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Sample records for nadp

  1. NADP - dependent isocritrate dehydrogenase from human kidney ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... properties of IDH1 purified from the mitochondria that was isolated from a ..... AMP could not activated NADP-IDH enzyme, even when some ... Influence of compartmental localization on the function of yeast NADP+-specific.

  2. Eucalypt NADP-Dependent Isocitrate Dehydrogenase1

    Science.gov (United States)

    Boiffin, Vincent; Hodges, Michael; Gálvez, Susana; Balestrini, Raffaella; Bonfante, Paola; Gadal, Pierre; Martin, Francis

    1998-01-01

    NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed. PMID:9662536

  3. Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP(+) oxidoreductase with NADP(+)/NADPH and ferredoxin.

    Science.gov (United States)

    Seo, Daisuke; Soeta, Takahiro; Sakurai, Hidehiro; Sétif, Pierre; Sakurai, Takeshi

    2016-06-01

    Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained as the major species at equilibrium even with about 50-fold molar excess NADP(+). In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~500 s(-1)) was much higher than the reverse one (reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

    Directory of Open Access Journals (Sweden)

    VERONIKA DOUBNEROVÁ

    2009-08-01

    Full Text Available The non-photosynthetic NADP-malic enzyme EC 1.1.1.40 (NADP-ME, which catalyzes the oxidative decarboxylation of L-malate and NADP+ to produce pyruvate and NADPH, respectively, and which could be involved in plant defense responses, was isolated from Nicotiana tabacum L. leaves. The mechanism of the enzyme reaction was studied by the initial rate method and was found to be an ordered sequential one. Regulation possibilities of purified cytosolic NADP-ME by cell metabolites were tested. Intermediates of the citric acid cycle (a-ketoglutarate, succinate, fumarate, metabolites of glycolysis (pyruvate, phosphoenolpyruvate, glucose-6-phosphate, compounds connected with lipogenesis (coenzyme A, acetyl-CoA, palmitoyl-CoA and some amino acids (glutamate, glutamine, aspartate did not significantly affect the NADP-ME activity from tobacco leaves. In contrast, macroergic compounds (GTP, ATP and ADP were strong inhibitors of NADP-ME; the type of inhibition and the inhibition constants were determined in the presence of the most effective cofactors (Mn2+ or Mg2+, required by NADP-ME. Predominantly non-competitive type of inhibitions of NADP-ME with respect to NADP+ and mixed type to L-malate were found.

  5. Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

    Science.gov (United States)

    Huppe, H. C.; Picaud, A.; Buchanan, B. B.; Miginiac-Maslow, M.

    1991-01-01

    The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

  6. NADP+ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

    International Nuclear Information System (INIS)

    Kawai, Y.; Whitsel, C.; Arinze, I.J.

    1986-01-01

    Cholera or pertussis toxin-catalyzed [ 32 P]ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD + , by endogenous enzymes such as NAD + -glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed [ 32 P]ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP + . The effect is concentration dependent; with 20 μM [ 32 P]NAD + as substrate maximal enhancement is obtained at 0.5-1.0 mM NADP + . The enhancement of [ 32 P]ADP-ribosylation by NADP + was much greater than that by other known effectors such as Mg 2+ , phosphate or isoniazid. The effect of NADP + on ADP-ribosylation may occur by inhibition of the degradation of NAD + probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP + , isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl 2 ) to suppress NADase activity, NADP + was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP + in the assay is necessary to obtain maximal ADP-ribosylation

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

    Science.gov (United States)

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

    2018-01-20

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

  8. Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney

    OpenAIRE

    Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V.; Park, Kwon Moo

    2008-01-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expressio...

  9. Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kil, In Sup; Huh, Tae Lin; Lee, Young Sup; Lee, You Mie; Park, Jeen-Woo

    2006-01-01

    The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.

  10. Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

    Science.gov (United States)

    Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

    2009-04-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

  11. Modulation of NADP(+)-dependent isocitrate dehydrogenase in aging.

    Science.gov (United States)

    Kil, In Sup; Lee, Young Sup; Bae, Young Seuk; Huh, Tae Lin; Park, Jeen-Woo

    2004-01-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46-48 population doubling level (PDL) and then gradually decreased at later PDL. 2',7'-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.

  12. A STD-NMR Study of the Interaction of the Anabaena Ferredoxin-NADP+ Reductase with the Coenzyme

    Directory of Open Access Journals (Sweden)

    Lara V. Antonini

    2014-01-01

    Full Text Available Ferredoxin-NADP+ reductase (FNR catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.

  13. What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

    Science.gov (United States)

    Wang, Xiao-Tao; Chan, Ting Fai; Lam, Veronica M S; Engel, Paul C

    2008-08-01

    Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP+ and no NADPH. This tightly bound NADP+ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP+ but, surprisingly, still fully active. Mr of the main component of "stripped" enzyme by gel filtration was approximately 100,000, suggesting a dimeric apoenzyme (subunit Mr = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate Mr of 150 kDa. Fluorescence titration of the stripped enzyme gave the K d for structural NADP+ as 37 nM, 200-fold lower than for "catalytic" NADP+. Structural NADP+ quenches 91% of protein fluorescence. At 37 degrees C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4 degrees C was partially reversed at room temperature, especially with added NADP+. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP+. Apparently, the true role of the second, tightly bound NADP+ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The Kd values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP+ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of Kd constant for structural NADP+ will now allow quantitative assessment of this defect in clinical G6PD mutations.

  14. UV-B induction of NADP-malic enzyme in etiolated and green maize seedlings

    International Nuclear Information System (INIS)

    Drincovich, M.F.; Casati, P.; Andreo, C.S.; Donahue, R.; Edwards, G.E.

    1998-01-01

    The effect of treatment of etiolated maize seedlings with UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400–700 nm), on the activity and quantity of NADP-malic enzyme (NADPME) and on RNA levels was determined. Under low levels of PAR (14 µmol m –2 s –1 ), exposure to UV-B radiation (9 µmol m –2 s –1 ) but not UV-A radiation (11 µmol m –2 s –1 ) for 6–24 h caused a marked increase in the activity of the enzyme similar to that observed under high PAR (300 µmol m –2 s –1 ) in the absence of UV-B. Western blot analysis indicated there was a specific increase of the photosynthetically active isoform of the enzyme. This increase was also measured at the RNA level by dot blot analysis, indicating that the induction is displayed at the level of NADP-ME transcription. UV-B treatment of green leaves after a 12 h dark period also caused an increase in the activity and level of NADP-ME. The UV-B induction of NADP-ME synthesis may reflect a mechanism for induction of photosynthetic processes in C4 photosynthesis. Alternatively, the relatively low intensity of UV-B radiation present under full sunlight might provide a signal that facilitates repair of UV-B-induced damage through the increased activity of different enzymes such as NADP-ME. It is speculated that the reducing power and pyruvate generated by activity of NADP-ME may be used for respiration in cellular repair processes and as substrates for the fatty acid synthesis required for membrane repair. (author)

  15. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

    1988-08-25

    The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by (14C)iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two (14C)carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated (14C)carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.

  16. Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

    International Nuclear Information System (INIS)

    Decottignies, P.; Schmitter, J.M.; Miginiac-Maslow, M.; Le Marechal, P.; Jacquot, J.P.; Gadal, P.

    1988-01-01

    The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15

  17. U.S. Forest Service Region 1 Lake Chemistry, NADP, and IMPROVE air quality data analysis

    Science.gov (United States)

    Jill Grenon; Mark Story

    2009-01-01

    This report was developed to address the need for comprehensive analysis of U.S. Forest Service (USFS) Region 1 air quality monitoring data. The monitoring data includes Phase 3 (long-term data) lakes, National Atmospheric Deposition Program (NADP), and Interagency Monitoring of Protected Visual Environments (IMPROVE). Annual and seasonal data for the periods of record...

  18. The regulation and catalytic mechanism of the NADP-malic enzyme from tobacco leaves

    Czech Academy of Sciences Publication Activity Database

    Doubnerová, V.; Potůčková, L.; Müller, Karel; Ryšlavá, H.

    2009-01-01

    Roč. 14, 8-9 (2009), s. 893-906 ISSN 0352-5139 Institutional research plan: CEZ:AV0Z50380511 Keywords : NADP-malic enzyme * macroergic compounds * Nicotiana tabacum L. Subject RIV: ED - Physiology Impact factor: 0.820, year: 2009

  19. The activity and isoforms of NADP-malic enzyme in Nicotiana benthamiana plants under biotic stress

    Czech Academy of Sciences Publication Activity Database

    Doubnerová, V.; Jirásková, A.; Janošková, M.; Müller, Karel; Baťková, Petra; Synková, Helena; Čeřovská, Noemi; Ryšlavá, H.

    2007-01-01

    Roč. 26, č. 4 (2007), s. 281-289 ISSN 0231-5882 Institutional research plan: CEZ:AV0Z50380511 Keywords : NADP * malic enzyme isoforms * Nicotiana benthamiana Subject RIV: EF - Botanics Impact factor: 1.286, year: 2007 http://www.gpb.sav.sk/2007-4.htm

  20. Concentration-dependent oligomerization of cross-linked complexes between ferredoxin and ferredoxin–NADP+ reductase

    International Nuclear Information System (INIS)

    Kimata-Ariga, Yoko; Kubota-Kawai, Hisako; Lee, Young-Ho; Muraki, Norifumi; Ikegami, Takahisa; Kurisu, Genji; Hase, Toshiharu

    2013-01-01

    Highlights: •Cross-linked complexes of ferredoxin (Fd) and Fd–NADP + reductase form oligomers. •In the crystal structures, Fd- and FNR moieties swap across the molecules. •The complexes exhibit concentration-dependent oligomerization at sub-milimolar order. -- Abstract: Ferredoxin–NADP + reductase (FNR) forms a 1:1 complex with ferredoxin (Fd), and catalyzes the electron transfer between Fd and NADP + . In our previous study, we prepared a series of site-specifically cross-linked complexes of Fd and FNR, which showed diverse electron transfer properties. Here, we show that X-ray crystal structures of the two different Fd–FNR cross-linked complexes form oligomers by swapping Fd and FNR moieties across the molecules; one complex is a dimer from, and the other is a successive multimeric form. In order to verify whether these oligomeric structures are formed only in crystal, we investigated the possibility of the oligomerization of these complexes in solution. The mean values of the particle size of these cross-linked complexes were shown to increase with the rise of protein concentration at sub-milimolar order, whereas the size of dissociable wild-type Fd:FNR complex was unchanged as analyzed by dynamic light scattering measurement. The oligomerization products were detected by SDS–PAGE after chemical cross-linking of these complexes at the sub-milimolar concentrations. The extent and concentration-dependent profile of the oligomerizaion were differentiated between the two cross-linked complexes. These results show that these Fd–FNR cross-linked complexes exhibit concentration-dependent oligomerization, possibly through swapping of Fd and FNR moieties also in solution. These findings lead to the possibility that some native multi-domain proteins may present similar phenomenon in vivo

  1. Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus.

    Science.gov (United States)

    Nguyen, Diep M N; Schut, Gerrit J; Zadvornyy, Oleg A; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L; Adams, Leslie A; Dinsmore, Jessica T; Nixon, William J; Boyd, Eric S; Bothner, Brian; Peters, John W; Adams, Michael W W

    2017-09-01

    Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP + oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Cellular defense against UVB-induced phototoxicity by cytosolic NADP+-dependent isocitrate dehydrogenase

    International Nuclear Information System (INIS)

    Jo, Seung-Hee; Lee, So-Hyun; Suk Chun, Hang; Min Lee, Su; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

    2002-01-01

    Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury

  3. Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

    2002-03-29

    Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

  4. Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kim, Sun Yee; Park, Jeen-Woo

    2003-03-01

    Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

  5. NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

    Directory of Open Access Journals (Sweden)

    Ekaterina Yu. Bezsudnova

    2016-01-01

    Full Text Available We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution, three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å, and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å. The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

  6. Engineering an NADPH/NADPRedox Biosensor in Yeast

    DEFF Research Database (Denmark)

    Zhang, Jie; Sonnenschein, Nikolaus; Pihl, Thomas Peter Boye

    2016-01-01

    Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biote......Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science...... in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP+ ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon...... NADPH deficiency by activation of NADPH regeneration. Finally, we couple the biosensor with an expression of dosage-sensitive genes (DSGs) and thereby create a novel tunable sensor-selector useful for synthetic selection of cells with higher NADPH/NADP+ ratios from mixed cell populations. We show...

  7. The differences between NAD-ME and NADP-ME subtypes of C4 photosynthesis: more than decarboxylating enzymes

    Directory of Open Access Journals (Sweden)

    Xiaolan Rao

    2016-10-01

    Full Text Available 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. 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.

  8. Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase

    NARCIS (Netherlands)

    Orru, Roberto; Torres Pazmino, Daniel; Fraaije, Marco W.; Mattevi, Andrea

    2010-01-01

    The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H)

  9. Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells.

    Science.gov (United States)

    Yang, Eun Sun; Park, Jeen-Woo

    2011-05-01

    1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.

  10. Decrease in the cytosolic NADP+-dependent isocitrate dehydrogenase activity through porcine sperm capacitation.

    Science.gov (United States)

    Katoh, Yuki; Tamba, Michiko; Matsuda, Manabu; Kikuchi, Kazuhiro; Okamura, Naomichi

    2018-02-26

    In order to understand the molecular mechanisms involved in the sperm capacitation, we have identified the proteins tyrosine-phosphorylated during the capacitation especially in conjunction with the regulation of the levels of reactive oxygen species (ROS) in sperm. In the present study, the effects of the tyrosine phosphorylation of cytosolic NADP + -dependent isocitrate dehydrogenase (IDPc) on its catalytic activity and on the levels of ROS in sperm have been studied. The tyrosine phosphorylated IDPc showed a significantly lowered enzymatic activity. The immunocytochemical analyses using the highly specific antisera against IDPc revealed that IDPc was mainly localized to the principal piece of the porcine sperm flagellum. As IDPc is one of the major NADPH regenerating enzymes in porcine sperm, it is strongly suggested that the decrease in IDPc activity is involved in the increased levels of ROS, which results in the induction of hyperactivated flagellar movement and capacitation. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Regulation of singlet oxygen-induced apoptosis by cytosolic NADP+-dependent isocitrate dehydrogenase.

    Science.gov (United States)

    Kim, Sun Yee; Lee, Su Min; Tak, Jean Kyoung; Choi, Kyeong Sook; Kwon, Taeg Kyu; Park, Jeen-Woo

    2007-08-01

    Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. Recently, we demonstrated that the control of redox balance and the cellular defense against oxidative damage are the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) through supplying NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPc activity in HL-60 cells regulates singlet oxygen-induced apoptosis. When we examined the protective role of IDPc against singlet oxygen-induced apoptosis with HL-60 cells transfected with the cDNA for mouse IDPc in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPc plays an important protective role in apoptosis of HL-60 cells induced by singlet oxygen.

  12. Isolation of a cotton NADP(H oxidase homologue induced by drought stress

    Directory of Open Access Journals (Sweden)

    NEPOMUCENO ALEXANDRE LIMA

    2000-01-01

    Full Text Available The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L. genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.

  13. Overexpression of the NADP+-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L.

    Science.gov (United States)

    Kobayashi, Keiichi; Hattori, Takasumi; Hayashi, Rie; Kirimura, Kohtaro

    2014-01-01

    In the tricarboxylic acid (TCA) cycle, NADP(+)-specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form α-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

  14. Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism.

    Science.gov (United States)

    Koh, Ho-Jin; Lee, Su-Min; Son, Byung-Gap; Lee, Soh-Hyun; Ryoo, Zae Young; Chang, Kyu-Tae; Park, Jeen-Woo; Park, Dong-Chan; Song, Byoung J; Veech, Richard L; Song, Hebok; Huh, Tae-Lin

    2004-09-17

    NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.

  15. Role of cytosolic NADP+-dependent isocitrate dehydrogenase in ischemia-reperfusion injury in mouse kidney.

    Science.gov (United States)

    Kim, Jinu; Kim, Ki Young; Jang, Hee-Seong; Yoshida, Takumi; Tsuchiya, Ken; Nitta, Kosaku; Park, Jeen-Woo; Bonventre, Joseph V; Park, Kwon Moo

    2009-03-01

    Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) synthesizes reduced NADP (NADPH), which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia-reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla, and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1 and S2 segments of proximal tubules was higher than in the S3 segment, which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondrion-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production and increased the ratio of oxidized glutathione to total glutathione [GSSG/(GSH+GSSG)], resulting in kidney dysfunction, tubular cell damage, and lipid peroxidation. In LLC-PK(1) cells, upregulation of IDPc by IDPc gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc downregulation by small interference RNA treatment presented results contrasting with the upregulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is downregulated with I/R.

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

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN (United Kingdom)

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

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

    International Nuclear Information System (INIS)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-01-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2 1 ; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3 2 . The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP

  18. Effect of X-rays and u.v.-light on the levels of NAD(P), NAD(P)H and hydroxyproline in Pinus silvestris pollen

    International Nuclear Information System (INIS)

    Zelles, L.

    1978-01-01

    Pollen grains of Pinus Silvestris were irradiated with stimulating and inhibiting doses of X-rays and u.v.-light and the levels of NAD(P), NAD(P)H and hydroxyproline determined during tube growth. Pollen grains irradiated with stimulating doses of X-rays and u.v.-light developed longer tubes, while grains irradiated with inhibiting doses of X-rays and u.v.-light developed shorter tubes than the unirradiated controls. After 32 hr of incubation, the levels of NAD(P), NAD(P)H and hydroxyproline were at their lowest compared with unirradiated pollen. In samples with stimulating doses of irradiation NAD(P) reached its maximum earlier than in samples with inhibiting irradiation. The ratio between the concentrations of NAD(P) and NAD(P)H in the irradiated samples was higher than in the unirradiated control. The hydroxyproline content was higher in irradiated than in unirradiated pollen. (author)

  19. Crystallization and preliminary crystallographic analysis of Gre2p, an NADP+-dependent alcohol dehydrogenase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Breicha, Klaus; Müller, Marion; Hummel, Werner; Niefind, Karsten

    2010-01-01

    The alcohol dehydrogenase Gre2p from S. cerevisiae catalyses the stereospecific reduction of a variety of different keto compounds and can therefore be applied as a valuable biocatalyst. The crystallization of the complex of Gre2p with NADP + and its preliminary X-ray analysis are described. Gre2p [Genes de respuesta a estres (stress-response gene)] from Saccharomyces cerevisiae is a monomeric enzyme of 342 amino acids with a molecular weight of 38.1 kDa. The enzyme catalyses both the stereospecific reduction of keto compounds and the oxidation of various hydroxy compounds and alcohols by the simultaneous consumption of the cofactor NADPH and formation of NADP + . Crystals of a Gre2p complex with NADP + were grown using PEG 8000 as a precipitant. They belong to the monoclinic space group P2 1 . The current diffraction resolution is 3.2 Å. In spite of the monomeric nature of Gre2p in solution, packing and self-rotation calculations revealed the existence of two Gre2p protomers per asymmetric unit related by a twofold noncrystallographic axis

  20. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

    Science.gov (United States)

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-09-02

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

  1. Expression of protein engineered NADP{sup +}-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki [National Institute of Advanced Industrial Science and Technology, Hiroshima (Japan). Biomass Technology Research Center; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke [Kyoto Univ. (Japan). Inst. of Advanced Energy

    2008-11-15

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD{sup +}-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP{sup +}. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP{sup +}-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP{sup +}-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. (orig.)

