea mays).

PubMed

Akinyemi, Ayodele Jacob; Faboya, Oluwabamise Lekan; Olayide, Israel; Faboya, Opeyemi Ayodeji; Ijabadeniyi, Tosin

2017-06-01

Cadmium (Cd) is one of the most toxic heavy metals that inhibit physiological processes of plants. Hence, the present study sought to investigate the effect of cadmium-contaminated seeds from two varieties of maize (Zea mays) on non-enzymatic antioxidant and nitric oxide levels. Seeds of yellow and white maize were exposed to different concentrations of Cd (0, 1, 3 and 5 ppm) for two weeks. The results from this study revealed that both varieties of maize bio-accumulate Cd in leaves in a dose-dependent manner. In addition, Cd exposure caused a significant (p < 0.05) decrease in total phenolic, GSH and nitric oxide (NO) levels at the highest concentration tested when compared with control. Therefore, the observed decrease in NO and endogenous antioxidant status by Cd treatment in maize plants could suggest some possible mechanism of action for Cd-induced oxidative stress and counteracting effect of the plants against Cd toxicity.

Physiological, Ultrastructural and Proteomic Responses in the Leaf of Maize Seedlings to Polyethylene Glycol-Stimulated Severe Water Deficiency

PubMed Central

Shao, Ruixin; Xin, Longfei; Mao, Jun; Li, Leilei; Kang, Guozhang; Yang, Qinghua

2015-01-01

After maize seedlings grown in full-strength Hoagland solution for 20 days were exposed to 20% polyethylene glycol (PEG)-stimulated water deficiency for two days, plant height, shoot fresh and dry weights, and pigment contents significantly decreased, whereas malondialdehyde (MDA) content greatly increased. Using transmission electron microscopy, we observed that chloroplasts of mesophyll cells in PEG-treated maize seedlings were swollen, with a disintegrating envelope and disrupted grana thylakoid lamellae. Using two-dimensional gel electrophoresis (2-DE) method, we were able to identify 22 protein spots with significantly altered abundance in the leaves of treated seedlings in response to water deficiency, 16 of which were successfully identified. These protein species were functionally classified into signal transduction, stress defense, carbohydrate metabolism, protein metabolism, and unknown categories. The change in the abundance of the identified protein species may be closely related to the phenotypic and physiological changes due to PEG-stimulated water deficiency. Most of the identified protein species were putatively located in chloroplasts, indicating that chloroplasts may be prone to damage by PEG stimulated-water deficiency in maize seedlings. Our results help clarify the molecular mechanisms of the responses of higher plants to severe water deficiency. PMID:26370980

Partitioning evapotranspiration via continuous sampling of water vapor isotopes over common row crops and candidate biofuel crops.

NASA Astrophysics Data System (ADS)

Miller, J. N.; Black, C. K.; Bernacchi, C.

2014-12-01

Global demand for renewable energy is accelerating land conversion from common row crops such as maize and soybean to cellulosic biofuel crops such as miscanthus and switchgrass. This land conversion is expected to alter ecohydrology via changes in evapotranspiration (ET). However, the direction in which evapotranspiration will shift, either partitioning more moisture through soil evaporation (E) or through plant transpiration (T) is uncertain. To investigate how land conversion from maize to miscanthus affects ET partitioning we measured the isotopic composition of water vapor via continuous air sampling. We obtained continuous diurnal measurements of δ2H and δ18O for miscanthus and maize on multiple days over the course of the growing season. Water vapor isotopes drawn from two heights were measured at 2 Hz using a cavity ringdown spectrometer and partitioned into components of E and T using a simple mixing equation. A second approach to partitioning was accomplished by subtracting transpiration measurements, obtained through sap flow sensors, from total ET, measured via eddy covariance. Preliminary results reveal that both methods compare favorably and that transpiration dominates variations in ET in miscanthus fields more so than in fields of maize.

[Effect of long-term application of NPK fertilizer on maize yield and yellow soil nutrients sustainability in Guizhou, China].

PubMed

Liu, Yan Ling; Li, Yu; Zhang, Ya Rong; Huang, Xing Cheng; Zhang, Wen An; Jiang, Tai Ming

2017-11-01

A long-term fertilization field experiment was conducted to investigate the effect of nitrogen (N), phosphorus (P), and potassium (K) fertilizer on maize relative yield, yield-increasing effect and the changes of nutrients in yellow soil in Guizhou Province. Five fertilizer combinations were evaluated, including balanced fertilization (NPK) and nutrient deficiency treatments (N, NK, NP, and PK). The maize relative yield, contribution efficiency of N, P, K fertilizer application, sustainability index of soil N, P, K nutrients, and other indicators were measured. The results revealed that the balanced fertilization (NPK) significantly increased maize yield, and the average yield under each treatment ranked as: NPK>NP>NK>PK>CK. The contribution efficiency and agronomic efficiency of N, P, K fertilizer application was N>P>K. The fertilization dependence was ranked as: combined application of N, P and K>N>P>K. But in the lack of P treatment (NK), the maize relative yield significantly decreased at a speed of 1.4% per year, with the contribution efficiency and fertilization dependence of applied P significantly increasing at a speed of 2.3% per year and 1.4% per year, respectively. Over time, the effect of P fertilizer on maize yield gradually became equal to that of N fertilizer. The pH and soil organic matter content were the lowest in the P-lack treatment (NK), while they were higher in the N-lack treatment (PK). The application of chemical P significantly improved the sustainability index of soil P, but the application of chemical N and K did not significantly change the sustainability index of soil N and K nutrients compared to the N- and K-lack treatments, respectively. In summary, the use of balanced fertilizer application is critical for achieving high maize yield in typical yellow soil regions in Guizhou Province. P and N fertilizers are equally important for improving maize yield, and long-term application of unbalanced chemical fertilizer, especially the lack of P, would not benefit the sustainable use of nutrients in yellow soil.

NAD+ in Aging: Molecular Mechanisms and Translational Implications.

PubMed

Fang, Evandro F; Lautrup, Sofie; Hou, Yujun; Demarest, Tyler G; Croteau, Deborah L; Mattson, Mark P; Bohr, Vilhelm A

2017-10-01

The coenzyme NAD + is critical in cellular bioenergetics and adaptive stress responses. Its depletion has emerged as a fundamental feature of aging that may predispose to a wide range of chronic diseases. Maintenance of NAD + levels is important for cells with high energy demands and for proficient neuronal function. NAD + depletion is detected in major neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, cardiovascular disease and muscle atrophy. Emerging evidence suggests that NAD + decrements occur in various tissues during aging, and that physiological and pharmacological interventions bolstering cellular NAD + levels might retard aspects of aging and forestall some age-related diseases. Here, we discuss aspects of NAD + biosynthesis, together with putative mechanisms of NAD + action against aging, including recent preclinical and clinical trials. Published by Elsevier Ltd.

The dynamic regulation of NAD metabolism in mitochondria

PubMed Central

Stein, Liana Roberts; Imai, Shin-ichiro

2012-01-01

Mitochondria are intracellular powerhouses that produce ATP and carry out diverse functions for cellular energy metabolism. While the maintenance of an optimal NAD/NADH ratio is essential for mitochondrial function, it has recently become apparent that the maintenance of the mitochondrial NAD pool also has critical importance. The biosynthesis, transport, and catabolism of NAD and its key intermediates play an important role in the regulation of NAD-consuming mediators, such as sirtuins, poly-ADP-ribose polymerases, and CD38/157 ectoenzymes, in intra- and extracellular compartments. Mitochondrial NAD biosynthesis is also modulated in response to nutritional and environmental stimuli. In this article, we discuss this dynamic regulation of NAD metabolism in mitochondria to shed light on the intimate connection between NAD and mitochondrial function. PMID:22819213

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

Inhibition of cereal rust fungi by both class I and II defensins derived from the flowers of Nicotiana alata.

PubMed

Dracatos, Peter M; van der Weerden, Nicole L; Carroll, Kate T; Johnson, Elizabeth D; Plummer, Kim M; Anderson, Marilyn A

2014-01-01

Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50 ) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5-10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad-spectrum resistance to pathogens. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Very high expander processing of maize on animal performance, digestibility and product quality of finishing pigs and broilers.

PubMed

Puntigam, R; Schedle, K; Schwarz, C; Wanzenböck, E; Eipper, J; Lechner, E-M; Yin, L; Gierus, M

2018-07-01

The present study investigated the effect of hydrothermic maize processing and supplementation of amino acids (AA) in two experiments. In total, 60 barrows and 384 broilers were fed four diets including either unprocessed (T1), or hydrothermically processed maize, that is short- (T2), or long-term conditioned (LC) (T3), and subsequently expanded maize of the same batch. Assuming a higher metabolizable energy (ME) content after processing, the fourth diet (T4) contains maize processed as treatment T3, but AA were supplemented to maintain the ideal protein value. Performance, digestibility and product quality in both species were assessed. Results show that in pigs receiving T4 the average daily feed intake was lower compared with the other treatments, whereas no difference was observed in broilers. The T3 improved the feed conversion rate compared with T1 (P<0.10) for both species. In contrast, average daily gain (ADG) (1277 g/day for T2 and 1267 g/day for T3 v. 971 g/day for T1) was only altered in pigs. The hydrothermic maize processing increased the apparent total tract digestibility (ATTD) of dry matter, starch and ether extract after acid hydrolysis. This may be a consequence of higher ATTD of gross energy in the finishing phase for both animal species, suggesting a higher ME content in diets with processed maize. The higher ME content of diets with processed maize is supported also by measurements of product quality. Supplementation of AA in T4 enhanced the loin depth in pigs as well as the amount of breast meat in broilers. Further effects of processing maize on meat quality were the reduced yellowness and antioxidative capacity (P<0.10) for broilers, likely due to the heat damage of xanthophylls and tocopherols. Processing also increased springiness and chewiness (P<0.10) of the broilers breast meat, whereas the loin meat of pigs showed a decreased lightness and yellowness (P<0.10) in meat when hydrothermic processed maize was used (for T2, T3 and T4). LC processed maize (T3) showed the lowest springiness in pork, however the supplementation of AA in T4 did not show differences between the treatments. Shown results demonstrated positive effects of hydrothermic processing of maize on animal performance and digestibility in both species. However, effects on carcass characteristics and product quality differed. The negative effects on product quality could be partly compensated with the AA supplementation, whereas a change in meat colour and reduced antioxidative capacity was observed in all groups fed hydrothermic maize processing.