  2. A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

    Science.gov (United States)

    Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

    2017-02-17

    The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

  3. Upregulation of cytosolic NADP+-dependent isocitrate dehydrogenase by hyperglycemia protects renal cells against oxidative stress.

    Science.gov (United States)

    Lee, Soh-Hyun; Ha, Sun-Ok; Koh, Ho-Jin; Kim, KilSoo; Jeon, Seon-Min; Choi, Myung-Sook; Kwon, Oh-Shin; Huh, Tae-Lin

    2010-02-28

    Hyperglycemia-induced oxidative stress is widely recognized as a key mediator in the pathogenesis of diabetic nephropathy, a complication of diabetes. We found that both expression and enzymatic activity of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) were upregulated in the renal cortexes of diabetic rats and mice. Similarly, IDPc was induced in murine renal proximal tubular OK cells by high hyperglycemia, while it was abrogated by co-treatment with the antioxidant N-Acetyl-Cysteine (NAC). In OK cells, increased expression of IDPc by stable transfection prevented hyperglycemia-mediated reactive oxygen species (ROS) production, subsequent cellular oxidative stress and extracellular matrix accumulation, whereas these processes were all stimulated by decreased IDPc expression. In addition, production of NADPH and GSH in the cytosol was positively correlated with the expression level of IDPc in OK cells. These results together indicate that upregulation of IDPc in response to hyperglycemia might play an essential role in preventing the progression of diabetic nephropathy, which is accompanied by ROS-induced cellular damage and fibrosis, by providing NADPH, the reducing equivalent needed for recycling reduced glutathione and low molecular weight antioxidant thiol proteins.

  4. Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

    Science.gov (United States)

    Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

    2010-04-01

    Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

  5. Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato

    Science.gov (United States)

    Green, L. S.; Yee, B. C.; Buchanan, B. B.; Kamide, K.; Sanada, Y.; Wada, K.

    1991-01-01

    Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.

  6. An Interbacterial NAD(P)+ Glycohydrolase Toxin Requires Elongation Factor Tu for Delivery to Target Cells

    Energy Technology Data Exchange (ETDEWEB)

    Whitney, John C.; Quentin, Dennis; Sawai, Shin; LeRoux, Michele; Harding, Brittany N.; Ledvina, Hannah E.; Tran, Bao Q.; Robinson, Howard; Goo, Young Ah; Goodlett, David R.; Raunser, Stefan; Mougous, Joseph D.

    2015-10-08

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

  7. Purification, kinetic behavior, and regulation of NAD(P)+ malic enzyme of tumor mitochondria.

    Science.gov (United States)

    Moreadith, R W; Lehninger, A L

    1984-05-25

    The purification and kinetic characterization of an NAD(P)+-malic enzyme from 22aH mouse hepatoma mitochondria are described. The enzyme was purified 328-fold with a final yield of 51% and specific activity of 38.1 units/mg of protein by employing DEAE-cellulose chromatography and an ATP affinity column. Sephadex G-200 chromatography yielded a native Mr = 240,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major subunit with Mr = 61,000, suggesting a tetrameric structure, and also showed that the preparation contained less than 10% polypeptide impurities. Use of the ATP affinity column required the presence of MnCl2 and fumarate (an allosteric activator) in the elution buffers. In the absence of fumarate, the Michaelis constants for malate, NAD+, and NADP+ were 3.6 mM, 55 microM, and 72 microM, respectively; in the presence of fumarate (2 mM), the constants were 0.34 mM, 9 microM, and 13 microM, respectively. ATP was shown to be an allosteric inhibitor, competitive with malate. However, the inhibition by ATP displayed hyperbolic competitive kinetics with a KI (ATP) of 80 microM (minus fumarate) and 0.5 mM (plus 2 mM fumarate). The allosteric properties of the enzyme are integrated into a rationale for its specific role in the pathways of malate and glutamate oxidation in tumor mitochondria.

  8. Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase gene enhances glioma radiosensitivity

    International Nuclear Information System (INIS)

    Kim, Sung Youl; Yoo, Young Hyun; Park, Jeen-Woo

    2013-01-01

    Highlights: •Silencing of the IDPm gene enhances IR-induced autophagy in glioma cells. •Autophagy inhibition augmented apoptosis of irradiated glioma cells. •Results offer a redox-active therapeutic strategy for the treatment of cancer. -- Abstract: Reactive oxygen species (ROS) levels are elevated in organisms that have been exposed to ionizing radiation and are protagonists in the induction of cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP + -dependent isocitrate dehydrogenase (IDPm) via the supply of NADPH for antioxidant systems. In the present study, we report an autophagic response to ionizing radiation in A172 glioma cells transfected with small interfering RNA (siRNA) targeting the IDPm gene. Autophagy in A172 transfectant cells was associated with enhanced autophagolysosome formation and GFP–LC3 punctuation/aggregation. Furthermore, we found that the inhibition of autophagy by chloroquine augmented apoptotic cell death of irradiated A172 cells transfected with IDPm siRNA. Taken together, our data suggest that autophagy functions as a survival mechanism in A172 cells against ionizing radiation-induced apoptosis and the sensitizing effect of IDPm siRNA and autophagy inhibitor on the ionizing radiation-induced apoptotic cell death of glioma cells offers a novel redox-active therapeutic strategy for the treatment of cancer

  9. Binding Thermodynamics of Ferredoxin:NADP+ Reductase: Two Different Protein Substrates and One Energetics

    Science.gov (United States)

    Martínez-Júlvez, Marta; Medina, Milagros; Velázquez-Campoy, Adrián

    2009-01-01

    Abstract The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP+ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His299 FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics. PMID:19527656

  10. Expression of cytosolic NADP(+)-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant.

    Science.gov (United States)

    Kim, Ji Young; Shin, Jae Yong; Kim, Miri; Hann, Seung-Kyung; Oh, Sang Ho

    2012-02-01

    Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2). This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  11. Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

    Science.gov (United States)

    Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

    2002-06-01

    NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

  12. Expression analysis of the fpr (ferredoxin-NADP+ reductase) gene in Pseudomonas putida KT2440

    International Nuclear Information System (INIS)

    Lee, Yunho; Pena-Llopis, Samuel; Kang, Yoon-Suk; Shin, Hyeon-Dong; Demple, Bruce; Madsen, Eugene L.; Jeon, Che Ok; Park, Woojun

    2006-01-01

    The ferredoxin-NADP + reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440

  13. Depletion of NADP(H) due to CD38 activation triggers endothelial dysfunction in the postischemic heart.

    Science.gov (United States)

    Reyes, Levy A; Boslett, James; Varadharaj, Saradhadevi; De Pascali, Francesco; Hemann, Craig; Druhan, Lawrence J; Ambrosio, Giuseppe; El-Mahdy, Mohamed; Zweier, Jay L

    2015-09-15

    In the postischemic heart, coronary vasodilation is impaired due to loss of endothelial nitric oxide synthase (eNOS) function. Although the eNOS cofactor tetrahydrobiopterin (BH4) is depleted, its repletion only partially restores eNOS-mediated coronary vasodilation, indicating that other critical factors trigger endothelial dysfunction. Therefore, studies were performed to characterize the unidentified factor(s) that trigger endothelial dysfunction in the postischemic heart. We observed that depletion of the eNOS substrate NADPH occurs in the postischemic heart with near total depletion from the endothelium, triggering impaired eNOS function and limiting BH4 rescue through NADPH-dependent salvage pathways. In isolated rat hearts subjected to 30 min of ischemia and reperfusion (I/R), depletion of the NADP(H) pool occurred and was most marked in the endothelium, with >85% depletion. Repletion of NADPH after I/R increased NOS-dependent coronary flow well above that with BH4 alone. With combined NADPH and BH4 repletion, full restoration of NOS-dependent coronary flow occurred. Profound endothelial NADPH depletion was identified to be due to marked activation of the NAD(P)ase-activity of CD38 and could be prevented by inhibition or specific knockdown of this protein. Depletion of the NADPH precursor, NADP(+), coincided with formation of 2'-phospho-ADP ribose, a CD38-derived signaling molecule. Inhibition of CD38 prevented NADP(H) depletion and preserved endothelium-dependent relaxation and NO generation with increased recovery of contractile function and decreased infarction in the postischemic heart. Thus, CD38 activation is an important cause of postischemic endothelial dysfunction and presents a novel therapeutic target for prevention of this dysfunction in unstable coronary syndromes.

  14. The ALDH21 gene found in lower plants and some vascular plants codes for a NADP+ -dependent succinic semialdehyde dehydrogenase.

    Science.gov (United States)

    Kopečná, Martina; Vigouroux, Armelle; Vilím, Jan; Končitíková, Radka; Briozzo, Pierre; Hájková, Eva; Jašková, Lenka; von Schwartzenberg, Klaus; Šebela, Marek; Moréra, Solange; Kopečný, David

    2017-10-01

    Lower plant species including some green algae, non-vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP + -dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ-aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD + is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP + binding induces a conformational change of the loop carrying Arg-228, which seals the NADP + in the coenzyme cavity via its 2'-phosphate and α-phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg-121 and Arg-457, and a hydrogen bond with Tyr-296. While both arginine residues are pre-formed for substrate/product binding, Tyr-296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  15. Isotope effect studies of chicken liver NADP malic enzyme: role of the metal ion and viscosity dependence

    International Nuclear Information System (INIS)

    Grissom, C.B.; Cleland, W.W.

    1988-01-01

    The role of the metal ion in the oxidative decarboxylation of malate by chicken liver NADP malic enzyme and details of the reaction mechanism have been investigated by 13 C isotope effects. With saturating NADP and the indicated metal ion at a total concentration 10-fold higher than its K/sub m/, the following primary 13 C kinetic isotope effects at C 4 of malate [ 13 (VK/sub mal/)] were observed at pH 8.0: Mg 2+ , 1.0336; Mn 2+ , 1.0365; Cd 2+ , 1.0366; Zn 2+ , 1.0337; Co 2+ , 1.0283; Ni 2+ , 1.025. Knowing the partitioning of the intermediate oxalacetate between decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation to pyuvate and reduction to malate allows calculation of the intrinsic carbon isotope effect for decarboxylation. For Mg 2+ as activator, this was 1.049 with NADP and 1.046 with 3-acetylpyridine adenine dinucleotide phosphate, although the intrinsic primary deuterium isotope effects on dehydrogenation were 5.6 and 4.2, and the partition ratios of the oxalacetate intermediate for decarboxylation as opposed to hydride transfer were 0.11 and 3.96. It was not possible to calculate reasonable intrinsic carbon isotope effects with the other metal ions by use of the partitioning ratio of oxalacetate because of decarboxylation by another mechanism. The variation of 13 (VK/sub mal/) with pH was used to dissect the total forward and external components. When the authors attempted to use the variation of 13 (VK/sub mal/) with solution viscosity to determine the internal and external commitments, incorrect values were obtained because of a specific effect of the viscosogen in decreasing the K/sub m/ for malate, so that VK/sub mal/ actually increased with viscosity instead of decreasing, as theory predicts

  16. The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme.

    OpenAIRE

    Davisson, V J; Schulz, A R

    1985-01-01

    The mitochondrial NAD(P)+ malic enzyme [EC 1.1.1.39, L-malate:NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrat...

  17. Ser95, Asn97, and Thr78 are important for the catalytic function of porcine NADP-dependent isocitrate dehydrogenase

    OpenAIRE

    Kim, Tae-Kang; Colman, Roberta F.

    2005-01-01

    The mammalian mitochondrial NADP-dependent isocitrate dehydrogenase is a citric acid cycle enzyme and an important contributor to cellular defense against oxidative stress. The Mn2+-isocitrate complex of the porcine enzyme was recently crystallized; its structure indicates that Ser95, Asn97, and Thr78 are within hydrogen-bonding distance of the γ-carboxylate of enzyme-bound isocitrate. We used site-directed mutagenesis to replace each of these residues by Ala and Asp. The wild-type and mutant...

  18. Determination by radioimmunoassay of the sum of oxidized and reduced forms of NAD and NADP in picomole quantities from the same acid extract

    International Nuclear Information System (INIS)

    Bredehorst, R.; Lengyel, H.; Hilz, H.

    1979-01-01

    The sum of the amounts of NAD + NADH was determined from the same acid tissue extract with the aid of a highly specific radioimmunoassay for 5'-AMP. NAD was converted to 5'-AMP via ADP-ribose by alkaline treatment while NADH was converted first to ADP-ribose by incubation of the acid extract at 25 0 C followed by alkaline conversion to 5'-AMP. Removal of phosphate groups in NADP and NADPH by treatment of the extracts with alkaline phosphates extended the procedure to the quantification of NADP(H). When combined with enzymic analyses of the oxidized coenzyme forms, NAD/NADH and NADP/NADPH ratios could also be obtained from the same extracts. The sensitivity of the test allows quantification of pyridine nucleotides in the range of 0.1-10 pmol. (orig.)

  19. Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.

    Science.gov (United States)

    Huang, D Y; Ichikawa, Y

    1997-03-07

    Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic

  20. Isolation and Characterization of NADP+ -Linked Isocitrate Dehydrogenase in Germinating Urd Bean Seeds (Phaseolus mungo

    Directory of Open Access Journals (Sweden)

    Pramod Kumar Srivastava

    2010-06-01

    Full Text Available Isocitrate dehydrogenase (EC 1.1.1.42 has been purified to homogeneity from germinating urd bean seeds. The enzyme NADP+ -linked isocitrate dehydrogenase is a tetrameric protein (molecular weight 130,000; gel filtration made up of four identical monomers (sub unit molecular weight about 32,000-33,000; PAGE in presence of sodium dodecyl sulphate. Thermal inactivation of purified enzyme at 40 °C, 45 °C and 50 °C shows single exponential loss of enzyme activity suggesting that the inactivation of this enzyme follows simple first order kinetics (rate constants for purified enzyme 0.020, 0.043 and 0.077 min–1 at 40 °C, 45 °C and 50 °C respectively. Thermal inactivation in presence of glutathione and dithiothretol at 45 °C and 50 °C also follows simple first order kinetics, but the presence of these compounds protects the loss of enzyme activity. The enzyme shows optimum activity at pH 7.3-8.0. The variation of Vmax and Km at different pH values (6.5-8.0 suggests that proton behaves as an "Uncompetitive Inhibitor". A basic group is present at the active site of enzyme which is accessible for protonation in this pH range in the presence of substrate only, with a pKa equal to 6.8. Successive dialysis against EDTA and phosphate buffer, pH 7.5 at 0-4 °C gives an enzymatically inactive protein. Thermal inactivation of this protein at 45 °C and 50 °C shows an exponential loss of enzyme activity as in the case of untreated (native enzyme. Full activity is restored on adding Mn2+ (3.75mM to a solution of this protein. Addition of Mg2+, Zn2+, Co2+ and Cu2+ brings about partial recovery. Alkali metal ions bring about 75% inhibition at 4mM concentration. The inhibition is stronger at high concentration of Na+ and K+ . Other metal ions are not effective.

  1. Molecular and functional characterization of ferredoxin NADP(H oxidoreductase from Gracilaria chilensis and its complex with ferredoxin

    Directory of Open Access Journals (Sweden)

    María Alejandra Vorphal

    Full Text Available Abstract Backgroud Ferredoxin NADP(H oxidoreductases (EC 1.18.1.2 (FNR are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP+ to NADPH using the electrons provided by Ferredoxin (Fd, a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd. Methods The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5′RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking. Results The kinetic analysis shows KMNADPH of 12.5 M and a kcat of 86 s−1, data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS, sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS. Conclusion The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.

  2. Phosphoenolpyruvate carboxylase, NADP-malic enzyme, and pyruvate, phosphate dikinase are involved in the acclimation of Nicotiana tabacum L. to drought stress

    Czech Academy of Sciences Publication Activity Database

    Doubnerová-Hýsková, V.; Miedzińska, L.; Dobrá, Jana; Vaňková, Radomíra; Ryšlavá, H.

    2014-01-01

    Roč. 171, č. 5 (2014), s. 19-25 ISSN 0176-1617 R&D Projects: GA MŠk 1M0505 Institutional support: RVO:61389030 Keywords : Drought * NADP-malic enzyme * Nicotiana tabacum L. Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.557, year: 2014

  3. Lake and bulk sampling chemistry, NADP, and IMPROVE air quality data analysis on the Bridger-Teton National Forest (USFS Region 4)

    Science.gov (United States)

    Jill Grenon; Terry Svalberg; Ted Porwoll; Mark Story

    2010-01-01

    Air quality monitoring data from several programs in and around the Bridger-Teton (B-T) National Forest - National Atmospheric Deposition Program (NADP), longterm lake monitoring, long-term bulk precipitation monitoring (both snow and rain), and Interagency Monitoring of Protected Visual Environments (IMPROVE) - were analyzed in this report. Trends were analyzed using...

  4. Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings

    Directory of Open Access Journals (Sweden)

    P. Casati

    1999-10-01

    Full Text Available The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME in etiolated maize (Zea mays seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.

  5. Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase gene enhances ethanol-induced toxicity in HepG2 cells.