Morphological and Physiological Alteration of Maize Root Architectures on Drought Stress.

USDA-ARS?s Scientific Manuscript database

Drought tolerance is a complex agronomic trait and root characteristics logically play an important role in determining the response of plants to drought stress. Research experiments were conducted to investigate genotypic variations in morphological and physiological responses of roots to drought s...

Effect of Environmental Density and Buoyancy on Growth and Gravitropic Response in Maize Roots

NASA Astrophysics Data System (ADS)

Robbins, J. L.; Mulkey, T. J.

2008-06-01

The mechanism by which plants sense gravity is not fully understood. The hydrostatic model was proposed as an alternative to the statolith model. These experiments are designed to provide further understanding about the underlying mechanism of the gravitropic sensing. Primary roots of maize with a length of about 1 cm were used. The roots were placed in environments of various density and buoyancy using air, water, sucrose, sucrose/polyethylene glycol 4000 (PEG), PEG 8000, and Ficoll PM 400. The rates of growth and gravitropic curvature were monitored using time-lapse video and digital recordings. Comparison of roots in air to roots in oxygenated water indicate that there is no significant difference in growth rates but the higher density of water and the other test solutions significantly slows the gravitropic response. Altering the environmental density and buoyancy of the solution surrounding the root does not appear to alter sedimentation of statoliths within the root tip.

Mitochondrial NAD(P)H In vivo: Identifying Natural Indicators of Oxidative Phosphorylation in the (31)P Magnetic Resonance Spectrum.

PubMed

Conley, Kevin E; Ali, Amir S; Flores, Brandon; Jubrias, Sharon A; Shankland, Eric G

2016-01-01

Natural indicators provide intrinsic probes of metabolism, biogenesis and oxidative protection. Nicotinamide adenine dinucleotide metabolites (NAD(P)) are one class of indicators that have roles as co-factors in oxidative phosphorylation, glycolysis, and anti-oxidant protection, as well as signaling in the mitochondrial biogenesis pathway. These many roles are made possible by the distinct redox states (NAD(P)(+) and NAD(P)H), which are compartmentalized between cytosol and mitochondria. Here we provide evidence for detection of NAD(P)(+) and NAD(P)H in separate mitochondrial and cytosol pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy ((31)P MRS). These NAD(P) pools are identified by chemical standards (NAD(+), NADP(+), and NADH) and by physiological tests. A unique resonance reflecting mitochondrial NAD(P)H is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(P)H with oxidation is matched by a stoichiometric rise in the NAD(P)(+) peak. This unique resonance also provides a measure of the improvement in mitochondrial oxidation that parallels the greater phosphorylation found after exercise training in these elderly subjects. The implication is that the dynamics of the mitochondrial NAD(P)H peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus, non-invasive detection of NAD(P)(+) and NAD(P)H in cytosol vs. mitochondria yields natural indicators of redox compartmentalization and sensitive intrinsic probes of the improvement of mitochondrial function with an intervention in human tissues in vivo. These natural indicators hold the promise of providing mechanistic insight into metabolism and mitochondrial function in vivo in a range of tissues in health, disease and with treatment.

The NAD+/PARP1/SIRT1 Axis in Aging.

PubMed

Mendelsohn, Andrew R; Larrick, James W

2017-06-01

NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism*

PubMed Central

Frederick, David W.; Davis, James G.; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A.; Nakamaru-Ogiso, Eiko; Baur, Joseph A.

2015-01-01

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

PubMed

Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

2015-01-16

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

New strategy for the determination of gliadins in maize- or rice-based foods matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: fractionation of gliadins from maize or rice prolamins by acidic treatment.

PubMed

Hernando, Alberto; Valdes, Israel; Méndez, Enrique

2003-08-01

A procedure for determining small quantities of gliadins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) in gluten-free foods containing relatively large amounts of prolamin proteins from maize or rice is described. We report for the first time that gliadins, the ethanol-soluble wheat prolamin fraction, can be quantitatively solubilized in 1.0 M acetic acid, while the corresponding ethanol-soluble maize or rice prolamin fraction remains insoluble in acetic acid. We describe a methodology for the detection of gliadins in maize and rice foods based on a two-step procedure of extraction (60% aqueous ethanol followed by 1 M acetic acid). Subsequent MALDI-TOFMS analysis of the resulting acidic extract from these gluten-free foods clearly confirms the presence of a typical mass pattern corresponding to gliadin components, ranging from 30 to 45 kDa. Depending on the percentages of maize or rice flours employed in the elaboration of these foods, the combined procedure enables levels of gliadins from 100 to 400 ppm to be detected. The efficiency of this combined procedure corroborates enzyme-linked immunosorbent assay data for a large number of maize/rice gluten-free foods by means of direct visualization of the characteristic gliadin mass pattern in maize or rice foods. Copyright 2003 John Wiley & Sons, Ltd.

A robust and efficient method for the extraction of plant extracellular surface lipids as applied to the analysis of silks and seedling leaves of maize

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

Loneman, Derek M.; Peddicord, Layton; Al-Rashid, Amani

Aerial plant organs possess a diverse array of extracellular surface lipids, including both non-polar and amphipathic constituents that collectively provide a primary line of defense against environmental stressors. Extracellular surface lipids on the stigmatic silks of maize are composed primarily of saturated and unsaturated linear hydrocarbons, as well as fatty acids, and aldehydes. To efficiently extract lipids of differing polarities from maize silks, five solvent systems (hexanes; hexanes:diethyl ether (95:5); hexanes:diethyl ether (90:10); chloroform:hexanes (1:1) and chloroform) were tested by immersing fresh silks in solvent for different extraction times. Surface lipid recovery and the relative composition of individual constituents weremore » impacted to varying degrees depending on solvent choice and duration of extraction. Analyses were performed using both silks and leaves to demonstrate the utility of the solvent- and time-optimized protocol in comparison to extraction with the commonly used chloroform solvent. Overall, the preferred solvent system was identified as hexanes:diethyl ether (90:10), based on its effectiveness in extracting surface hydrocarbons and fatty acids as well as its reduced propensity to extract presumed internal fatty acids. Metabolite profiling of wildtype and glossy1 seedlings, which are impaired in surface lipid biosynthesis, demonstrated the ability of the preferred solvent to extract extracellular surface lipids rich in amphipathic compounds (aldehydes and alcohols). In addition to the expected deficiencies in dotriacontanal and dotriacontan-1-ol for gl1 seedlings, an unexpected increase in fatty acid recovery was observed in gl1 seedlings extracted in chloroform, suggesting that chloro-form extracts lipids from internal tissues of gl1 seedlings. This highlights the importance of extraction method when evaluating mutants that have altered cuticular lipid compositions. Lastly, metabolite profiling of silks from maize inbreds B73 and Mo17, exposed to different environments and harvested at different ages, revealed differences in hydrocarbon and fatty acid composition, demonstrating the dynamic nature of surface lipid accumulation on silks.« less

A robust and efficient method for the extraction of plant extracellular surface lipids as applied to the analysis of silks and seedling leaves of maize

DOE PAGES

Loneman, Derek M.; Peddicord, Layton; Al-Rashid, Amani; ...

2017-07-11

Aerial plant organs possess a diverse array of extracellular surface lipids, including both non-polar and amphipathic constituents that collectively provide a primary line of defense against environmental stressors. Extracellular surface lipids on the stigmatic silks of maize are composed primarily of saturated and unsaturated linear hydrocarbons, as well as fatty acids, and aldehydes. To efficiently extract lipids of differing polarities from maize silks, five solvent systems (hexanes; hexanes:diethyl ether (95:5); hexanes:diethyl ether (90:10); chloroform:hexanes (1:1) and chloroform) were tested by immersing fresh silks in solvent for different extraction times. Surface lipid recovery and the relative composition of individual constituents weremore » impacted to varying degrees depending on solvent choice and duration of extraction. Analyses were performed using both silks and leaves to demonstrate the utility of the solvent- and time-optimized protocol in comparison to extraction with the commonly used chloroform solvent. Overall, the preferred solvent system was identified as hexanes:diethyl ether (90:10), based on its effectiveness in extracting surface hydrocarbons and fatty acids as well as its reduced propensity to extract presumed internal fatty acids. Metabolite profiling of wildtype and glossy1 seedlings, which are impaired in surface lipid biosynthesis, demonstrated the ability of the preferred solvent to extract extracellular surface lipids rich in amphipathic compounds (aldehydes and alcohols). In addition to the expected deficiencies in dotriacontanal and dotriacontan-1-ol for gl1 seedlings, an unexpected increase in fatty acid recovery was observed in gl1 seedlings extracted in chloroform, suggesting that chloro-form extracts lipids from internal tissues of gl1 seedlings. This highlights the importance of extraction method when evaluating mutants that have altered cuticular lipid compositions. Lastly, metabolite profiling of silks from maize inbreds B73 and Mo17, exposed to different environments and harvested at different ages, revealed differences in hydrocarbon and fatty acid composition, demonstrating the dynamic nature of surface lipid accumulation on silks.« less

Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae.