    Science.gov (United States)

    Yang, Eun Sun; Lee, Su-Min; Park, Jeen-Woo

    2010-07-01

    It has been shown that acute and chronic alcohol administrations increase the production of reactive oxygen species, lower cellular antioxidant levels and enhance oxidative stress in many tissues. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme by supplying NADPH to the cytosol. Upon exposure to ethanol, IDPc was susceptible to the loss of its enzyme activity in HepG2 cells. Transfection of HepG2 cells with an IDPc small interfering RNA noticeably downregulated IDPc and enhanced the cells' vulnerability to ethanol-induced cytotoxicity. Our results suggest that suppressing the expression of IDPc enhances ethanol-induced toxicity in HepG2 cells by further disruption of the cellular redox status.

  6. Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells.

    Science.gov (United States)

    Yang, Eun Sun; Yang, Joon-Hyuck; Park, Ji Eun; Park, Jeen-Woo

    2005-01-01

    Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.

  7. NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

    Directory of Open Access Journals (Sweden)

    Beatriz González

    Full Text Available Mammalian methionine adenosyltransferase II (MAT II is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+ with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

  8. Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

    Science.gov (United States)

    Skubatz, Hanna; Howald, William N

    2013-03-01

    A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

  9. Crystal structure studies of NADP{sup +} dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, S.M. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Pampa, K.J. [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India); Manjula, M. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Abdoh, M.M.M. [Department of Physics, Faculty of Science, An-Najah National University, Nablus, West Bank, Palestine (Country Unknown); Kunishima, Naoki [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148 (Japan); Lokanath, N.K., E-mail: lokanath@physics.uni-mysore.ac.in [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India)

    2014-06-20

    Highlights: • We determined the structure of isocitrate dehydrogenase with citrate and cofactor. • The structure reveals a unique novel terminal domain involved in dimerization. • Clasp domain shows significant difference, and catalytic residues are conserved. • Oligomerization of the enzyme is quantized with subunit-subunit interactions. • Novel domain of this enzyme is classified as subfamily of the type IV. - Abstract: NADP{sup +} dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP{sup +} was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.

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

    Science.gov (United States)

    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

  11. J-GLOBAL MeSH Dictionary: グルタミン酸デヒドロゲナーゼ(NADP+) [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term グルタミン酸デヒドロゲナーゼ(NADP+) 名詞 一般 * * * * グルタミン酸デヒドロゲナーゼ(NAD...P+) ... MeSH D018086 200906075423936874 C LS38 UNKNOWN_2 グルタミン酸 デヒドロゲナーゼ ( NADP + )

  12. Metabolismo intermedio e patogenicità microbica: studio della regolazione e dell’espressione differenziale del gene gdhA, codificante la L-glutammato deidrogenasi NADP-specifica, in isolati clinici di Neisseria meningitidis

    OpenAIRE

    Colicchio, Roberta

    2006-01-01

    Neisseria meningitidis (meningococco) è un batterio a ristretto spettro d’ospite e presenta “stili di vita” alternativi: commensale-patogeno, intracellulare-extracellulare. Un fattore cruciale per tale comportamento risiede nella capacità di sintetizzare ed utilizzare nutrienti essenziali per la propria sopravvivenza nei diversi microambienti dell’ospite durante un naturale ciclo infettivo. Dati di letteratura indicano che gdhA, codificante la L-glutammato deidrogenasi NADP-specifica (NADP...

  13. Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

    OpenAIRE

    Lee, Su Jeong; Park, Jeen-Woo

    2014-01-01

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocyte...

  14. Different rates of synthesis and degradation of two chloroplastic ammonium-inducible NADP-specific glutamate dehydrogenase isoenzymes during induction and deinduction in Chlorella sorokiniana cells

    International Nuclear Information System (INIS)

    Bascomb, N.F.; Prunkard, D.E.; Schmidt, R.R.

    1987-01-01

    The kinetics of accumulation (per milliliter of culture) of the α- and β-subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level. From pulse-chase experiments, using 35 SO 4 and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t/sub 1/2/ = 50 versus 150 minutes, respectively) and during the deinduction period (t/sub 1/2/ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed

  15. Cytosolic NADP(+)-dependent isocitrate dehydrogenase protects macrophages from LPS-induced nitric oxide and reactive oxygen species.

    Science.gov (United States)

    Maeng, Oky; Kim, Yong Chan; Shin, Han-Jae; Lee, Jie-Oh; Huh, Tae-Lin; Kang, Kwang-il; Kim, Young Sang; Paik, Sang-Gi; Lee, Hayyoung

    2004-04-30

    Macrophages activated by microbial lipopolysaccharides (LPS) produce bursts of nitric oxide and reactive oxygen species (ROS). Redox protection systems are essential for the survival of the macrophages since the nitric oxide and ROS can be toxic to them as well as to pathogens. Using suppression subtractive hybridization (SSH) we found that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is strongly upregulated by nitric oxide in macrophages. The levels of IDPc mRNA and of the corresponding enzymatic activity were markedly increased by treatment of RAW264.7 cells or peritoneal macrophages with LPS or SNAP (a nitric oxide donor). Over-expression of IDPc reduced intracellular peroxide levels and enhanced the survival of H2O2- and SNAP-treated RAW264.7 macrophages. IDPc is known to generate NADPH, a cellular reducing agent, via oxidative decarboxylation of isocitrate. The expression of enzymes implicated in redox protection, superoxide dismutase (SOD) and catalase, was relatively unaffected by LPS and SNAP. We propose that the induction of IDPc is one of the main self-protection mechanisms of macrophages against LPS-induced oxidative stress.

  16. RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

    Science.gov (United States)

    Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

    2012-08-01

    A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

  17. Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 is Involved in Detoxification of Nitrite in Roots.

    Science.gov (United States)

    Hachiya, Takushi; Ueda, Nanae; Kitagawa, Munenori; Hanke, Guy; Suzuki, Akira; Hase, Toshiharu; Sakakibara, Hitoshi

    2016-11-01

    Ferredoxin:NADP(H) oxidoreductase (FNR) plays a key role in redox metabolism in plastids. Whereas leaf FNR (LFNR) is required for photosynthesis, root FNR (RFNR) is believed to provide electrons to ferredoxin (Fd)-dependent enzymes, including nitrite reductase (NiR) and Fd-glutamine-oxoglutarate aminotransferase (Fd-GOGAT) in non-photosynthetic conditions. In some herbal species, however, most nitrate reductase activity is located in photosynthetic organs, and ammonium in roots is assimilated mainly by Fd-independent NADH-GOGAT. Therefore, RFNR might have a limited impact on N assimilation in roots grown with nitrate or ammonium nitrogen sources. AtRFNR genes are rapidly induced by application of toxic nitrite. Thus, we tested the hypothesis that RFNR could contribute to nitrite reduction in roots by comparing Arabidopsis thaliana seedlings of the wild type with loss-of-function mutants of RFNR2 When these seedlings were grown under nitrate, nitrite or ammonium, only nitrite nutrition caused impaired growth and nitrite accumulation in roots of rfnr2 Supplementation of nitrite with nitrate or ammonium as N sources did not restore the root growth in rfnr2 Also, a scavenger for nitric oxide (NO) could not effectively rescue the growth impairment. Thus, nitrite toxicity, rather than N depletion or nitrite-dependent NO production, probably causes the rfnr2 root growth defect. Our results strongly suggest that RFNR2 has a major role in reduction of toxic nitrite in roots. A specific set of genes related to nitrite reduction and the supply of reducing power responded to nitrite concomitantly, suggesting that the products of these genes act co-operatively with RFNR2 to reduce nitrite in roots. © 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.

  18. Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

    Science.gov (United States)

    Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

    2013-11-08

    SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

  19. Crystal structure of the NADP+ and tartrate-bound complex of L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis.

    Science.gov (United States)

    Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

    2018-05-01

    A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP + /sulfate ion at 1.18 Å and the structure in complex with NADP + /L-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP + and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.

  20. Effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme in bean (Phaseolus vulgaris L.) grown under different nitrogen conditions.

    Science.gov (United States)

    Pinto, M E; Casati, P; Hsu, T P; Ku, M S; Edwards, G E

    1999-02-01

    The effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme have been examined in different cultivars of Phaseolous vulgaris L. grown under 1 and 12 mM nitrogen. Low nitrogen nutrition reduces chlorophyll and soluble protein contents in the leaves and thus the photosynthesis rate and dry-matter accumulation. Chlorophyll, soluble protein and Rubisco contents and photosynthesis rate are not significantly altered by ambient levels of UV-B radiation (17 microW m-2, 290-320 nm, 4 h/day for one week). Comparative studies show that under high nitrogen, UV-B radiation slightly enhances leaf expansion and dry-matter accumulation in cultivar Pinto, but inhibits these parameters in Vilmorin. These results suggest that the UV-B effect on growth is mediated through leaf expansion, which is particularly sensitive to UV-B, and that Pinto is more tolerant than Vilmorin. The effect of UV-B radiation on UV-B-absorbing compounds and on NADP-malic enzyme (NADP-ME) activity is also examined. Both UV-B radiation and low-nitrogen nutrition enhance the content of UV-B-absorbing compounds, and among the three cultivars used, Pinto exhibits the highest increases and Arroz the lowest. The same trend is observed for the specific activity and content of NADP-ME. On a leaf-area basis, the amount of UV-B-absorbing compounds is highly correlated with the enzyme activity (r2 = 0.83), suggesting that NADP-ME plays a key role in biosynthesis of these compounds. Furthermore, the higher sensitivity of Vilmorin than Pinto to UV-B radiation appears to be related to the activity of NADP-ME and the capacity of the plants to accumulate UV-B-absorbing compounds.

  1. J-GLOBAL MeSH Dictionary: ジヒドロウラシルデヒドロゲナーゼ(NADP) [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term ジヒドロウラシルデヒドロゲナーゼ(NADP) 名詞 一般 * * * * ジヒドロウラシルデヒドロゲナーゼ(NAD...P) ... MeSH D042943 200906021340471184 C LS38 UNKNOWN_2 ジヒドロウラシルデヒドロゲナーゼ ( NADP )

  2. Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

    Science.gov (United States)

    Lee, Su-Min; Park, Sin Young; Shin, Seoung Woo; Kil, In Sup; Yang, Eun Sun; Park, Jeen-Woo

    2009-02-01

    Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

  3. Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells.

    Science.gov (United States)

    Lee, Su Jeong; Park, Jeen-Woo

    2014-04-01

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells.

  4. Effects of lindane on the photosynthetic apparatus of the cyanobacterium Anabaena: fluorescence induction studies and immunolocalization of ferredoxin-NADP+ reductase.

    Science.gov (United States)

    Bueno, Marta; Fillat, Maria F; Strasser, Reto J; Maldonado-Rodriguez, Ronald; Marina, Nerea; Smienk, Henry; Gómez-Moreno, Carlos; Barja, Francisco

    2004-01-01

    Cyanobacteria have the natural ability to degrade moderate amounts of organic pollutants. However, when pollutant concentration exceeds the level of tolerance, bleaching of the cells and death occur within 24 hours. Under stress conditions, cyanobacterial response includes the short-term adaptation of the photosynthetic apparatus to light quality, named state transitions. Moreover, prolonged stresses produce changes in the functional organization of phycobilisomes and in the core-complexes of both photosystems, which can result in large changes in the PS II fluorescence yield. The localization of ferredoxin-NADP+ reductase (FNR) at the ends of some peripheral rods of the cyanobacterial phycobilisomes, makes this protein a useful marker to check phycobilisome integrity. The goal of this work is to improve the knowledge of the mechanism of action of a very potent pesticide, lindane (gamma-hexaclorociclohexane), in the cyanobacterium Anabaena sp., which can be considered a potential candidate for bioremediation of pesticides. We have studied the effect of lindane on the photosynthetic apparatus of Anabaena using fluorescence induction studies. As ferredoxin-NADP+ reductase plays a key role in the response to oxidative stress in several systems, changes in synthesis, degradation and activity of FNR were analyzed. Immunolocalization of this enzyme was used as a marker of phycobilisome integrity. The knowledge of the changes caused by lindane in the photosynthetic apparatus is essential for rational further design of genetically-modified cyanobacteria with improved biorremediation abilities. Polyphasic chlorophyll a fluorescence rise measurements (OJIP) have been used to evaluate the vitality and stress adaptation of the nitrogen-fixing cyanobacterium Anabaena PCC 7119 in the presence of increasing concentrations of lindane. Effects of the pesticide on the ultrastructure have been investigated by electron microscopy, and FNR has been used as a marker of phycobilisome

  5. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

    Science.gov (United States)

    Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

  6. Two global conformation states of a novel NAD(P) reductase like protein of the thermogenic appendix of the Sauromatum guttatum inflorescence.

    Science.gov (United States)

    Skubatz, Hanna; Howald, William N

    2013-06-01

    A novel NAD(P) reductase like protein (RL) belonging to a class of reductases involved in phenylpropanoid synthesis was previously purified to homogeneity from the Sauromatum guttatum appendix. The Sauromatum appendix raises its temperature above ambient temperature to ~30 °C on the day of inflorescence opening (D-day). Changes in the charge state distribution of the protein in electrospray ionization-mass spectrometry spectra were observed during the development of the appendix. RL adopted two conformations, state A (an extended state) that appeared before heat-production (D - 4 to D - 2), and state B (a compact state) that began appearing on D - 1 and reached a maximum on D-day. RL in healthy leaves of Arabidopsis is present in state A, whereas in thermogenic sporophylls of male cones of Encephalartos ferox is present in state B. These conformational changes strongly suggest an involvement of RL in heat-production. The biophysical properties of this protein are remarkable. It is self-assembled in aqueous solutions into micrometer sizes of organized morphologies. The assembly produces a broad range of cyclic and linear morphologies that resemble micelles, rods, lamellar micelles, as well as vesicles. The assemblies could also form network structures. RL molecules entangle with each other and formed branched, interconnected networks. These unusual assemblies suggest that RL is an oligomer, and its oligomerization can provide additional information needed for thermoregulation. We hypothesize that state A controls the plant basal temperature and state B allows a shift in the temperature set point to above ambient temperature.

  7. Compounds from the Fruits of the Popular European Medicinal Plant Vitex agnus-castus in Chemoprevention via NADP(H):Quinone Oxidoreductase Type 1 Induction.

    Science.gov (United States)

    Li, Shenghong; Qiu, Shengxiang; Yao, Ping; Sun, Handong; Fong, Harry H S; Zhang, Hongjie

    2013-01-01

    As part of our continuing efforts in the search for potential biologically active compounds from medicinal plants, we have isolated 18 compounds including two novel nitrogen containing diterpenes from extracts of the fruits of Vitex agnus-castus. These isolates, along with our previously obtained novel compound vitexlactam A (1), were evaluated for potential biological effects, including cancer chemoprevention. Chemically, the nitrogenous isolates were found to be two labdane diterpene alkaloids, each containing an α , β -unsaturated γ -lactam moiety. Structurally, they were elucidated to be 9 α -hydroxy-13(14)-labden-16,15-amide (2) and 6 β -acetoxy-9 α -hydroxy-13(14)-labden-15,16-amide (3), which were named vitexlactams B and C, respectively. The 15 known isolates were identified as vitexilactone (4), rotundifuran (5), 8-epi-manoyl oxide (6), vitetrifolin D (7), spathulenol (8), cis-dihydro-dehydro-diconiferylalcohol-9-O- β -D-glucoside (9), luteolin-7-O-glucoside (10), 5-hydroxy-3,6,7,4'-tetramethoxyflavone (11), casticin (12), artemetin (13), aucubin (14), agnuside (15), β -sitosterol (16), p-hydroxybenzoic acid (17), and p-hydroxybenzoic acid glucose ester (18). All compound structures were determined/identified on the basis of 1D and/or 2D NMR and mass spectrometry techniques. Compounds 6, 8, 9, and 18 were reported from a Vitex spieces for the first time. The cancer chemopreventive potentials of these isolates were evaluated for NADP(H):quinone oxidoreductase type 1 (QR1) induction activity. Compound 7 demonstrated promising QR1 induction effect, while the new compound vitexlactam (3) was only slightly active.

  8. Compounds from the Fruits of the Popular European Medicinal Plant Vitex agnus-castus in Chemoprevention via NADP(H:Quinone Oxidoreductase Type 1 Induction

    Directory of Open Access Journals (Sweden)

    Shenghong Li

    2013-01-01

    Full Text Available As part of our continuing efforts in the search for potential biologically active compounds from medicinal plants, we have isolated 18 compounds including two novel nitrogen containing diterpenes from extracts of the fruits of Vitex agnus-castus. These isolates, along with our previously obtained novel compound vitexlactam A (1, were evaluated for potential biological effects, including cancer chemoprevention. Chemically, the nitrogenous isolates were found to be two labdane diterpene alkaloids, each containing an α, β-unsaturated γ-lactam moiety. Structurally, they were elucidated to be 9α-hydroxy-13(14-labden-16,15-amide (2 and 6β-acetoxy-9α-hydroxy-13(14-labden-15,16-amide (3, which were named vitexlactams B and C, respectively. The 15 known isolates were identified as vitexilactone (4, rotundifuran (5, 8-epi-manoyl oxide (6, vitetrifolin D (7, spathulenol (8, cis-dihydro-dehydro-diconiferylalcohol-9-O-β-D-glucoside (9, luteolin-7-O-glucoside (10, 5-hydroxy-3,6,7,4′-tetramethoxyflavone (11, casticin (12, artemetin (13, aucubin (14, agnuside (15, β-sitosterol (16, p-hydroxybenzoic acid (17, and p-hydroxybenzoic acid glucose ester (18. All compound structures were determined/identified on the basis of 1D and/or 2D NMR and mass spectrometry techniques. Compounds 6, 8, 9, and 18 were reported from a Vitex spieces for the first time. The cancer chemopreventive potentials of these isolates were evaluated for NADP(H:quinone oxidoreductase type 1 (QR1 induction activity. Compound 7 demonstrated promising QR1 induction effect, while the new compound vitexlactam (3 was only slightly active.

  9. Data for rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H-preferring xylose reductase–xylitol dehydrogenase pathway

    Directory of Open Access Journals (Sweden)

    Biao Zhang

    2015-12-01

    Full Text Available A thermo-tolerant NADP(H-preferring xylose pathway was constructed in Kluyveromyces marxianus for ethanol production with xylose at elevated temperatures (Zhang et al., 2015 [25]. Ethanol production yield and efficiency was enhanced by pathway engineering in the engineered strains. The constructed strain, YZJ088, has the ability to co-ferment glucose and xylose for ethanol and xylitol production, which is a critical step toward enabling economic biofuel production from lignocellulosic biomass. This study contains the fermentation results of strains using the metabolic pathway engineering procedure. The ethanol-producing abilities of various yeast strains under various conditions were compared, and strain YZJ088 showed the highest production and fastest productivity at elevated temperatures. The YZJ088 xylose fermentation results indicate that it fermented well with xylose at either low or high inoculum size. When fermented with an initial cell concentration of OD600=15 at 37 °C, YZJ088 consumed 200 g/L xylose and produced 60.07 g/L ethanol; when the initial cell concentration was OD600=1 at 37 °C, YZJ088 consumed 98.96 g/L xylose and produced 33.55 g/L ethanol with a productivity of 0.47 g/L/h. When fermented with 100 g/L xylose at 42 °C, YZJ088 produced 30.99 g/L ethanol with a productivity of 0.65 g/L/h, which was higher than that produced at 37 °C.

  10. Purification and characteristics of an inducible by polycyclic aromatic hydrocarbons NADP(+)-dependent naphthalenediol dehydrogenase (NDD) in Mucor circinelloides YR-1.

    Science.gov (United States)

    Camacho-Morales, Reyna Lucero; Zazueta-Novoa, Vanesa; Casillas, Juana Lizbeth González; Ballesteros, Elizabeth Aranda; Bote, Juan Antonio Ocampo; Zazueta-Sandoval, Roberto

    2014-05-01

    We detected NADP(+)-dependent dihydrodiol dehydrogenase (DD) activity in a cell-free extract from Mucor circinelloides YR-1, after high-speed centrifugation. We analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different DD activity bands were revealed. Five constitutive DD activities (DD1-5) were present when glucose was used as carbon source and three inducible activities (NDD, PDD1 and PDD2) when aromatic hydrocarbon compounds were used. NDD activity was induced all of the aromatic hydrocarbon compounds. The highest DD activity inducer was naphthalene and the lowest was pyrene. One of the enzymes showed higher activity with cis-naphthalene-diol rather than with trans-nahthalenediol as a substrate. We purified this particular enzyme to homogeneity and found that it had an isoelectric point of 4.6. The molecular weight for the native protein was 197.4kDa and 49.03±0.5kDa for the monomer that conforms it, suggesting a homotetrameric structure for the complete enzyme. Polyclonal antibodies were raised against it and obtained. NDD activity was almost totally inhibited when antibodies were used at low concentrations, and in native immunoblots only one band, which corresponds to the activity band detected in the zymograms, could be detected. In denaturing PAGE immunoblots only one band was detected. This band corresponds to the purified protein band of 49kDa detected in SDS-PAGE gels. The other two inducible enzymes PDD1 and PDD2 were present only when phenanthrene was used as sole carbon source in the culture media. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP+-dependent dehydrogenases of the pentose phosphate pathway

    International Nuclear Information System (INIS)

    Rodrigues, Juan; Branco, Vasco; Lu, Jun; Holmgren, Arne; Carvalho, Cristina

    2015-01-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP + -dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI 50 : 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg 2+ > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates the

  12. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, Juan, E-mail: juanricardorodrigues@gmail.com [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, Central University of Venezuela (Venezuela, Bolivarian Republic of); Branco, Vasco [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal); Lu, Jun; Holmgren, Arne [Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet (Sweden); Carvalho, Cristina, E-mail: cristina.carvalho@ff.ulisboa.pt [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal)

    2015-08-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates

  13. Cloning, Expression and Characterization of NAD Kinase from Staphylococcus aureus Involved in the Formation of NADP (H: A Key Molecule in the Maintaining of Redox Status and Biofilm Formation

    Directory of Open Access Journals (Sweden)

    U Venkateswara Prasad

    2017-01-01

    Full Text Available Background:Staphylococcus aureus has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK and this gene of S. aureus ATCC 12600 was cloned, sequenced, expressed and characterized. Materials and Methods: The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in Escherichia coli DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the S. aureus was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure. Results: The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814 revealed presence of only one kind of NADK in all S. aureus strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Š observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity. Conclusion: The results of this study suggest increased NADPH concentration in S. aureus plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.

  14. Biological roles of crop NADP-malic enzymes and molecular ...

    African Journals Online (AJOL)

    刘增辉

    2011-06-08

    Jun 8, 2011 ... catalyze the oxidative decarboxylation of malate to produce pyruvate, CO2 and .... causes dryness of soil and atmosphere, which can limit the plant ... closing through regulate the degradation of malic in day. In addition, there ...

  15. Role of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) on cellular defence against oxidative injury by gamma-rays.

    Science.gov (United States)

    Lee, S H; Jo, S H; Lee, S M; Koh, H J; Song, H; Park, J W; Lee, W H; Huh, T L

    2004-09-01

    To investigate the regulation of NADPH-producing isocitrate dehydrogenase (ICDH) in cytosol (IDPc) and mitochondria (IDPm) upon gamma-ray irradiation, and the roles of IDPc and IDPm in the protection against cellular damage induced by gamma-ray irradiation. Changes of IDPc and IDPm proteins upon gamma-ray irradiation to NIH3T3 cells were analysed by immunoblotting. To increase or decrease the expression of IDPc or IDPm, NIH3T3 cells were stably transfected with mouse IDPc or IDPm cDNA in either the sense or the antisense direction. The transfected cells with either increased or decreased IDPc or IDPm were exposed to gamma-rays, and the levels of reactive oxygen species generation, protein oxidation and lipid peroxidation were measured. Both IDPc and IDPm activities were induced by gamma-ray in NIH3T3 cells. Cells with decreased expression of IDPc or IDPm had elevated reactive oxygen species generation, lipid peroxidation and protein oxidation. Conversely, overproduction of IDPc or IDPm protein partially protected the cells from oxidative damage induced by gamma-ray irradiation. The protective role of IDPc and IDPm against gamma-ray-induced cellular damage can be attributed to elevated NADPH, reducing equivalents needed for recycling reduced glutathione in the cytosol and mitochondria. Thus, a primary biological function of the ICDHs may be production of NADPH, which is a prerequisite for some cellular defence systems against oxidative damage.

  16. Elements Required for an Efficient NADP-Malic Enzyme Type C4 Photosynthesis1[C][W][OPEN

    Science.gov (United States)

    Wang, Yu; Long, Stephen P.; Zhu, Xin-Guang

    2014-01-01

    C4 photosynthesis has higher light, nitrogen, and water use efficiencies than C3 photosynthesis. Although the basic anatomical, cellular, and biochemical features of C4 photosynthesis are well understood, the quantitative significance of each element of C4 photosynthesis to the high photosynthetic efficiency are not well defined. Here, we addressed this question by developing and using a systems model of C4 photosynthesis, which includes not only the Calvin-Benson cycle, starch synthesis, sucrose synthesis, C4 shuttle, and CO2 leakage, but also photorespiration and metabolite transport between the bundle sheath cells and mesophyll cells. The model effectively simulated the CO2 uptake rates, and the changes of metabolite concentrations under varied CO2 and light levels. Analyses show that triose phosphate transport and CO2 leakage can help maintain a high photosynthetic rate by balancing ATP and NADPH amounts in bundle sheath cells and mesophyll cells. Finally, we used the model to define the optimal enzyme properties and a blueprint for C4 engineering. As such, this model provides a theoretical framework for guiding C4 engineering and studying C4 photosynthesis in general. PMID:24521879

  17. Nitrogen Control in Pseudomonas aeruginosa : A Role for Glutamine in the Regulation of the Synthesis of NADP-Dependent Glutamate Dehydrogenase, Urease and Histidase

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1981-01-01

    In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress

  18. Decreased insulin secretory response of pancreatic islets during culture in the presence of low glucose is associated with diminished 45Ca2+ net uptake, NADPH/NADP+ and GSH/GSSG ratios

    International Nuclear Information System (INIS)

    Verspohl, E.J.; Kaiser, P.; Wahl, M.; Ammon, H.P.T.

    1988-01-01

    In isolated rat pancreatic islets maintained at a physiologic glucose concentration (5.6 mM) the effect of glucose on parameters which are known to be involved in the insulin secretion coupling such as NADPH, reduced glutathione (GSH), 86 Rb + efflux, and 45 Ca ++ net uptake were investigated. The insulinotropic effect of 16.7 mM glucose was decreased with the period of culturing during the first 14 days being significant after 2 days though in control experiments both protein content and ATP levels per islet were not affected and insulin content was only slightly decreased. Both NADPH and GSH decreased with time of culture. 86 Rb + efflux which is decreased by enhancing the glucose concentration from 3 to 5.6 mM in freshly isolated islets was not affected by culturing whatsoever, even not after 14 days of culture when there was not longer any insulin responsiveness to glucose. The 45 Ca ++ net uptake was decreased during culturing. The data indicate (1) that the diminished glucose-stimulated release of insulin during culturing is not due to cell loss or simple energy disturbances, (2) that more likely it is the result of a diminished 45 Ca ++ net uptake as a consequence of the inability of islet cells to maintain proper NADPH and GSH levels, and (3) that potassium ( 86 Rb + ) efflux may not be related to changes of NADPH and GSH

  19. Electron Microscopic Analysis and Structural Characterization of Novel NADP(H)-Containing Methanol : N,N'-Dimethyl-4-Nitrosoaniline Oxidoreductases from the Gram-Positive Methylotrophic Bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19

    NARCIS (Netherlands)

    Bystrykh, Leonid V.; Vonck, Janet; Bruggen, Ernst F.J. van; Beeumen, Jozef van; Samyn, Bart; Govorukhina, Natalya I.; Arfman, Nico; Duine, Johannis A.; Dijkhuizen, Lubbert

    The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry)

  20. Estudio biofísico y enzimológico de las reacciones fotorreducción de compuestos oxidados de nitrógeno (Nitrato y Nitrito) NADP y glutatión en cianobacterias

    OpenAIRE

    Serrano Delgado, Aurelio

    1985-01-01

    La vida en nuestro planeta depende admirablemente y de f orma casi absoluta de la energía radiante de la luz solar, la cual llega incesantemente a la biosfera en cantidades enormes (1017 W = 3 * 1024 J por año). La energía luminosa es convertida en otras formas de energía mediante la función clorofílica ó Fotosíntesis que llevan a cabo los organismos fotoergónicos (algas verdeazuladas ó cianobacterias, algas verdes y plantas superiores). Este proceso consiste esencialmente en la transformació...

  1. "v ne-, pod- nebo nadpřirozených silách": Ustanovení světa hry Toma Stopparda Rosencrantz a Guildenstern jsou mrtvi

    OpenAIRE

    Krtička, Filip

    2010-01-01

    The atmosphere of Stoppard's tour de force is one of confusion from the beginning to end. Guildenstern is confused about the outcome of the coin tossing and, thus, about the ruling principle of the world, Rosencrantz is confused about Guildenstern's role-playing practice of questioning Hamlet, everyone is confused about which one of the pair is Rosencrantz and which one is Guildenstern, and they are in turn confused about everyone else. Determinism is confused with absurdity, fate is confused...

  2. Deficiency of the Mitochondrial NAD Kinase Causes Stress-Induced Hepatic Steatosis in Mice

    NARCIS (Netherlands)

    Zhang, Kezhong; Kim, Hyunbae; Fu, Zhiyao; Qiu, Yining; Yang, Zhao; Wang, Jiemei; Zhang, Deqiang; Tong, Xin; Yin, Lei; Li, Jing; Wu, Jianmei; Qi, Nathan R.; Houten, Sander M.; Zhang, Ren

    2018-01-01

    The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP

  3. The effect of night temperature and leaf inorganic nitrogen status on ...

    African Journals Online (AJOL)

    Themeda triandra, Hyparrhenia hirta and Zea mays have a single bundle sheath with agranal, centrifugal chloroplasts suggesting a NADP-ME-type photosynthetic pathway. Plants grown in liquid cultures had a higher NADP malic enzyme activity than those grown in sand with nutrients being supplied. Decreased minimum ...

  4. NCBI nr-aa BLAST: CBRC-DYAK-06-0066 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-06-0066 ref|YP_482035.1| NAD(P) transhydrogenase, beta subunit [Frankia s...p. CcI3] gb|ABD12306.1| NAD(P) transhydrogenase, beta subunit [Frankia sp. CcI3] YP_482035.1 0.077 25% ...

  5. Microbial production of 3-hydroxypropionic acid

    DEFF Research Database (Denmark)

    2014-01-01

    A yeast cell havinga reduced level of activity of NAD dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has at least one exogenous gene encoding NADP dependent GAPDH and/or has up-regulation of at least one endogenous gene expressing NADP dependent GAPDH, wherein combined expression of t...

  6. Nadpřirozené bytosti v knihách o Harry Potterovi a jejich srovnání s pohádkovými příběhy/Supernatural Creatures in the Harry Potter Series and Their Comparison with Fairy Tale Stories

    OpenAIRE

    JEŘÁBKOVÁ, Vanda

    2013-01-01

    This bachelor thesis deals with selected supernatural creatures of Harry Potter?s series. It is primarily focused on a dragon, basilisk, phoenix, unicorn, merpeople, werewolf, giant and wizard. Each of these creatures is depicted from author?s point of view at first, then it is compared with its original depiction and finally it draws a comparison between itself and its fairy tale?s portrayal. The emphasis is put on comprehension of the chosen creature?s main meaning, its role and functioning...

  7. Fluorimetric study of the interaction between nicotinamide adenine dinucleotide phosphate and tetracycline-europium complex and its application

    International Nuclear Information System (INIS)

    Peng Qian; Hou Faju; Ge Xiaoxia; Jiang Chongqiu; Gong Shubo

    2005-01-01

    A new spectrofluorimetric method was developed for the determination of trace amount of nicotinamide adenine dinucleotide phosphate (NADP). Using europium (Eu 3+ )-tetracycline (TC) complex as a fluorescent probe, in the buffer solution of pH 7.60. NADP can remarkably enhance the fluorescence intensity of the Eu 3+ -TC complex at λ = 612 nm and the enhanced fluorescence intensity of Eu 3+ ion is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 4.4 x 10 -7 to 2.2 x 10 -6 mol l -1 with detection limit of 6.9 x 10 -8 mol l -1 . This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples and in serum samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Eu 3+ -TC system and the Eu 3+ -TC-NADP system have been also discussed

  8. Spectrofluorimetric determination of trace amount of coenzyme II using ciprofloxacin-terbium complex as a fluorescent probe

    International Nuclear Information System (INIS)

    Bian Weiwei; Wang Yusheng; Zhu Xiaojing; Jiang Chongqiu

    2006-01-01

    A new spectrofluorimetric method was developed for the determination of trace amount of nicotinamide adenine dinucleotide phosphate (NADP). Using terbium ion (Tb 3+ )-ciprofloxacin (CIP) complex as a fluorescent probe, in the buffer solution of pH=9.00, NADP can remarkably enhance the fluorescence intensity of the Tb 3+ -CIP complex at λ=545nm and the enhanced fluorescence intensity of Tb 3+ ion is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 4.9x10 -7 -3.7x10 -6 molL -1 with detection limit of 1.3x10 -7 molL -1 . This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Tb 3+ -CIP system and the Tb 3+ -CIP-NADP system have been also discussed

  9. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    International Nuclear Information System (INIS)

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-01-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  10. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  11. Fulltext PDF

    Indian Academy of Sciences (India)

    tendency of CIB1 expression from nontumourous liver tissue to tumour centre. Knockdown of ... Asia due to a high prevalence of chronic HBV and HCV ..... Alcohol dehydrogenase [NADP+] ..... effects on HCC tumour growth in vivo, through the.

  12. Main: 1JB9 [RPSD[Archive

    Lifescience Database Archive (English)

    Full Text Available 1JB9 トウモロコシ Corn Zea mays L. Ferredoxin-Nadp Reductase Precursor Zea Mays Molecule:...SLLYDEEFTSYLKQYPDNFRYDKALSREQKNRSGGKMYVQDKIEEYSDEIFKLLDGGAHIYFCGLKGMMPGIQDTLKKVAERRGESWDQKLAQLKKNKQWHVEVY corn_1JB9.jpg ...

  13. Expression, purification and characterization of Oryza sativa L. NAD ...

    African Journals Online (AJOL)

    Jane

    2011-10-17

    Oct 17, 2011 ... yeast, fungi, plants, animals and humans. MEs catalyze ... Plant NADP-. MEs were induced by many biotic or abiotic stresses, such as pathogen (Sutherland, 1991), wounding, UV-B ..... (2006) showed that the single mutation.

  14. Metabolic turnover of pyridine nucleotides in ascites cells of sarcoma Sa 180 and in the liver tissue of rats before and after ionizing radiation

    International Nuclear Information System (INIS)

    Kunz, K.; Musil, J.

    1979-01-01

    The metabolic turnover of NADP + labeled with 14 C in the ribose moiety of their molecules was determined in the ascites cells of sarcoma Sa 180 and in the rat liver tissue. The half-lives of NAD + and NADP + in the Sa 180 sarcoma cells were 60 mins (NAD + ) and 90 mins (NADP + ), the corresponding values in the liver cells were 80 mins (NAD + ) and 120 mins (NADP + ). Experiments were conducted on animals aimed at ascertaining the time-dependent effect of ionizing radiation on the values of pooled NAD + and NADP + after 0.129 C/kg (500 R) and 0.387 C/kg (1500 R) whole-body irradiation, and the metabolic turnover of these nucleotides 5 h after whole-body irradiation with 0.387 C/kg (1500 R). Exposure to 0.129 C/kg (500 R) whole-body irradiation induced no apparent changes compared with the controls. Within 5 h of irradiation the whole-body dose of 0.387 C/kg (1500 R) produced changes in rat liver cells characterized by a reduction in the nucleotide biological half-lives (NAD + from 80 to 60 mins and NADP + from 120 to 70 mins). No such changes in the pyridine nucleotide turnover were detected in the Sa 180 ascites sarcoma cells. (author)

  15. Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry).

    Science.gov (United States)

    Botman, Dennis; Tigchelaar, Wikky; Van Noorden, Cornelis J F

    2014-11-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM. © The Author(s) 2014.

  16. Mitochondrial NAD(PH in vivo: identifying natural indicators of oxidative phosphorylation in the 31P magnetic resonance spectrum.

    Directory of Open Access Journals (Sweden)

    Kevin eConley

    2016-03-01

    Full Text Available 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(PH, which are compartmentalized between cell and mitochondria. Here we provide evidence for detection of NAD(P+ and NAD(PH in separate mitochondrial and cell pools in vivo in human tissue by phosphorus magnetic resonance spectroscopy (31P MRS. These NAD(P pools are identified by chemical standards (NAD+, NADP+ and NADH and by physiological tests. A unique resonance reflecting mitochondrial NAD(PH is revealed by the changes elicited by elevation of mitochondrial oxidation. The decline of NAD(PH 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(PH peak provides an intrinsic probe of the reversal of mitochondrial dysfunction in elderly muscle. Thus non-invasive detection of NAD(P+ and NAD(PH in cell vs. mitochondria yield 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.