PubMed

Kato, Michiko; Lin, Su-Ju

2014-11-01

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD(+) homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae

PubMed Central

Kato, Michiko; Lin, Su-Ju

2014-01-01

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD+ is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD+ homeostasis is essential for proper cellular function and aberrant NAD+ metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD+ metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD+ metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD+ metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD+ metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD+ metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD+-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD+ intermediates, and their potential roles in NAD+ homeostasis. To date, it remains unclear how NAD+ and NAD+ intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD+ homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability. PMID:25096760

Contribution of NAD 2D-NMR in liquid crystals to the determination of hydrogen isotope profile of methyl groups in miliacin

NASA Astrophysics Data System (ADS)

Berdagué, Philippe; Lesot, Philippe; Jacob, Jérémy; Terwilliger, Valery J.; Le Milbeau, Claude

2016-01-01

The hydrogen isotopic composition (δD or (D/H) value) of molecular biomarkers preserved in sedimentary archives is increasingly used to provide clues about the evolution of past climatic conditions. The rationale is that intact biomarkers retain isotopic information related to the climatic conditions that prevailed at the time of their synthesis. Some of these biomarkers may be degraded during diagenesis, however. The extent to which these degradations alter the original δD value of the source biomarker is presently debated and the capacity to resolve this question by determination of compound-specific δD values alone is limited. The ;bulk; or ;global; δD value of any molecule is in fact a composite of δD values at each site within this molecule (δDi or (D/H)i with i = number of hydrogen/deuterium atoms in the considered molecule). Determination of this site-specific δDi value in biomarkers could not only yield outstanding paleoenvironmental information but also help forecast the impacts of diagenesis and define essential steps in biosynthetic pathways. This task is analytically challenging. Here, we examined the capabilities of natural abundance deuterium 2D-NMR (NAD 2D-NMR) using homopolypeptide liquid crystals as an NMR solvent to: (i) analyze the NAD spectra of biomakers; (ii) determine the site-specific distribution of hydrogen in the nine methyl groups (δDMei with i = 23-31) of miliacin, a pentacyclic triterpene of the amyrin family and key biomarker for broomcorn millet in sedimentary archives. Relative (D/H)Mei values were established by anisotropic NAD 2D-NMR. Then absolute δDMei values were obtained by determining δDMei value of the methoxy group of miliacin using two independent approaches: isotropic NAD NMR (SNIF-NMR™) and GC-irMS. The resulting isotope profile for miliacin shows, for the first time, large variations in δDMei values that can directly be explained by biosynthetic processes. This approach has also the potential to permit predicting fractionations during pentacyclic triterpene diagenesis.

High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules

PubMed Central

Baker, Bo Y; Shi, Wuxian; Wang, Benlian; Palczewski, Krzysztof

2014-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4, and bGAPDH(NAD)3. The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54–56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies. PMID:25176140

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.

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

Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

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

Thoden, James B.; Holden, Hazel M.

2010-09-08

2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to usemore » {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.« less

Highly Stable l-Lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression

PubMed Central

Heydari, Mojgan; Ohshima, Toshihisa; Nunoura-Kominato, Naoki; Sakuraba, Haruhiko

2004-01-01

l-Lysine dehydrogenase, which catalyzes the oxidative deamination of l-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ1-piperideine-6-carboxylate, indicating that the enzyme is l-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM l-lysine. The enzyme was relatively selective for l-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for l-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da. PMID:14766574

Highly stable L-lysine 6-dehydrogenase from the thermophile Geobacillus stearothermophilus isolated from a Japanese hot spring: characterization, gene cloning and sequencing, and expression.

PubMed

Heydari, Mojgan; Ohshima, Toshihisa; Nunoura-Kominato, Naoki; Sakuraba, Haruhiko

2004-02-01

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.

Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol.

PubMed

Damgaard, S E

1981-09-29

The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways

PubMed Central

Ma, Menggen; Song, Mingzhou

2010-01-01

Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)+ for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress. PMID:19517136

SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury-induced SARM1 activation.

PubMed

Summers, Daniel W; Gibson, Daniel A; DiAntonio, Aaron; Milbrandt, Jeffrey

2016-10-11

Axon injury in response to trauma or disease stimulates a self-destruction program that promotes the localized clearance of damaged axon segments. Sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is an evolutionarily conserved executioner of this degeneration cascade, also known as Wallerian degeneration; however, the mechanism of SARM1-dependent neuronal destruction is still obscure. SARM1 possesses a TIR domain that is necessary for SARM1 activity. In other proteins, dimerized TIR domains serve as scaffolds for innate immune signaling. In contrast, dimerization of the SARM1 TIR domain promotes consumption of the essential metabolite NAD + and induces neuronal destruction. This activity is unique to the SARM1 TIR domain, yet the structural elements that enable this activity are unknown. In this study, we identify fundamental properties of the SARM1 TIR domain that promote NAD + loss and axon degeneration. Dimerization of the TIR domain from the Caenorhabditis elegans SARM1 ortholog TIR-1 leads to NAD + loss and neuronal death, indicating these activities are an evolutionarily conserved feature of SARM1 function. Detailed analysis of sequence homology identifies canonical TIR motifs as well as a SARM1-specific (SS) loop that are required for NAD + loss and axon degeneration. Furthermore, we identify a residue in the SARM1 BB loop that is dispensable for TIR activity yet required for injury-induced activation of full-length SARM1, suggesting that SARM1 function requires multidomain interactions. Indeed, we identify a physical interaction between the autoinhibitory N terminus and the TIR domain of SARM1, revealing a previously unrecognized direct connection between these domains that we propose mediates autoinhibition and activation upon injury.