  17. Comparison of precipitation chemistry measurements obtained by the Canadian Air and Precipitation Monitoring Network and National Atmospheric Deposition Program for the period 1995-2004

    Science.gov (United States)

    Wetherbee, Gregory A.; Shaw, Michael J.; Latysh, Natalie E.; Lehmann, Christopher M.B.; Rothert, Jane E.

    2010-01-01

    Precipitation chemistry and depth measurements obtained by the Canadian Air and Precipitation Monitoring Network (CAPMoN) and the US National Atmospheric Deposition Program/National Trends Network (NADP/NTN) were compared for the 10-year period 1995–2004. Colocated sets of CAPMoN and NADP instrumentation, consisting of precipitation collectors and rain gages, were operated simultaneously per standard protocols for each network at Sutton, Ontario and Frelighsburg, Ontario, Canada and at State College, PA, USA. CAPMoN samples were collected daily, and NADP samples were collected weekly, and samples were analyzed exclusively by each network’s laboratory for pH, H + , Ca2+  , Mg2+  , Na + , K + , NH+4 , Cl − , NO−3 , and SO2−4 . Weekly and annual precipitation-weighted mean concentrations for each network were compared. This study is a follow-up to an earlier internetwork comparison for the period 1986–1993, published by Alain Sirois, Robert Vet, and Dennis Lamb in 2000. Median weekly internetwork differences for 1995–2004 data were the same to slightly lower than for data for the previous study period (1986–1993) for all analytes except NO−3 , SO2−4 , and sample depth. A 1994 NADP sampling protocol change and a 1998 change in the types of filters used to process NADP samples reversed the previously identified negative bias in NADP data for hydrogen-ion and sodium concentrations. Statistically significant biases (α = 0.10) for sodium and hydrogen-ion concentrations observed in the 1986–1993 data were not significant for 1995–2004. Weekly CAPMoN measurements generally are higher than weekly NADP measurements due to differences in sample filtration and field instrumentation, not sample evaporation, contamination, or analytical laboratory differences.

  18. Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids.

    Science.gov (United States)

    Kozuleva, Marina A; Ivanov, Boris N

    2010-07-01

    The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP(+). The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 microM in the absence of NADP(+) increased the proportion of oxygen reduction by Fd from 25-35 to 40-60% in different experiments. This proportion decreased with increasing light intensity. When NADP(+) was added in the presence of 15 microM Fd, which was optimal for the NADP(+) reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP(+) and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.

  19. Shikimate dehydrogenase from Pinu sylvestris L. needles

    International Nuclear Information System (INIS)

    Osipov, V.I.; Shein, I.V.

    1986-01-01

    Shikimate dehydrogenase was isolated by extraction from pine needles and partially purified by fractionation with ammonium sulfate. In conifers, in contrast to other plants, all three isoenzymes of shikimate dehydrogenase exhibit activity not only with NADP + , but also with NAD + . The values of K/sub m/ for shikimate, when NADP + and NAD + are used as cofactors, are 0.22 and 1.13 mM, respectively. The enzyme is maximally active at pH 10 with both cofactors. It is suggested that NAD-dependent shikimate dehydrogenase catalyzes the initial reaction of the alternative pathway of the conversion of shikimic acid to hydroxybenzoic acid. The peculiarities of the organization and regulation of the initial reactions of the shikimate pathway in conifers and in plants with shikimate dehydrogenase absolutely specific for NADP are discussed

  20. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    Science.gov (United States)

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Arsenic-induced stress activates sulfur metabolism in different organs of garlic (Allium sativum L.) plants accompanied by a general decline of the NADPH-generating systems in roots.

    Science.gov (United States)

    Ruíz-Torres, Carmelo; Feriche-Linares, Rafael; Rodríguez-Ruíz, Marta; Palma, José M; Corpas, Francisco J

    2017-04-01

    Arsenic (As) contamination is a major environmental problem which affects most living organisms from plants to animals. This metalloid poses a health risk for humans through its accumulation in crops and water. Using garlic (Allium sativum L.) plants as model crop exposed to 200μM arsenate, a comparative study among their main organs (roots and shoots) was made. The analysis of arsenic, glutathione (GSH), phytochelatins (PCs) and lipid peroxidation contents with the activities of antioxidant enzymes (catalase, superoxide dismutase, ascorbate-glutathione cycle), and the main components of the NADPH-generating system, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH) was carried out. Data showed a correlation among arsenic accumulation in the different organs, PCs content and the antioxidative response, with a general decline of the NADPH-generating systems in roots. Overall, our results demonstrate that there are clear connections between arsenic uptake, increase of their As-chelating capacity in roots and a decline of antioxidative enzyme activities (catalase and the ascorbate peroxidase) whose alteration provoked As-induced oxidative stress. Thus, the data suggest that roots act as barrier of arsenic mediated by a prominent sulfur metabolism which is characterized by the biosynthesis of high amount of PCs. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. Aluminium effects on pyridine nucleotide redox state in roots of Scots pine

    Directory of Open Access Journals (Sweden)

    Gabriela Lorenc-Plucińska

    2014-01-01

    Full Text Available After prolonged (3-9 weeks hydroponic treatment of Scots pine seedlings with different concentrations (0.5-4.0 mM of Al (AI(N033, the levels of pyridine nucleotides were determined in root homogenates. After 3 weeks of Al stress, a significant decrease of the anabolic reduction charge (ARC: NADPH/(NADP+ + NADPH and an increase of the redox status (NAD(PH/NAD(P+, catabolic reduction charge (CRC: NADH/(NAD+ + NADH and phosphorylation capacity expressed as NADP+/NAD+ ratio was found in the 4.0 mM Al treatment. After 6 weeks, Al at concentrations of 0.5 and 1.0 mM induced an enhancement of the NADH level and a reduction of NADPH level, but the redox ratios were not changed significantly. After 9 weeks treatment with Al concentrations ranging from 0.5 to 4.0 mM, decreases of the relative level of NADP+, NADPH and NADH and increases of NAD+ were found. Consequently, the CRC, NAD(PH/NAD(P+ and NADP+/NAD+ ratios reached a minimum and ARC a maximum as compared to previous measurements.

  3. Measurement Of Technical Efficiency In Irrigated Vegetable ...

    African Journals Online (AJOL)

    This study measured technical efficiency and identified its determinants in irrigated vegetable production in Nasarawa State of Nigeria using a stochastic frontier model. A complete enumeration of 193 NADP-registered vegetable farmers was done. The predicted farm technical efficiency ranges from 25.94 to 96.24 per cent ...

  4. Chronic antioxidant therapy fails to ameliorate hypertension: potential mechanisms behind

    Czech Academy of Sciences Publication Activity Database

    Pecháňová, Olga; Šimko, F.

    2009-01-01

    Roč. 27, Suppl.6 (2009), S32-S36 ISSN 0263-6352 R&D Projects: GA ČR(CZ) GA305/08/0139 Institutional research plan: CEZ:AV0Z50110509 Keywords : endothelial dysfunction * NADP(H) oxidase * reactive oxygen species Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 4.988, year: 2009

  5. Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Edman, J C; Edman, U; Cao, Mi-Mi

    1989-01-01

    Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi...

  6. Evolutionary history of a dispersal-associated locus across sympatric and allopatric divergent populations of a wing-polymorphic beetle across Atlantic Europe

    NARCIS (Netherlands)

    van Belleghem, S.M.; Roelofs, D.; Hendrickx, F.

    2015-01-01

    Studying the evolutionary history of trait divergence, in particular those related to dispersal capacity, is of major interest for the process of local adaptation and metapopulation dynamics. Here, we reconstruct the evolution of different alleles at the nuclear-encoded mitochondrial NADP

  7. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John

    2016-01-01

    alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

  8. Antioxidative response mechanisms in halophytes: their role in ...

    Indian Academy of Sciences (India)

    changes in ionic and water balance cause molecular damage and growth arrest. ... An optimal supply of CO2 determines the availability of. NADP to leaves via the ...... plasts in the plant cell, but could also leak into the cytosol, resulting in ...

  9. Enzyme Activities in Oleaginous Yeasts Accumulating and Utilizing Exogenous or Endogenous Lipids

    NARCIS (Netherlands)

    Holdsworth, Jane E.; Veenhuis, Marten; Ratledge, Colin

    1988-01-01

    The activities of ATP:citrate lyase (ACL; EC 4.1.3.8), carnitine acetyltransferase (CAT; EC 2.3.1.7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42), isocitrate lyase (ICL; EC 4.1.3.1) and malic enzyme (malate dehydrogenase; EC 1.1.1.40) were measured in four oleaginous yeasts, Candida

  10. Beyond the Protein Matrix : Probing Cofactor Variants in a Baeyer-Villiger Oxygenation Reaction

    NARCIS (Netherlands)

    Martinoli, Christian; Dudek, Hanna M.; Orru, Roberto; Edmondson, Dale E.; Fraaije, Marco W.; Mattevi, Andrea

    2013-01-01

    A general question in biochemistry is the interplay between the chemical properties of cofactors and the surrounding protein matrix. Here, the functions of NADP(+) and FAD are explored by investigation of a representative monooxygenase reconstituted with chemically modified cofactor analogues. Like

  11. Annual Frequency of Malaria Attack in Different Haemoglobin ...

    African Journals Online (AJOL)

    GREG F. FOMBO

    believed to be due to the enzyme deficiency advantage against fatal malaria. However, the mechanism of this .... Fluorescence was produced due to the reduction of NADP+ to. NADPH. ... Presence of fluorescence indicated normal cells while weak fluorescence ..... Molecular Monitoring of Malaria Vaccine Trial. Trends in.

  12. Effect of Potato virusY on activities of antioxidant and anaplerotic enzymes in Nicotina tabacum L. transgenic plants with the gene for p3 protein

    Czech Academy of Sciences Publication Activity Database

    Doubnerová, V.; Janoušková, M.; Synková, Helena; Šubr, Z.; Čeřovská, Noemi; Ryšlavá, H.

    2007-01-01

    Roč. 33, 3-4 (2007), s. 123-140 ISSN 1312-8183 Institutional research plan: CEZ:AV0Z50380511 Keywords : antioxidant enzymes * NADP-malic enzyme * PEPC Subject RIV: EB - Genetics ; Molecular Biology www.bio21.bas.bg/ipp/gapbfiles/v-33/07_3-4_123-140.pdf

  13. Trends in bromide wet deposition concentrations in the contiguous United States, 2001-2016.

    Science.gov (United States)

    Wetherbee, Gregory A; Lehmann, Christopher M B; Kerschner, Brian M; Ludtke, Amy S; Green, Lee A; Rhodes, Mark F

    2018-02-01

    Bromide (Br - ) and other solute concentration data from wet deposition samples collected and analyzed by the National Atmospheric Deposition Program (NADP) from 2001 to 2016, were statistically analyzed for trends both geographically and temporally by precipitation type. Analysis was limited to NADP sites in the contiguous 48 United States. The Br - concentrations for this time period had a high number of values censored at the detection limits with greater than 86 percent of sample concentrations below analytical detection. Bromide was more frequently detected at NADP sites in coastal regions. Analysis using specialized statistical techniques for censored data revealed that Br - concentrations varied by precipitation type with higher concentrations usually observed in liquid versus precipitation containing snow. Negative temporal trends in Br - wet deposition concentrations were observed at a majority of NADP sites; approximately 25 percent of these trend values were statistically significant at less than 0.05 to 0.10 significance levels. Potential causes for the negative trends were explored, including annual and seasonal changes in precipitation depth, reduced emissions of methyl bromide (CH 3 Br) from coastal wetlands, and declining industrial use of bromine compounds. The results indicate that Br - in non-coastal wet-deposition comes mainly from long-range transport, not local sources. Correlations between Br - , chloride, and nitrate concentrations also were evaluated. Published by Elsevier Ltd.

  14. Determination of glutamate dehydrogenase activity and its kinetics in mouse tissues using metabolic mapping (quantitative enzyme histochemistry)

    NARCIS (Netherlands)

    Botman, Dennis; Tigchelaar, Wikky; van Noorden, Cornelis J. F.

    2014-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we

  15. 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

    Science.gov (United States)

    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

  16. A two-dimensional microscale model of gas exchange during photosynthesis in maize (Zea mays L.) leaves

    NARCIS (Netherlands)

    Retta, Moges; Ho, Quang Tri; Yin, Xinyou; Verboven, Pieter; Berghuijs, Herman N.C.; Struik, Paul C.; Nicolaï, Bart M.

    2016-01-01

    CO2 exchange in leaves of maize (Zea mays L.) was examined using a microscale model of combined gas diffusion and C4 photosynthesis kinetics at the leaf tissue level. Based on a generalized scheme of photosynthesis in NADP-malic enzyme type C4 plants, the model

  17. A hydrogen fuel cell for rapid, enzyme-catalysed organic synthesis with continuous monitoring.

    Science.gov (United States)

    Wan, Lei; Megarity, Clare F; Siritanaratkul, Bhavin; Armstrong, Fraser A

    2018-01-23

    A one-pot fuel cell for specific, enzyme-catalysed organic synthesis, with continuous monitoring of rate and reaction progress, combines an electrode catalysing rapid, reversible and diffusion-controlled interconversion of NADP + and NADPH with a Pt electrode catalysing 2H + /H 2 interconversion. This Communication demonstrates its performance and characteristics using the reductive amination of 2-oxoglutarate as a test system.

  18. Coupled reactions by coupled enzymes : alcohol to lactone cascade with alcohol dehydrogenase-cyclohexanone monooxygenase fusions

    NARCIS (Netherlands)

    Aalbers, Friso S; Fraaije, Marco W

    2017-01-01

    The combination of redox enzymes for redox-neutral cascade reactions has received increasing appreciation. An example is the combination of an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO). The ADH can use NADP(+) to oxidize cyclohexanol to form cyclohexanone and NADPH. Both

  19. Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development

    LENUS (Irish Health Repository)

    Storm, Janet

    2011-07-14

    Abstract Background Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. Methods The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. Results No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10Δgdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of α-ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. Conclusions First, the data support the conclusion that D10Δgdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra

  20. Unique photosynthetic phenotypes in Portulaca (Portulacaceae): C3-C4 intermediates and NAD-ME C4 species with Pilosoid-type Kranz anatomy.

    Science.gov (United States)

    Voznesenskaya, Elena V; Koteyeva, Nuria K; Edwards, Gerald E; Ocampo, Gilberto

    2017-01-01

    Portulacaceae is a family that has considerable diversity in photosynthetic phenotypes. It is one of 19 families of terrestrial plants where species having C 4 photosynthesis have been found. Most species in Portulaca are in the alternate-leaved (AL) lineage, which includes one clade (Cryptopetala) with taxa lacking C 4 photosynthesis and three clades having C 4 species (Oleracea, Umbraticola and Pilosa). All three species in the Cryptopetala clade lack Kranz anatomy, the leaves have C 3 -like carbon isotope composition and they have low levels of C 4 cycle enzymes. Anatomical, biochemical and physiological analyses show they are all C 3 -C 4 intermediates. They have intermediate CO 2 compensation points, enrichment of organelles in the centripetal position in bundle sheath (BS) cells, with selective localization of glycine decarboxylase in BS mitochondria. In the three C 4 clades there are differences in Kranz anatomy types and form of malic enzyme (ME) reported to function in C 4 (NAD-ME versus NADP-ME): Oleracea (Atriplicoid, NAD-ME), Umbraticola (Atriplicoid, NADP-ME) and Pilosa (Pilosoid, NADP-ME). Structural and biochemical analyses were performed on Pilosa clade representatives having Pilosoid-type leaf anatomy with Kranz tissue enclosing individual peripheral vascular bundles and water storage in the center of the leaf. In this clade, all species except P. elatior are NADP-ME-type C 4 species with grana-deficient BS chloroplasts and grana-enriched M chloroplasts. Surprisingly, P. elatior has BS chloroplasts enriched in grana and NAD-ME-type photosynthesis. The results suggest photosynthetic phenotypes were probably derived from an ancestor with NADP-ME-type C 4 , with two independent switches to NAD-ME type. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  1. Spectroscopy and Speciation Studies on the Interactions of Aluminum (III with Ciprofloxacin and β-Nicotinamide Adenine Dinucleotide Phosphate in Aqueous Solutions

    Directory of Open Access Journals (Sweden)

    Xiaodi Yang

    2012-08-01

    Full Text Available In this study, both experimental and theoretical approaches, including absorption spectra, fluorescence emission spectra, 1H- and 31P-NMR, electrospray ionization mass spectrometry (ESI-MS, pH-potentiometry and theoretical approaches using the BEST & SPE computer programs were applied to study the competitive complexation between ciprofloxacin (CIP and b-nicotinamide adenine dinucleotide phosphate (NADP with aluminum (III in aqueous solutions. Rank annihilation factor analysis (RAFA was used to analyze the absorption and fluorescence emission spectra of the ligands, the binary complexes and the ternary complexes. It is found, at the mM total concentration level and pH = 7.0, the bidentate mononuclear species [Al(CIP]2+ and [Al(NADP] predominate in the aqueous solutions of the Al(III-CIP and Al(III-NADP systems, and the two complexes have similar conditional stability constants. However, the pH-potentiometry results show at the mM total concentration level and pH = 7.0, the ternary species [Al(CIP(HNADP] predominates in the ternary complex system. Comparing predicted NMR spectra with the experimental NMR results, it can be concluded that for the ternary complex, CIP binds to aluminum ion between the 3-carboxylic and 4-carbonyl groups, while the binding site of oxidized coenzyme II is through the oxygen of phosphate, which is linked to adenosine ribose, instead of pyrophosphate. The results also suggested CIP has the potential to be a probe molecular for the detection of NADP and the Al(III-NADP complexes under physiological condition.

  2. Comparison of snowpack and winter wet-deposition chemistry in the Rocky Mountains, USA: implications for winter dry deposition

    Science.gov (United States)

    Clow, David W.; Ingersoll, George P.; Mast, M. Alisa; Turk, John T.; Campbell, Donald H.