Redox imbalance and mitochondrial abnormalities in the diabetic lung.

PubMed

Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

2017-04-01

Although the lung is one of the least studied organs in diabetes, increasing evidence indicates that it is an inevitable target of diabetic complications. Nevertheless, the underlying biochemical mechanisms of lung injury in diabetes remain largely unexplored. Given that redox imbalance, oxidative stress, and mitochondrial dysfunction have been implicated in diabetic tissue injury, we set out to investigate mechanisms of lung injury in diabetes. The objective of this study was to evaluate NADH/NAD + redox status, oxidative stress, and mitochondrial abnormalities in the diabetic lung. Using STZ induced diabetes in rat as a model, we measured redox-imbalance related parameters including aldose reductase activity, level of poly ADP ribose polymerase (PAPR-1), NAD + content, NADPH content, reduced form of glutathione (GSH), and glucose 6-phophate dehydrogenase (G6PD) activity. For assessment of mitochondrial abnormalities in the diabetic lung, we measured the activities of mitochondrial electron transport chain complexes I to IV and complex V as well as dihydrolipoamide dehydrogenase (DLDH) content and activity. We also measured the protein content of NAD + dependent enzymes such as sirtuin3 (sirt3) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Our results demonstrate that NADH/NAD + redox imbalance occurs in the diabetic lung. This redox imbalance upregulates the activities of complexes I to IV, but not complex V; and this upregulation is likely the source of increased mitochondrial ROS production, oxidative stress, and cell death in the diabetic lung. These results, together with the findings that the protein contents of DLDH, sirt3, and NQO1 all are decreased in the diabetic lung, demonstrate that redox imbalance, mitochondrial abnormality, and oxidative stress contribute to lung injury in diabetes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

NAD+ salvage pathway in cancer metabolism and therapy.

PubMed

Kennedy, Barry E; Sharif, Tanveer; Martell, Emma; Dai, Cathleen; Kim, Youra; Lee, Patrick W K; Gujar, Shashi A

2016-12-01

Nicotinamide adenine dinucleotide (NAD + ) is an essential coenzyme for various physiological processes including energy metabolism, DNA repair, cell growth, and cell death. Many of these pathways are typically dysregulated in cancer cells, making NAD + an intriguing target for cancer therapeutics. NAD + is mainly synthesized by the NAD + salvage pathway in cancer cells, and not surprisingly, the pharmacological targeting of the NAD + salvage pathway causes cancer cell cytotoxicity in vitro and in vivo. Several studies have described the precise consequences of NAD + depletion on cancer biology, and have demonstrated that NAD+ depletion results in depletion of energy levels through lowered rates of glycolysis, reduced citric acid cycle activity, and decreased oxidative phosphorylation. Additionally, depletion of NAD + causes sensitization of cancer cells to oxidative damage by disruption of the anti-oxidant defense system, decreased cell proliferation, and initiation of cell death through manipulation of cell signaling pathways (e.g., SIRT1 and p53). Recently, studies have explored the effect of well-known cancer therapeutics in combination with pharmacological depletion of NAD + levels, and found in many cases a synergistic effect on cancer cell cytotoxicity. In this context, we will discuss the effects of NAD + salvage pathway inhibition on cancer cell biology and provide insight on this pathway as a novel anti-cancer therapeutic target. Copyright © 2016 Elsevier Ltd. All rights reserved.

A [32P]-NAD+-based method to identify and quantitate long residence time enoyl-ACP reductase inhibitors

PubMed Central

Yu, Weixuan; Neckles, Carla; Chang, Andrew; Bommineni, Gopal Reddy; Spagnuolo, Lauren; Zhang, Zhuo; Liu, Nina; Lai, Christina; Truglio, James; Tonge, Peter J.

2015-01-01

The classical methods for quantifying drug-target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-ACP reductase as a model system, we developed a Penefsky column-based method for direct measurement of tR, where the off-rate of the drug was determined with radiolabeled [adenylate-32P] NAD(P+) cofactor. Twenty-three FabI inhibitors were analyzed and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percent drug-target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady state kinetic methods for compounds with tR values of 10-100 min. In addition, we were able to identify seven long tR inhibitors (100-1500 min) and to accurately determine their tR values. The method was then used to measure tR as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in tR was observed when the temperature was increased from 25 °C to 37 °C . PMID:25684450

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

Inhibition of Inflammasome-Dependent Interleukin 1β Production by Streptococcal NAD+-Glycohydrolase: Evidence for Extracellular Activity

PubMed Central

Hancz, Dóra; Westerlund, Elsa; Bastiat-Sempe, Benedicte; Sharma, Onkar; Valfridsson, Christine; Meyer, Lena; Love, John F.; O’Seaghdha, Maghnus; Wessels, Michael R.

2017-01-01

ABSTRACT Group A Streptococcus (GAS) is a common human pathogen and the etiologic agent of a large number of diseases ranging from mild, self-limiting infections to invasive life-threatening conditions. Two prominent virulence factors of this bacterium are the genetically and functionally linked pore-forming toxin streptolysin O (SLO) and its cotoxin NAD+-glycohydrolase (NADase). Overexpression of these toxins has been linked to increased bacterial virulence and is correlated with invasive GAS disease. NADase can be translocated into host cells by a SLO-dependent mechanism, and cytosolic NADase has been assigned multiple properties such as protection of intracellularly located GAS bacteria and induction of host cell death through energy depletion. Here, we used a set of isogenic GAS mutants and a macrophage infection model and report that streptococcal NADase inhibits the innate immune response by decreasing inflammasome-dependent interleukin 1β (IL-1β) release from infected macrophages. Regulation of IL-1β was independent of phagocytosis and ensued also under conditions not allowing SLO-dependent translocation of NADase into the host cell cytosol. Thus, our data indicate that NADase not only acts intracellularly but also has an immune regulatory function in the extracellular niche. PMID:28720729

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria

PubMed Central

Sultan, Laure D.; Grewe, Felix; Rolle, Katarzyna; Abudraham, Sivan; Shevtsov, Sofia; Klipcan, Liron; Barciszewski, Jan; Dietrich, André

2016-01-01

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory. PMID:27760804

Cloning, expression, and biochemical characterization of a novel NADP+-dependent 7α-hydroxysteroid dehydrogenase from Clostridium difficile and its application for the oxidation of bile acids.

PubMed

Bakonyi, Daniel; Hummel, Werner

2017-04-01

A gene encoding a novel 7α-specific NADP + -dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP + using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type. Copyright © 2016. Published by Elsevier Inc.

Altered expression of polyketide biosynthetic gene clusters in fumonisin-deficient mutants of Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a pathogen of maize and produces fumonisins, a group of polyketide derived secondary metabolites. Fumonisins cause diseases in animals, and they have been correlated epidemiologically with esophageal cancer and birth defects in humans. Fumonisin biosynthetic genes are c...

Gene evolutionary trajectories and GC patterns driven by recombination in Zea mays

USDA-ARS?s Scientific Manuscript database

Recombination occurring during meiosis is critical for creating genetic variation and plays an essential role in plant evolution. In addition to creating novel gene combinations, recombination can affect genome structure through altering GC patterns. In maize (Zea mays) and other grasses, another in...

Morphological and Biological alteration of maize root architectures on drought stress

USDA-ARS?s Scientific Manuscript database

Drought tolerance is a complex agronomic trait and root characteristics logically play an important role in determining the response of plants to drought stress. Studies were conducted to investigate genotypic variations in morphological and physiological responses of roots to drought stress in corn...

Morphological and biological alteration of maize root architectures on drought stress

USDA-ARS?s Scientific Manuscript database

Drought tolerance is a complex agronomic trait and root characteristics logically play an important role in determining the response of plants to drought stress. Studies were conducted to investigate genotypic variations in morphological and physiological responses of roots to drought stress in corn...

Plant exudates may stabilize or weaken soil depending on species, origin and time

PubMed Central

Naveed, M.; Brown, L. K.; Raffan, A. C.; George, T. S.; Bengough, A. G.; Roose, T.; Sinclair, I.; Koebernick, N.; Cooper, L.; Hackett, C. A.

2017-01-01

Summary We hypothesized that plant exudates could either gel or disperse soil depending on their chemical characteristics. Barley (Hordeum vulgare L. cv. Optic) and maize (Zea mays L. cv. Freya) root exudates were collected using an aerated hydroponic method and compared with chia (Salvia hispanica L.) seed exudate, a commonly used root exudate analogue. Sandy loam soil was passed through a 500‐μm mesh and treated with each exudate at a concentration of 4.6 mg exudate g−1 dry soil. Two sets of soil samples were prepared. One set of treated soil samples was maintained at 4°C to suppress microbial processes. To characterize the effect of decomposition, the second set of samples was incubated at 16°C for 2 weeks at −30 kPa matric potential. Gas chromatography–mass spectrometry (GC–MS) analysis of the exudates showed that barley had the largest organic acid content and chia the largest content of sugars (polysaccharide‐derived or free), and maize was in between barley and chia. Yield stress of amended soil samples was measured by an oscillatory strain sweep test with a cone plate rheometer. When microbial decomposition was suppressed at 4°C, yield stress increased 20‐fold for chia seed exudate and twofold for maize root exudate compared with the control, whereas for barley root exudate decreased to half. The yield stress after 2 weeks of incubation compared with soil with suppressed microbial decomposition increased by 85% for barley root exudate, but for chia and maize it decreased by 87 and 54%, respectively. Barley root exudation might therefore disperse soil and this could facilitate nutrient release. The maize root and chia seed exudates gelled soil, which could create a more stable soil structure around roots or seeds. Highlights Rheological measurements quantified physical behaviour of plant exudates and effect on soil stabilization.Barley root exudates dispersed soil, which could release nutrients and carbon.Maize root and chia seed exudates had a stabilizing effect on soil.Physical engineering of soil in contact with plant roots depends on the nature and origin of exudates. PMID:29263712