    Depth-integrated snowpack chemistry was measured just prior to maximum snowpack depth during the winters of 1992-1999 at 12 sites co-located with National Atmospheric Deposition Program/National Trend Network (NADP/NTN) sites in the central and southern Rocky Mountains, USA. Winter volume-weighted mean wet-deposition concentrations were calculated for the NADP/NTN sites, and the data were compared to snowpack concentrations using the paired t-test and the Wilcoxon signed-rank test. No statistically significant differences were indicated in concentrations of SO 42- or NO 3- ( p>0.1). Small, but statistically significant differences ( p⩽0.03) were indicated for all other solutes analyzed. Differences were largest for Ca 2+ concentrations, which on average were 2.3 μeq l -1 (43%) higher in the snowpack than in winter NADP/NTN samples. Eolian carbonate dust appeared to influence snowpack chemistry through both wet and dry deposition, and the effect increased from north to south. Dry deposition of eolian carbonates was estimated to have neutralized an average of 6.9 μeq l -1 and a maximum of 12 μeq l -1 of snowpack acidity at the southernmost sites. The good agreement between snowpack and winter NADP/NTN SO 42- and NO 3- concentrations indicates that for those solutes the two data sets can be combined to increase data density in high-elevation areas, where few NADP/NTN sites exist. This combination of data sets will allow for better estimates of atmospheric deposition of SO 42- and NO 3- across the Rocky Mountain region.

  3. External quality-assurance results for the national atmospheric deposition program/national trends network, 2000-2001

    Science.gov (United States)

    Wetherbee, Gregory A.; Latysh, Natalie E.; Gordon, John D.

    2004-01-01

    Five external quality-assurance programs were operated by the U.S. Geological Survey for the National Atmospheric Deposition Program/National Trends Network (NADP/NTN) from 2000 through 2001 (study period): the intersite-comparison program, the blind-audit program, the field-audit program, the interlaboratory-comparison program, and the collocated-sampler program. Each program is designed to measure specific components of the total error inherent in NADP/NTN wet-deposition measurements. The intersite-comparison program assesses the variability and bias of pH and specific-conductance determinations made by NADP/NTN site operators with respect to accuracy goals. The accuracy goals are statistically based using the median of all of the measurements obtained for each of four intersite-comparison studies. The percentage of site operators responding on time that met the pH accuracy goals ranged from 84.2 to 90.5 percent. In these same four intersite-comparison studies, 88.9 to 99.0 percent of the site operators met the accuracy goals for specific conductance. The blind-audit program evaluates the effects of routine sample handling, processing, and shipping on the chemistry of weekly precipitation samples. The blind-audit data for the study period indicate that sample handling introduced a small amount of sulfate contamination and slight changes to hydrogen-ion content of the precipitation samples. The magnitudes of the paired differences are not environmentally significant to NADP/NTN data users. The field-audit program (also known as the 'field-blank program') was designed to measure the effects of field exposure, handling, and processing on the chemistry of NADP/NTN precipitation samples. The results indicate potential low-level contamination of NADP/NTN samples with calcium, ammonium, chloride, and nitrate. Less sodium contamination was detected by the field-audit data than in previous years. Statistical analysis of the paired differences shows that contaminant ions

  4. pH-tuneable binding of 2'-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study.

    Science.gov (United States)

    Ciulli, Alessio; Lobley, Carina M C; Tuck, Kellie L; Smith, Alison G; Blundell, Tom L; Abell, Chris

    2007-02-01

    The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

  5. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    Science.gov (United States)

    Ciulli, Alessio; Lobley, Carina M. C.; Tuck, Kellie L.; Smith, Alison G.; Blundell, Tom L.; Abell, Chris

    2007-01-01

    The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes. PMID:17242510

  6. Kinetic studies of the inhibition of a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme by bile acids and anti-inflammatory drugs.

    Science.gov (United States)

    Miyabe, Y; Amano, T; Deyashiki, Y; Hara, A; Tsukada, F

    1995-01-01

    We have investigated the steady-state kinetics for a cytosolic 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme of human liver and its inhibition by several bile acids and anti-inflammatory drugs such as indomethacin, flufemanic acid and naproxen. Initial velocity and product inhibition studies performed in the NADP(+)-linked (S)-1-indanol oxidation at pH 7.4 were consistent with a sequential ordered mechanism in which NADP+ binds first and leaves last. The bile acids and drugs, competitive inhibitors with respect to the alcohol substrate, exhibited uncompetitive inhibition with respect to the coenzyme, with Ki values less than 1 microM, whereas indomethacin exhibited noncompetitive inhibition (Ki < 24 microM). The kinetics of the inhibition by a mixture of the two inhibitors suggests that bile acids and drugs, except indomethacin, bind to overlapping sites at the active center of the enzyme-coenzyme binary complex.

  7. Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions.

    Science.gov (United States)

    Liu, Jiaheng; Li, Huiling; Zhao, Guangrong; Caiyin, Qinggele; Qiao, Jianjun

    2018-05-01

    NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

  8. Bleaching herbicide norflurazon inhibits phytoene desaturase by competition with the cofactors.

    Science.gov (United States)

    Breitenbach, J; Zhu, C; Sandmann, G

    2001-11-01

    Cofactor requirement was determined for the heterologous expressed phytoene desaturases from the cyanobacterium Synechococcus and the higher plant Gentiana lutea. The cyanobacterial enzyme is dependent on either NAD(P) or plastoquinone, whereas only quinones such as plastoquinone can function as a cofactor for the phytoene desaturase from G. lutea. Enzyme kinetic studies were carried out to determine a possible competition between the cofactors and the bleaching herbicide norflurazon. For the Synechococcus enzyme, competition between norflurazon and NADP, as well as plastoquinone, could be demonstrated. The K(m) values for these cofactors were 6.6 mM and 0.23 microM, respectively. Inhibition of the phytoene desaturase from G. lutea by norflurazon was also competitive with respect to plastoquinone. The K(m) values of both enzymes for plastoquinone were very close.

  9. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    International Nuclear Information System (INIS)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-01-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [ 14 C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined

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

    Science.gov (United States)

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

    2015-09-01

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

  11. Identification and Overexpression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter mathranii

    DEFF Research Database (Denmark)

    Yao, Shuo; Just Mikkelsen, Marie

    2010-01-01

    Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (Adh....... Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield. Copyright © 2010 S. Karger AG, Basel...

  12. The effect of exhaustive exercise on the concentration of purine nucleotides and their metabolites in erythrocytes

    OpenAIRE

    E Skotnicka; I Baranowska-Bosiacka; W Dudzińska; M Suska; R Nowak; K Krupecki; AJ Hłyńczak

    2008-01-01

    In this study we tried to obtain a complete overview of purine nucleotide metabolism in erythrocytes before and during an incremental, intermittent exhaustive exercise bout protocol for sportsmen (high-performance rowers) and untrained, healthy, active volunteers. Erythrocyte levels of the main nucleotides (ATP, ADP, AMP, GTP, GDP, GMP, IMP, NAD and NADP ), nucleosides (Ado, Guo, Ino) and the base Hyp were measured using the HPLC method. The parameters that can be deducted from their concent...

  13. Physiological and Environmental Aspects of Photosynthesis

    OpenAIRE

    Ricardo Alfredo Kluge; Universidade de São Paulo; Jaqueline V. Tezotto-Uliana; Universidade de São Paulo; Paula P. M. da Silva; Universidade de São Paulo

    2015-01-01

    Undoubtedly, photosynthesis is one of the most important process for the life planet maintenance. The sun releases radiant energy that is able to boost the photosynthetic apparatus of the plants, which produce carbohydrates that will be used in the respiration. Among the most important reactions of photosynthesis is the release of oxygen, essential for respiration, which happens in photosystem II. The products generated in the first phase of photosynthesis or photochemical phase (ATP and NADP...

  14. Enzyme study of the separate stages in alcohol fermentation

    Energy Technology Data Exchange (ETDEWEB)

    Mar Monux, D

    1968-01-01

    The precise roles of ATP, DNA, and NADP in interaction with enzymes in certain of the 11 phases of fermentation are outlined. Individual enzymes which take part in the 11 phases are: (1) hexose transferase; (2) phosphohexoseisomerase; (3) fructosinase; (4) aldolase; (5) an SH-enzyme; (6) 3-phosphoglycero-1-phosphotransferase; (7) ghosphoglyceromutosase; (8) 2-phosphoglycerohydrolase; (9) pyruvic transferase; (10) pyruvic decarboxylase; (11) alcohol dehydrogenase.

  15. Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration

    OpenAIRE

    Park, Jung Hyun; Ku, Hyeong Jun; Lee, Jin Hyup; Park, Jeen-Woo

    2017-01-01

    Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants, such as reduced glutathione and protein thiols, and production of reactive oxygen species. Mitochondrial NADP+-dependent isocitrate dehydrogenase (idh2) regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the ro...

  16. Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

    Science.gov (United States)

    de Freitas-Silva, Larisse; Rodríguez-Ruiz, Marta; Houmani, Hayet; da Silva, Luzimar Campos; Palma, José M; Corpas, Francisco J

    2017-11-01

    Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20μM glyphosate, we analyzed H 2 O 2 , ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H 2 0 2 , nitric oxide or peroxynitrite. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. A quality-assurance assessment for constituents reported by the National Atmospheric Deposition Program and the National Trends Network

    Science.gov (United States)

    See, R.B.; Schroder, L.J.; Willoughby, T.C.

    1989-01-01

    A continuing quality-assurance program has been operated by the U.S. Geographical Survey to evaluate any bias introduced by routine handling, shipping, and laboratory analyses of wet-deposition samples collected in the National Atmospheric Deposition Program (NADP) and National Trends Network (NTN). Blind-audit samples having a variety of constituent concentrations and values were selected. Only blind-audit samples with constituent concentrations and values less than the 95th-percentile concentration for natural wet-deposition samples were included in the analysis. Of the major ions, there was a significant increase of Ca2+, Mg2+, K+ SO42+ and Cl- in samples handled according to standard protocols and shipped in NADP/NTN sample-collection buckets. For 1979-1987, graphs of smoothed data showing the estimated contaminations in blind-audit samples indicate a decrease in the median concentration and ranges of Ca2+, Mg2+ and SO42- contamination of blind-audit samples shipped in sample-collection buckets. Part of the contamination detected in blind-audit samples can be attributed to contact with the sample-collection bucket and lid; however, additional sources also seem to contaminate the blind-audit sample. Apparent decreases in the magnitude and range of sample contamination may be caused by differences in sample-collection bucket- and lid-washing procedures by the NADP/NTN Central Analytical Laboratory. Although the degree of bias is minimal for most constituents, summaries of the NADP/NTN data base may contain overestimates of Ca2+, Mg2+, Na-, K+, SO42- and Cl- concentrations, and underestimates of H+ concentrations.

  18. Revealing diversity in structural and biochemical forms of C4 photosynthesis and a C3-C4 intermediate in genus Portulaca L. (Portulacaceae).

    Science.gov (United States)

    Voznesenskaya, Elena V; Koteyeva, Nuria K; Edwards, Gerald E; Ocampo, Gilberto

    2010-08-01

    Portulacaceae is one of 19 families of terrestrial plants in which species having C(4) photosynthesis have been found. Representative species from major clades of the genus Portulaca were studied to characterize the forms of photosynthesis structurally and biochemically. The species P. amilis, P. grandiflora, P. molokiniensis, P. oleracea, P. pilosa, and P. umbraticola belong to the subgenus Portulaca and are C(4) plants based on leaf carbon isotope values, Kranz anatomy, and expression of key C(4) enzymes. Portulaca umbraticola, clade Umbraticola, is NADP-malic enzyme (NADP-ME)-type C(4) species, while P. oleracea and P. molokiniensis in clade Oleracea are NAD-ME-type C(4) species, all having different forms of Atriplicoid-type leaf anatomy. In clade Pilosa, P. amilis, P. grandiflora, and P. pilosa are NADP-ME-type C(4) species. They have Pilosoid-type anatomy in which Kranz tissues enclose peripheral vascular bundles with water storage in the centre of the leaf. Portulaca cf. bicolor, which belongs to subgenus Portulacella, is an NADP-ME C(4) species with Portulacelloid-type anatomy; it has well-developed Kranz chlorenchyma surrounding lateral veins distributed in one plane under the adaxial epidermis with water storage cells underneath. Portulaca cryptopetala (clade Oleracea), an endemic species from central South America, was identified as a C(3)-C(4) based on its intermediate CO(2) compensation point and selective localization of glycine decarboxylase of the photorespiratory pathway in mitochondria of bundle sheath cells. The C(4) Portulaca species which were examined also have cotyledons with Kranz-type anatomy, while the stems of all species have C(3)-type photosynthetic cells. The results indicate that multiple structural and biochemical forms of C(4) photosynthesis evolved in genus Portulaca.

  19. Revealing diversity in structural and biochemical forms of C4 photosynthesis and a C3–C4 intermediate in genus Portulaca L. (Portulacaceae)

    Science.gov (United States)

    Voznesenskaya, Elena V.; Koteyeva, Nuria K.; Edwards, Gerald E.; Ocampo, Gilberto

    2010-01-01

    Portulacaceae is one of 19 families of terrestrial plants in which species having C4 photosynthesis have been found. Representative species from major clades of the genus Portulaca were studied to characterize the forms of photosynthesis structurally and biochemically. The species P. amilis, P. grandiflora, P. molokiniensis, P. oleracea, P. pilosa, and P. umbraticola belong to the subgenus Portulaca and are C4 plants based on leaf carbon isotope values, Kranz anatomy, and expression of key C4 enzymes. Portulaca umbraticola, clade Umbraticola, is NADP-malic enzyme (NADP-ME)-type C4 species, while P. oleracea and P. molokiniensis in clade Oleracea are NAD-ME-type C4 species, all having different forms of Atriplicoid-type leaf anatomy. In clade Pilosa, P. amilis, P. grandiflora, and P. pilosa are NADP-ME-type C4 species. They have Pilosoid-type anatomy in which Kranz tissues enclose peripheral vascular bundles with water storage in the centre of the leaf. Portulaca cf. bicolor, which belongs to subgenus Portulacella, is an NADP-ME C4 species with Portulacelloid-type anatomy; it has well-developed Kranz chlorenchyma surrounding lateral veins distributed in one plane under the adaxial epidermis with water storage cells underneath. Portulaca cryptopetala (clade Oleracea), an endemic species from central South America, was identified as a C3–C4 based on its intermediate CO2 compensation point and selective localization of glycine decarboxylase of the photorespiratory pathway in mitochondria of bundle sheath cells. The C4 Portulaca species which were examined also have cotyledons with Kranz-type anatomy, while the stems of all species have C3-type photosynthetic cells. The results indicate that multiple structural and biochemical forms of C4 photosynthesis evolved in genus Portulaca. PMID:20591900

  20. Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of “Desulfomonile tiedjei”

    OpenAIRE

    DeWeerd, Kim A.; Suflita, Joseph M.

    1990-01-01

    We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, “Desulfomonile tiedjei.” We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c3, or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, C...

  1. External quality-assurance project report for the National Atmospheric Deposition Program/National Trends Network and Mercury Deposition Network, 2009-2010

    Science.gov (United States)

    Wetherbee, Gregory A.; Martin, RoseAnn; Rhodes, Mark F.; Chesney, Tanya A.

    2014-01-01

    The U.S. Geological Survey operated six distinct programs to provide external quality-assurance monitoring for the National Atmospheric Deposition Program/National Trends Network (NTN) and Mercury Deposition Network (MDN) during 2009–2010. The field-audit program assessed the effects of onsite exposure, sample handling, and shipping on the chemistry of NTN samples; a system-blank program assessed the same effects for MDN. Two interlaboratory-comparison programs assessed the bias and variability of the chemical analysis data from the Central Analytical Laboratory (CAL) and Mercury (Hg) Analytical Laboratory (HAL). The blind-audit program was also implemented for the MDN to evaluate analytical bias in total Hg concentration data produced by the HAL. The co-located-sampler program was used to identify and quantify potential shifts in NADP data resulting from replacement of original network instrumentation with new electronic recording rain gages (E-gages) and precipitation collectors that use optical sensors. The results indicate that NADP data continue to be of sufficient quality for the analysis of spatial distributions and time trends of chemical constituents in wet deposition across the United States. Results also suggest that retrofit of the NADP networks with the new precipitation collectors could cause –8 to +14 percent shifts in NADP annual precipitation-weighted mean concentrations and total deposition values for ammonium, nitrate, sulfate, and hydrogen ion, and larger shifts (+13 to +74 percent) for calcium, magnesium, sodium, potassium, and chloride. The prototype N-CON Systems bucket collector is more efficient in the catch of precipitation in winter than Aerochem Metrics Model 301 collector, especially for light snowfall.

  2. J-GLOBAL MeSH Dictionary: NADP依存性HMG‐COA‐レダクターゼ [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term NADP依存性HMG‐COA‐レダクターゼ 名詞 一般 * * * * NAD...P依存性HMG‐COA‐レダクターゼ ... MeSH D025782 200906087882493851 C LS38 UNKNOWN_2 NADP 依存 性 H MG ‐ COA ‐ レダクターゼ

  3. J-GLOBAL MeSH Dictionary: フェレドキシンNADPレダクターゼ [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term フェレドキシンNADPレダクターゼ 名詞 一般 * * * * フェレドキシンNAD...Pレダクターゼ ... MeSH D005287 200906031562191487 C LS38 UNKNOWN_2 フェレドキシン NADP レダクターゼ

  4. Kinetics of carbonyl reductase from human brain.

    OpenAIRE

    Bohren, K M; von Wartburg, J P; Wermuth, B

    1987-01-01

    Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. D...

  5. Effects of physical characteristics of carbon black on metabolic regulation in mice

    International Nuclear Information System (INIS)

    Chuang, Hsiao-Chi; Hsiao, Ta-Chih; Lee, Chii-Hong; Chun-Te Lin, Justin; Chuang, Kai-Jen; Feng, Po-Hao; Cheng, Tsun-Jen

    2018-01-01

    Potential adverse effects of human exposure to carbon black (CB) have been reported, but limited knowledge regarding CB-regulated metabolism is currently available. To evaluate how physical parameters of CB influence metabolism, we investigated CB and diesel exhaust particles (DEPs) and attempted to relate various physical parameters, including the hydrodynamic diameter, zeta potential, and particle number concentrations, to lung energy metabolism in female BALB/c mice. A body weight increase was arrested by 3 months of exposure to CB of smaller-size fractions, which was negatively correlated with pyruvate in plasma. There were no significant differences in cytotoxic lactate dehydrogenase (LDH) or total protein in bronchoalveolar lavage fluid (BALF) after 3 months of CB exposure. However, we observed alterations in acetyl CoA and the NADP/NADPH ratio in lung tissues with CB exposure. Additionally, the NADP/NADPH ratio was associated with the zeta potential of CB. Mild peribronchiovascular and interstitial inflammation and multinucleated giant cells (macrophages) with a transparent and rhomboid appearance and containing foreign bodies were observed in lung sections. We suggest that physical characteristics of CB, such as the zeta potential, may disrupt metabolism after pulmonary exposure. These results, therefore, provide the first evidence of a link between pulmonary exposure to CB and metabolism. - Highlights: • We evaluated how physical parameters of CB influence metabolism in mice lungs. • Body weight was arrested by exposure to CB in mice. • CB-induced peribronchiovascular and interstitial inflammation in the lungs. • Disruption in functions of acetyl CoA and NADP/NADPH was observed in lungs. • NADP/NADPH ratio was associated with the zeta potentials of CB.

  6. Two new glucose 6-phosphate dehydrogenase variants associated with congenital nonspherocytic hemolytic anemia found in Japan: GD(-) Tokushima and GD(-) Tokyo.

    Science.gov (United States)

    Miwa, S; Ono, J; Nakashima, K; Abe, S; Kageoka, T

    1976-01-01

    Two new variants of glucose 6-phosphate dehydrogenase (G6PD) deficiency associated with chronic nonspherocytic hemolytic anemia were discovered in Japan. Gd(-) Tokushima was found in a 17-years-old male whose erythrocytes contained 4.4% of normal enzyme activity. Partially purified enzyme revealed a main band of normal electrophoretic mobility with additional two minor bands of different mobility; normal Km G6P, and Km NADP five-to sixfold higher than normal; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; marked thermal instability; a normal pH curve; and normal Ki NADPH. The hemolytic anemia was moderate to severe. Gd(-) Tokyo was characterized from a 15-year-old male who had chronic nonspherocytic hemolytic anemia of mild degree. The erythrocytes contained 3% of normal enzyme activity, and partially purified enzyme revealed slow electrophoretic mobility (90% of normal for both a tris-hydrochloride buffer system and a tris-EDTA-borate buffer system, and 70% of normal for a phosphate buffer system); normal Km G6P and Km NADP; normal utilization of 2-deoxy-G6P, galactose-6P, and deamino-NADP; greatly increased thermal instability; a normal pH curve; and normal Ki NADPH. These two variants are clearly different from hitherto described G6PD variants, including the Japanese variants Gd(-) Heian and Gd(-) Kyoto. The mothers of both Gd(-) Tokushima and Gd(-) Tokoyo were found to be heterozygote by an ascorbate-cyanide test.

  7. [In vivo and in vitro actions of biscarbamates on the photosynthetic activity of chloroplasts].

    Science.gov (United States)

    Chueca, A; Barón, M; López-Gorgé, J

    1982-01-01

    The "photosynthetic inhibition" component in the whole context of plant toxicity, when different concentrations of the bis-carbamate phenmedipham are supplied through the roots or foliar application to spinach plants grown in hydroponic media have been determined. Chloroplasts were isolated after eight days of the herbicide addition, and then determined: electron transport H2O leads to NADP+, H2O leads to ferrycyanide and ascorbate/DPIP leads to NADP+, cyclic and non cyclic photophosphorilation, CO2 assimilation rate and intermediate patterns of CO2 fixation. We have also determined in foliar disks the O2 evolving and the CO2 assimilation capabilities. Type A and type B chloroplasts showed increased inhibition, respectively, of the Phot. II dependent electron transport chains H2O leads to NADP+ and H2O leads to ferricyanide, to the extent that the phenmedipham concentration increased in the hydroponic media and the spraying solution, so that a 50% inhibition of both processes was obtained at 100 microM and 10 microM, respectively, against 0.2 microM in the in vitro experiments. Non cyclic photophosphorylation showed a stronger inhibition than the cyclic one. Concerning the Phot. I dependent electron transport ascorbate/DPIP leads to NADP+, the chloroplast preparations showed a negligible inhibition. We have found a synergistic effect of the above two factors on the CO2 assimilation. The intermediate patterns of CO2 assimilation showed a decrease of the 3C-compounds P-glycerate and trioses-P, with a parallel increase of the sugar mono and diphosphates as well as disaccharides and amino acids.

  8. Europe/Latin America Report, Science and Technology

    Science.gov (United States)

    1997-01-16

    to transform E. coli. Another source of genese for the fermentation of cellulose is the bacterium of the rumen Bacteriodes succinogenes: from a...was made in the transformation of bacteria of the genus Clostridia was achieved by using protoplasts and regeneration systems. Hemicellulose: only...complete transformation (200 mg of initial sterioids) in 3 days and the turnover number for NADP is 470. 15 Also studied was anaerobic reoxidation in

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

    Science.gov (United States)

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

    2018-01-20

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

  10. 17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

    Science.gov (United States)

    Blomquist, Charles H; Lima, P H; Tarrant, A M; Atkinson, M J; Atkinson, S

    2006-04-01

    Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.

  11. Photosynthetic carbon fixation characteristics of fruiting structures of Brassica campestris L

    International Nuclear Information System (INIS)

    Singal, H.R.; Sheoran, I.S.; Singh, R.

    1987-01-01

    Activities of key enzymes of the Calvin cycle and C 4 metabolism, rates of CO 2 fixation, and the initial products of photosynthetic 14 CO 2 fixation were determined in the podwall, seed coat (fruiting structures), and the subtending leaf (leaf below a receme) of Brassica campestris L. cv Toria. Compared to activities of ribulose-1,5-bisphosphate carboxylase and other Calvin cycle enzymes, e.g. NADP-glyceraldehyde-3-phosphate-dehydrogenase and ribulose-5-phosphate kinase, the activities of phosphoenol pyruvate carboxylase and other enzymes of C 4 metabolism, viz. NADP-malate dehydrogenase, NADP-malic enzyme, glutamate pyruvate transaminase, and glutamate oxaloacetate transaminase, were generally much higher in seed than in podwall and leaf. Podwall and leaf were comparable to each other. Pulse-chase experiments showed that in seed the major product of 14 CO 2 assimilation was malate (in short time), whereas in podwall and leaf, the label initially appeared in 3-PGA. With time, the label moved to sucrose. In contrast to legumes, Brassica pods were able to fix net CO 2 during light. However, respiratory losses were very high during the dark period

  12. USGS Tracks Acid Rain

    Science.gov (United States)

    Gordon, John D.; Nilles, Mark A.; Schroder, LeRoy J.

    1995-01-01

    The U.S. Geological Survey (USGS) has been actively studying acid rain for the past 15 years. When scientists learned that acid rain could harm fish, fear of damage to our natural environment from acid rain concerned the American public. Research by USGS scientists and other groups began to show that the processes resulting in acid rain are very complex. Scientists were puzzled by the fact that in some cases it was difficult to demonstrate that the pollution from automobiles and factories was causing streams or lakes to become more acidic. Further experiments showed how the natural ability of many soils to neutralize acids would reduce the effects of acid rain in some locations--at least as long as the neutralizing ability lasted (Young, 1991). The USGS has played a key role in establishing and maintaining the only nationwide network of acid rain monitoring stations. This program is called the National Atmospheric Deposition Program/National Trends Network (NADP/NTN). Each week, at approximately 220 NADP/NTN sites across the country, rain and snow samples are collected for analysis. NADP/NTN site in Montana. The USGS supports about 72 of these sites. The information gained from monitoring the chemistry of our nation's rain and snow is important for testing the results of pollution control laws on acid rain.

  13. 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.

    Science.gov (United States)

    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.

  14. C/sub 4/ photosynthesis in Euphorbia degeneri and E. remyi: a comparison of photosynthetic carbon metabolism in leaves, callus cultures and regenerated plants

    Energy Technology Data Exchange (ETDEWEB)

    Ruzin, S.E.

    1984-04-01

    Based on analysis of /sup 14/CO/sub 2/ fixation kinetics and assays of enzymes related to C/sub 4/ metabolism (NAD-ME, NADP-ME, NAD-MDH, NADP-MDH, AST, ALT), leaves and regenerated plants of Euphorbia degeneri exhibit a modified NADP-ME-type photosynthesis. Apparently, both aspartate and malate are used for transport of CO/sub 2/ to bundle sheath cells. Callus grown on either non-shoot-forming or shoot-forming media fixes CO/sub 2/ into RPP-cycle intermediates and sucrose, as well as malate and aspartate. /sup 14/CO/sub 2/ pulse/chase kinetics show no significant loss of label from C/sub 4/ acids throughout a one minute chase. Analysis of PEPCase revealed the presence of 2 isoenzymes in both leaf and regenerated plant tissues (K/sub m/ (PEP) = 0.080 and 0.550) but only one isoenzyme in callus (K/sub m/ = 0.100). It appears that C/sub 4/ photosynthesis does not occur in callus derived from this C/sub 4/ dicot but is regenerated concomitant with shoot regeneration, and ..beta..-carboxylation of PEP in callus, mediated by the low K/sub m/ isoenzyme of PEPCase, produces C/sub 4/ acids that are not involved in the CO/sub 2/ shuttle mechanism characteristic of C/sub 4/ photosynthesis. 161 references, 19 figures, 12 tables.

  15. Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges.

    Science.gov (United States)

    Chánique, Andrea M; Parra, Loreto P

    2018-01-01

    Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa . Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

  16. C4 photosynthesis in Euphorbia degeneri and E. remyi: a comparison of photosynthetic carbon metabolism in leaves, callus cultures and regenerated plants

    International Nuclear Information System (INIS)

    Ruzin, S.E.

    1984-04-01

    Based on analysis of 14 CO 2 fixation kinetics and assays of enzymes related to C 4 metabolism (NAD-ME, NADP-ME, NAD-MDH, NADP-MDH, AST, ALT), leaves and regenerated plants of Euphorbia degeneri exhibit a modified NADP-ME-type photosynthesis. Apparently, both aspartate and malate are used for transport of CO 2 to bundle sheath cells. Callus grown on either non-shoot-forming or shoot-forming media fixes CO 2 into RPP-cycle intermediates and sucrose, as well as malate and aspartate. 14 CO 2 pulse/chase kinetics show no significant loss of label from C 4 acids throughout a one minute chase. Analysis of PEPCase revealed the presence of 2 isoenzymes in both leaf and regenerated plant tissues (K/sub m/ [PEP] = 0.080 and 0.550) but only one isoenzyme in callus (K/sub m/ = 0.100). It appears that C 4 photosynthesis does not occur in callus derived from this C 4 dicot but is regenerated concomitant with shoot regeneration, and β-carboxylation of PEP in callus, mediated by the low K/sub m/ isoenzyme of PEPCase, produces C 4 acids that are not involved in the CO 2 shuttle mechanism characteristic of C 4 photosynthesis. 161 references, 19 figures, 12 tables

  17. The effect of Walterinnesia aegyptia venom proteins on TCA cycle activity and mitochondrial NAD(+)-redox state in cultured human fibroblasts.

    Science.gov (United States)

    Ghneim, Hazem K; Al-Sheikh, Yazeed A; Aboul-Soud, Mourad A M

    2015-01-01

    Fibroblast cultures were used to study the effects of crude Walterinnesia aegyptia venom and its F1-F7 protein fractions on TCA cycle enzyme activities and mitochondrial NAD-redox state. Confluent cells were incubated with 10 μg of venom proteins for 4 hours at 37°C. The activities of all studied TCA enzymes and the non-TCA mitochondrial NADP(+)-dependent isocitrate dehydrogenase underwent significant reductions of similar magnitude (50-60% of control activity) upon incubation of cells with the crude venom and fractions F4, F5, and F7 and 60-70% for fractions F3 and F6. In addition, the crude and fractions F3-F7 venom proteins caused a drop in mitochondrial NAD(+) and NADP(+) levels equivalent to around 25% of control values. Whereas the crude and fractions F4, F5, and F7 venom proteins caused similar magnitude drops in NADH and NADPH (around 55% of control levels), fractions F3 and F6 caused a more drastic drop (60-70% of control levels) of both reduced coenzymes. Results indicate that the effects of venom proteins could be directed at the mitochondrial level and/or the rates of NAD(+) and NADP(+) biosynthesis.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Palmer, T; Jackson, J B

    1992-02-21

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

  20. Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

    Science.gov (United States)

    Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

    1995-08-01

    Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

  1. Changing regional emissions of airborne pollutants reflected in the chemistry of snowpacks and wetfall in the Rocky Mountain region, USA, 1993–2012

    Science.gov (United States)

    Ingersoll, George P.; Miller, Debra C.; Morris, Kristi H.; McMurray, Jill A.; Port, Garrett M.; Caruso, Brian

    2016-01-01

    Wintertime precipitation sample data from 55 Snowpack sites and 17 National Atmospheric Deposition Program (NADP)/National Trends Network Wetfall sites in the Rocky Mountain region were examined to identify long-term trends in chemical concentration, deposition, and precipitation using Regional and Seasonal Kendall tests. The Natural Resources Conservation Service snow-telemetry (SNOTEL) network provided snow-water-equivalent data from 33 sites located near Snowpack- and NADP Wetfall-sampling sites for further comparisons. Concentration and deposition of ammonium, calcium, nitrate, and sulfate were tested for trends for the period 1993–2012. Precipitation trends were compared between the three monitoring networks for the winter seasons and downward trends were observed for both Snowpack and SNOTEL networks, but not for the NADP Wetfall network. The dry-deposition fraction of total atmospheric deposition, relative to wet deposition, was shown to be considerable in the region. Potential sources of regional airborne pollutant emissions were identified from the U.S. Environmental Protection Agency 2011 National Emissions Inventory, and from long-term emissions data for the period 1996–2013. Changes in the emissions of ammonia, nitrogen oxides, and sulfur dioxide were reflected in significant trends in snowpack and wetfall chemistry. In general, ammonia emissions in the western USA showed a gradual increase over the past decade, while ammonium concentrations and deposition in snowpacks and wetfall showed upward trends. Emissions of nitrogen oxides and sulfur dioxide declined while regional trends in snowpack and wetfall concentrations and deposition of nitrate and sulfate were downward.

  2. Fecal hydroxysteroid dehydrogenase activities in vegetarian Seventh-Day Adventists, control subjects, and bowel cancer patients.

    Science.gov (United States)

    Macdonald, I A; Webb, G R; Mahony, D E

    1978-10-01

    Cell-free extracts were prepared from mixed fecal anaerobic bacteria grown from stools of 14 vegetarian Seventh-Day Adventists, 16 omnivorous control subjects, and eight patients recently diagnosed with cancer of the large bowel. Preparations were assayed for NAD- and NADP-dependent 3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenases with bile salts and androsterone as substrates (eight substrate-cofactor combinations were tested). A significant intergroup difference was observed in the amounts of NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenase produced: bowel cancer patients exceeded controls, and controls exceeded Seventh-Day Adventists. Other enzyme activity comparisons were not significant. The pH values of the stools were significantly higher in cancer patients compared to Seventh-Day Adventists; values were 7.03 +/- 0.60 and 6.46 +/- 0.58 respectively. The pH value for controls was 6.66 +/- 0.62. A plot of pH value versus NADP-dependent 7alpha-hydroxysteroid dehydrogenase tended to separate the cancer patients from the other groups. Comparative data suggest that much of the 3alpha-hydroxysteroid dehydrogenase active against bile salt is also active against androsterone.

  3. A reductive aminase from Aspergillus oryzae

    Science.gov (United States)

    Aleku, Godwin A.; France, Scott P.; Man, Henry; Mangas-Sanchez, Juan; Montgomery, Sarah L.; Sharma, Mahima; Leipold, Friedemann; Hussain, Shahed; Grogan, Gideon; Turner, Nicholas J.

    2017-10-01

    Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l-1 d-1.

  4. Spatial and temporal variation in sources of atmospheric nitrogen deposition in the Rocky Mountains using nitrogen isotopes

    Science.gov (United States)

    Nanus, Leora; Campbell, Donald H.; Lehmann, Christopher M. B.; Mast, M. Alisa

    2018-03-01

    Variation in source areas and source types of atmospheric nitrogen (N) deposition to high-elevation ecosystems in the Rocky Mountains were evaluated using spatially and temporally distributed N isotope data from atmospheric deposition networks for 1995-2016. This unique dataset links N in wet deposition and snowpack to mobile and stationary emissions sources, and enhances understanding of the impacts of anthropogenic activities and environmental policies that mitigate effects of accelerated N cycling across the Rocky Mountain region. δ15N-NO3- at 50 U.S. Geological Survey Rocky Mountain Snowpack (Snowpack) sites ranged from -3.3‰ to +6.5‰, with a mean value of +1.4‰. At 15 National Atmospheric Deposition Program (NADP)/National Trends Network wet deposition (NADP Wetfall) sites, summer δ15N-NO3- is significantly lower ranging from -7.6‰ to -1.3‰ while winter δ15N-NO3- ranges from -2.6‰ to +5.5‰, with a mean value of +0.7‰ during the cool season. The strong seasonal difference in NADP Wetfall δ15N-NO3- is due in part to variation in the proportion of N originating from source regions at different times of the year due to seasonal changes in weather patterns. Snowpack NO3- and δ15N-NO3- are significantly related to NADP Wetfall (fall and winter) suggesting that bulk snowpack samples provide a reliable estimate at high elevations. Spatial trends show higher NO3- concentrations and δ15N-NO3- in the Southern Rocky Mountains located near larger anthropogenic N emission sources compared to the Northern Rocky Mountains. NADP Wetfall δ15N-NH4+ ranged from -10‰ to 0‰, with no observed spatial pattern. However, the lowest δ15N-NH4+(-9‰), and the highest NH4+ concentration (35 μeq/L) were observed at a Utah site dominated by local agricultural activities, whereas the higher δ15N-NH4+ observed in Colorado and Wyoming are likely due to mixed sources, including fossil fuel combustion and agricultural sources. These findings show spatial and

  5. Spatial and temporal variation in sources of atmospheric nitrogen deposition in the Rocky Mountains using nitrogen isotopes

    Science.gov (United States)

    Nanus, Leora; Campbell, Donald H.; Lehmann, Christopher M.B.; Mast, M. Alisa

    2018-01-01

    Variation in source areas and source types of atmospheric nitrogen (N) deposition to high-elevation ecosystems in the Rocky Mountains were evaluated using spatially and temporally distributed N isotope data from atmospheric deposition networks for 1995-2016. This unique dataset links N in wet deposition and snowpack to mobile and stationary emissions sources, and enhances understanding of the impacts of anthropogenic activities and environmental policies that mitigate effects of accelerated N cycling across the Rocky Mountain region. δ15N−NO3− at 50 U.S. Geological Survey Rocky Mountain Snowpack (Snowpack) sites ranged from −3.3‰ to +6.5‰, with a mean value of +1.4‰. At 15 National Atmospheric Deposition Program (NADP)/National Trends Network wet deposition (NADP Wetfall) sites, summer δ15N−NO3− is significantly lower ranging from −7.6‰ to −1.3‰ while winter δ15N−NO3− ranges from −2.6‰ to +5.5‰, with a mean value of +0.7‰ during the cool season. The strong seasonal difference in NADP Wetfall δ15N−NO3− is due in part to variation in the proportion of N originating from source regions at different times of the year due to seasonal changes in weather patterns. Snowpack NO3− and δ15N−NO3− are significantly related to NADP Wetfall (fall and winter) suggesting that bulk snowpack samples provide a reliable estimate at high elevations. Spatial trends show higher NO3−concentrations and δ15N−NO3− in the Southern Rocky Mountains located near larger anthropogenic N emission sources compared to the Northern Rocky Mountains. NADP Wetfall δ15N−NH4+ ranged from −10‰ to 0‰, with no observed spatial pattern. However, the lowest δ15N−NH4+(−9‰), and the highest NH4+ concentration (35 μeq/L) were observed at a Utah site dominated by local agricultural activities, whereas the higher δ15N−NH4+observed in Colorado and Wyoming are likely due to mixed sources, including fossil fuel combustion and

  6. Pathways of primary CO2 fixation in C-4 plants of the family chenopodiaceae from the arid zone of central Asia

    International Nuclear Information System (INIS)

    P'yankov, V.I.; Vakhrusheva, D.V.