33 CFR 110.1 - General.

Code of Federal Regulations, 2010 CFR

2010-07-01

... North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83...

7 CFR 11.21 - Organization.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Organization. NAD was established on October 13, 1994. Delegation of authority to the Director, NAD, appears at..., Colorado; and the headquarters staff located in Alexandria, Virginia. NAD is headed by a Director. NAD is...

33 CFR 165.8 - Geographic coordinates.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83 only after application...

Reagentless D-sorbitol biosensor based on D-sorbitol dehydrogenase immobilized in a sol-gel carbon nanotubes-poly(methylene green) composite.

PubMed

Wang, Zhijie; Etienne, Mathieu; Urbanova, Veronika; Kohring, Gert-Wieland; Walcarius, Alain

2013-04-01

A reagentless D-sorbitol biosensor based on NAD-dependent D-sorbitol dehydrogenase (DSDH) immobilized in a sol-gel carbon nanotubes-poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD(+) cofactor with DSDH in a sol-gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of D-sorbitol at 0.2 V with a sensitivity of 8.7 μA mmol(-1) L cm(-2) and a detection limit of 0.11 mmol L(-1). Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions.

Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

PubMed

Inoue, Hiroyuki; Hashimoto, Seitaro; Matsushika, Akinori; Watanabe, Seiya; Sawayama, Shigeki

2014-12-01

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD(+)-dependent XDH or engineered NADP(+)-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD(+)-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.

Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

PubMed

Beauchamp, Justin; Vieille, Claire

2015-01-01

N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

33 CFR 67.50-1 - Scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

... North American Datum of 1983 (NAD 83), unless such geographic coordinates are expressly labeled NAD 83. Geographic coordinates without the NAD 83 reference may be plotted on maps or charts referenced to NAD 83...

Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

PubMed Central

Al-Shabany, Abbas Jawad; Moody, Alan John; Foey, Andrew David; Billington, Richard Andrew

2016-01-01

Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD+ has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD+ homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD+ levels and expression levels of NAD+ homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD+ levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD+ synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD+ homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD+ levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD+. The agonist-induced rise in NAD+ shows striking parallels to well-known second messengers and raises the possibility that NAD+ is acting in a similar manner in this model. PMID:26764408

SIRT1 is a Highly Networked Protein That Mediates the Adaptation to Chronic Physiological Stress

PubMed Central

Clark-Knowles, Katherine V.; Caron, Annabelle Z.; Gray, Douglas A.

2013-01-01

SIRT1 is a NAD+-dependent protein deacetylase that has a very large number of established protein substrates and an equally impressive list of biological functions thought to be regulated by its activity. Perhaps as notable is the remarkable number of points of conflict concerning the role of SIRT1 in biological processes. For example, evidence exists suggesting that SIRT1 is a tumor suppressor, is an oncogene, or has no effect on oncogenesis. Similarly, SIRT1 is variably reported to induce, inhibit, or have no effect on autophagy. We believe that the resolution of many conflicting results is possible by considering recent reports indicating that SIRT1 is an important hub interacting with a complex network of proteins that collectively regulate a wide variety of biological processes including cancer and autophagy. A number of the interacting proteins are themselves hubs that, like SIRT1, utilize intrinsically disordered regions for their promiscuous interactions. Many studies investigating SIRT1 function have been carried out on cell lines carrying undetermined numbers of alterations to the proteins comprising the SIRT1 network or on inbred mouse strains carrying fixed mutations affecting some of these proteins. Thus, the effects of modulating SIRT1 amount and/or activity are importantly determined by the genetic background of the cell (or the inbred strain of mice), and the effects attributed to SIRT1 are synthetic with the background of mutations and epigenetic differences between cells and organisms. Work on mice carrying alterations to the Sirt1 gene suggests that the network in which SIRT1 functions plays an important role in mediating physiological adaptation to various sources of chronic stress such as calorie restriction and calorie overload. Whether the catalytic activity of SIRT1 and the nuclear concentration of the co-factor, NAD+, are responsible for modulating this activity remains to be determined. However, the effect of modulating SIRT1 activity must be interpreted in the context of the cell or tissue under investigation. Indeed, for SIRT1, we argue that context is everything. PMID:24020004