    1991-01-01

    The authors determined the kinetics of 14 C incorporation after a 10-sec gulp of 14 CO 2 to study formation of primary photosynthetic products in 35 C-4 species of the family chenopodiaceae in the desert zone of central Asia. Representatives of the main life forms of desert plants with various types of anatomical structure of the assimilating organs (salsoloid, kochioid, atriplercoid, and suaedoid groups) were among the studied species. It was demonstrated that the ratio of pathways of formation of C-4 acids (malate and aspartate) depends upon the structural type affiliation and the ecological conditions of the habitat. Plants of the suaedoid and atriplecoid groups (which usually dwell under conditions of salination) exhibit the aspartate NAD-malic enzyme (NAD-ME) type of C-4 photosynthesis. The malate and aspartate types of metabolism are detected in species of the kochioid group. All biochemical variants (NADP-ME, NAD-ME, and mixed) are represented in the salsoloid group. Psammophilous species of this group have the malate type of acid metabolism, whereas halophilic species have the aspartate type. Quantitative criteria are proposed for referring C-4 plants to the NAD- and NADP-ME types on the basis of formation of primary products. It is demonstrated that the role of the aspartate pathway of CO 2 transfer in plants increases in proportion to worsening of ecological conditions of the habitat. The evolutionary sequence of metabolic systems was formed in the C-3-NAD-ME-NADP-ME direction, the origin of the C-4 syndrome having been associated with colonization of arid territories, especially saline localities

  7. Substrate specific effects of calcium on metabolism of rat heart mitochondria.

    Science.gov (United States)

    Panov, A V; Scaduto, R C

    1996-04-01

    Oxidative metabolism in the heart is tightly coupled to mechanical work. Because this coupling process is believed to involve Ca2+, the roles of mitochondrial Ca2+ in the regulation of oxidative phosphorylation was studied in isolated rat heart mitochondria. The electrical component of the mitochondrial membrane potential (delta psi) and the redox state of the pyridine nucleotides were determined during the oxidation of various substrates under different metabolic states. In the absence of added adenine nucleotides, the NADP+ redox couple was almost completely reduced, regardless of the specific substrate and the presence of Ca2+, whereas NAD+ couple redox state was highly dependent on the substrate type and the presence of Ca2+. Titration of respiration with ADP, in the presence of excess hexokinase and glucose, showed that both respiration and NAD(P)+ reduction were very sensitive to ADP. The maximal enzyme reaction rate of ADP-stimulated respiration Michaelis constants (Km) for ADP were dependent on the particular substrate employed. delta psi was much less sensitive to ADP. With either alpha-ketoglutarate or glutamate as substrate, Ca2+ significantly increased reduction of NAD(P)+.Ca2+ did not influence NAD(P)+ reduction with either acetylcarnitine or pyruvate as substrate. In the presence of ADP, delta psi was increased by Ca2+ at all metabolic states with glutamate plus malate, 0.5 mM alpha-ketoglutarate plus malate, or pyruvate plus malate as substrates. The data presented support the hypothesis that cardiac respiration is controlled by the availability of both Ca2+ and ADP to mitochondria. The data indicate that an increase in substrate supply to mitochondria can increase mitochondrial respiration at given level of ADP. This effect can be produced by Ca2+ with substrates such as glutamate, which utilize alpha-ketoglutarate dehydrogenase activity for oxidation. Increases in respiration by Ca2+ may mitigate an increase in ADP during periods of increased

  8. Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

    Science.gov (United States)

    Chocklett, Samuel W; Sobrado, Pablo

    2010-08-10

    Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism.

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

    Directory of Open Access Journals (Sweden)

    Gerda Venter

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

  10. Conserved and divergent rhythms of crassulacean acid metabolism-related and core clock gene expression in the cactus Opuntia ficus-indica.

    Science.gov (United States)

    Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

    2011-08-01

    The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional

  11. Conserved and Divergent Rhythms of Crassulacean Acid Metabolism-Related and Core Clock Gene Expression in the Cactus Opuntia ficus-indica1[C][W

    Science.gov (United States)

    Mallona, Izaskun; Egea-Cortines, Marcos; Weiss, Julia

    2011-01-01

    The cactus Opuntia ficus-indica is a constitutive Crassulacean acid metabolism (CAM) species. Current knowledge of CAM metabolism suggests that the enzyme phosphoenolpyruvate carboxylase kinase (PPCK) is circadian regulated at the transcriptional level, whereas phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), NADP-malic enzyme (NADP-ME), and pyruvate phosphate dikinase (PPDK) are posttranslationally controlled. As little transcriptomic data are available from obligate CAM plants, we created an expressed sequence tag database derived from different organs and developmental stages. Sequences were assembled, compared with sequences in the National Center for Biotechnology Information nonredundant database for identification of putative orthologs, and mapped using Kyoto Encyclopedia of Genes and Genomes Orthology and Gene Ontology. We identified genes involved in circadian regulation and CAM metabolism for transcriptomic analysis in plants grown in long days. We identified stable reference genes for quantitative polymerase chain reaction and found that OfiSAND, like its counterpart in Arabidopsis (Arabidopsis thaliana), and OfiTUB are generally appropriate standards for use in the quantification of gene expression in O. ficus-indica. Three kinds of expression profiles were found: transcripts of OfiPPCK oscillated with a 24-h periodicity; transcripts of the light-active OfiNADP-ME and OfiPPDK genes adapted to 12-h cycles, while transcript accumulation patterns of OfiPEPC and OfiMDH were arrhythmic. Expression of the circadian clock gene OfiTOC1, similar to Arabidopsis, oscillated with a 24-h periodicity, peaking at night. Expression of OfiCCA1 and OfiPRR9, unlike in Arabidopsis, adapted best to a 12-h rhythm, suggesting that circadian clock gene interactions differ from those of Arabidopsis. Our results indicate that the evolution of CAM metabolism could be the result of modified circadian regulation at both the transcriptional and posttranscriptional

  12. Highly Stable l-Lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression

    Science.gov (United States)

    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

  13. Teaching the Z-Scheme of electron transport in photosynthesis: a perspective.

    Science.gov (United States)

    Mohapatra, Pradipta Kumar; Singh, Nihar Ranjan

    2015-01-01

    This paper deals with how Govindjee taught the Z-Scheme of electron transport in oxygenic photosynthesis at Ravenshaw University, Cuttack, Odisha, India, in 2014, in a unique and highly effective fashion-using students to act as molecules, representing the entire electron transport chain from water to nicotinamide adenine dinucleotide phosphate (NADP(+)). It culminated in a show by B.Sc. students in the garden of the Department of Botany, Ravenshaw University. The first author (PKM) personally acted as Ferredoxin NADP Reductase (FNR) catalyzing the reduction of NADP(+) to NADPH, taking electrons from reduced ferredoxin at the end of Photosystem I. On the other hand, the Q-cycle was played by M.Sc. students, who acted as molecules running this ingenious cycle that produces extra protons. An interesting event was when a student, acting as a herbicide, who was dressed like a devil (fierce looking, in black clothes with a sword; "Yamaraj: The God of Death", as he called himself), stopped all reactions by throwing out QB, the second plastoquinone molecule of Photosystem II, and that too aggressively, taking its position instead. The second author was the major organizer of the Z-scheme show. We provide here a basic background on the process, a bit on Govindjee's teaching, and some selected pictures from the drama played in March, 2014 at Ravenshaw University. Here, we also recognize the teacher Govindjee for his ingenious and fun-filled teaching methods that touched the hearts and the souls of the students as well as the teachers of Ravenshaw University. He was rated as one of the most-admired teachers of plant biology at our university.

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

    Science.gov (United States)

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

    2014-01-01

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

  15. Metabolic reconstruction of Setaria italica: a systems biology approach for integrating tissue-specific omics and pathway analysis of bioenergy grasses

    Directory of Open Access Journals (Sweden)

    Cristiana Gomes De Oliveira Dal'molin

    2016-08-01

    Full Text Available The urgent need for major gains in industrial crops productivity and in biofuel production from bioenergy grasses have reinforced attention on understanding C4 photosynthesis. Systems biology studies of C4 model plants may reveal important features of C4 metabolism. Here we chose foxtail millet (Setaria italica, as a C4 model plant and developed protocols to perform systems biology studies. As part of the systems approach, we have developed and used a genome-scale metabolic reconstruction in combination with the use of multi-omics technologies to gain more insights into the metabolism of S.italica. mRNA, protein and metabolite abundances, were measured in mature and immature stem/leaf phytomers and the multi-omics data were integrated into the metabolic reconstruction framework to capture key metabolic features in different developmental stages of the plant. RNA-Seq reads were mapped to the S. italica resulting for 83% coverage of the protein coding genes of S. italica. Besides revealing similarities and differences in central metabolism of mature and immature tissues, transcriptome analysis indicates significant gene expression of two malic enzyme isoforms (NADP- ME and NAD-ME. Although much greater expression levels of NADP-ME genes are observed and confirmed by the correspondent protein abundances in the samples, the expression of multiple genes combined to the significant abundance of metabolites that participates in C4 metabolism of NAD-ME and NADP-ME subtypes suggest that S. italica may use mixed decarboxylation modes of C4 photosynthetic pathways under different plant developmental stages. The overall analysis also indicates different levels of regulation in mature and immature tissues in carbon fixation, glycolysis, TCA cycle, amino acids, fatty acids, lignin and cellulose syntheses. Altogether, the multi-omics analysis reveals different biological entities and their interrelation and regulation over plant development. With this study

  16. Metabolic Reconstruction of Setaria italica: A Systems Biology Approach for Integrating Tissue-Specific Omics and Pathway Analysis of Bioenergy Grasses.

    Science.gov (United States)

    de Oliveira Dal'Molin, Cristiana G; Orellana, Camila; Gebbie, Leigh; Steen, Jennifer; Hodson, Mark P; Chrysanthopoulos, Panagiotis; Plan, Manuel R; McQualter, Richard; Palfreyman, Robin W; Nielsen, Lars K

    2016-01-01

    The urgent need for major gains in industrial crops productivity and in biofuel production from bioenergy grasses have reinforced attention on understanding C4 photosynthesis. Systems biology studies of C4 model plants may reveal important features of C4 metabolism. Here we chose foxtail millet (Setaria italica), as a C4 model plant and developed protocols to perform systems biology studies. As part of the systems approach, we have developed and used a genome-scale metabolic reconstruction in combination with the use of multi-omics technologies to gain more insights into the metabolism of S. italica. mRNA, protein, and metabolite abundances, were measured in mature and immature stem/leaf phytomers, and the multi-omics data were integrated into the metabolic reconstruction framework to capture key metabolic features in different developmental stages of the plant. RNA-Seq reads were mapped to the S. italica resulting for 83% coverage of the protein coding genes of S. italica. Besides revealing similarities and differences in central metabolism of mature and immature tissues, transcriptome analysis indicates significant gene expression of two malic enzyme isoforms (NADP- ME and NAD-ME). Although much greater expression levels of NADP-ME genes are observed and confirmed by the correspondent protein abundances in the samples, the expression of multiple genes combined to the significant abundance of metabolites that participates in C4 metabolism of NAD-ME and NADP-ME subtypes suggest that S. italica may use mixed decarboxylation modes of C4 photosynthetic pathways under different plant developmental stages. The overall analysis also indicates different levels of regulation in mature and immature tissues in carbon fixation, glycolysis, TCA cycle, amino acids, fatty acids, lignin, and cellulose syntheses. Altogether, the multi-omics analysis reveals different biological entities and their interrelation and regulation over plant development. With this study, we demonstrated

  17. An in silico analysis of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

    Directory of Open Access Journals (Sweden)

    SIMON BROWN

    2014-04-01

    Full Text Available Nematode glutamate dehydrogenase (GDH amino acid sequences are very highly conserved (68-99% identity and are also very similar to those of the bovine and human enzymes (54-60% identity. The residues involved in binding nucleotides or substrates are completely conserved and tend to be located in highly conserved regions of the sequence. Based on the strong homology between the bovine, Teladorsagia circumcincta and Haemonchus contortus GDH sequences, models of the structure of the T. circumcincta and H. contortus monomers were constructed. The structure of the T. circumcincta monomer obtained using SWISS-MODEL was very similar to that of the bovine enzyme monomer and the backbone of the polypetide deviated very little from that of the bovine enzyme monomer. Despite the sequence differences between the bovine and T. circumcincta enzymes, the relative positions and orientations of the residues involved in ligand binding were very similar. The reported Km for NADP+ of T. circumcincta is about 35 and times that of the bovine enzyme, whereas the Kms of the two enzymes for glutamate, -ketoglutarate and NAD(PH are much more similar. The residue corresponding to S267 of the bovine enzyme is involved in binding the 2′-phosphate of NADP+ and is replaced in the T. circumcincta and H. contortus sequences by a tryptophan. The partial occlusion of the NAD(P-binding site by the tryptophan sidechain and the loss of at least one potential H-bond provided by the serine may explain the lower affinity of the T. circumcincta for NADP+.

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

    Science.gov (United States)

    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.

  19. RDH13L, an enzyme responsible for the aldehyde-alcohol redox coupling reaction (AL-OL coupling reaction) to supply 11-cis retinal in the carp cone retinoid cycle.

    Science.gov (United States)

    Sato, Shinya; Miyazono, Sadaharu; Tachibanaki, Shuji; Kawamura, Satoru

    2015-01-30

    Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Effects of equipment performance on data quality from the National Atmospheric Deposition Program/National Trends Network and the Mercury Deposition Network

    Science.gov (United States)

    Wetherbee, Gregory A.; Rhodes, Mark F.

    2013-01-01

    The U.S. Geological Survey Branch of Quality Systems operates the Precipitation Chemistry Quality Assurance project (PCQA) to provide independent, external quality-assurance for the National Atmospheric Deposition Program (NADP). NADP is composed of five monitoring networks that measure the chemical composition of precipitation and ambient air. PCQA and the NADP Program Office completed five short-term studies to investigate the effects of equipment performance with respect to the National Trends Network (NTN) and Mercury Deposition Network (MDN) data quality: sample evaporation from NTN collectors; sample volume and mercury loss from MDN collectors; mercury adsorption to MDN collector glassware, grid-type precipitation sensors for precipitation collectors, and the effects of an NTN collector wind shield on sample catch efficiency. Sample-volume evaporation from an NTN Aerochem Metrics (ACM) collector ranged between 1.1–33 percent with a median of 4.7 percent. The results suggest that weekly NTN sample evaporation is small relative to sample volume. MDN sample evaporation occurs predominantly in western and southern regions of the United States (U.S.) and more frequently with modified ACM collectors than with N-CON Systems Inc. collectors due to differences in airflow through the collectors. Variations in mercury concentrations, measured to be as high as 47.5 percent per week with a median of 5 percent, are associated with MDN sample-volume loss. Small amounts of mercury are also lost from MDN samples by adsorption to collector glassware irrespective of collector type. MDN 11-grid sensors were found to open collectors sooner, keep them open longer, and cause fewer lid cycles than NTN 7-grid sensors. Wind shielding an NTN ACM collector resulted in collection of larger quantities of precipitation while also preserving sample integrity.

  1. Characterization and expression of the maize β-carbonic anhydrase gene repeat regions.

    Science.gov (United States)

    Tems, Ursula; Burnell, James N

    2010-12-01

    In maize, carbonic anhydrase (CA; EC 4.2.1.1) catalyzes the first reaction of the C(4) photosynthetic pathway; it catalyzes the hydration of CO(2) to bicarbonate and provides an inorganic carbon source for the primary carboxylation reaction catalyzed by phosphoenolpyruvate (PEP) carboxylase. The β-CA isozymes from maize, as well as other agronomically important NADP-malic enzyme (NADP-ME) type C(4) crops, have remained relatively uncharacterized but differ significantly from the β-CAs of other C(4) monocot species primarily due to transcript length and the presence of repeat sequences. This research confirmed earlier findings of repeat sequences in maize CA transcripts, and demonstrated that the gene encoding these transcripts is also composed of repeat sequences. One of the maize CA genes was sequenced and found to encode two domains, with distinct groups of exons corresponding to the repeat regions of the transcript. We have also shown that expression of a single repeat region of the CA transcript produced active enzyme that associated as a dimer and was composed primarily of α-helices, consistent with that observed for other plant CAs. As the presence of repeat regions in the CA gene is unique to NADP-ME type C(4) monocot species, the implications of these findings in the context of the evolution of the location and function of this C(4) pathway enzyme are strongly suggestive of CA gene duplication resulting in an evolutionary advantage and a higher photosynthetic efficiency. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  2. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States); Totah, Rheem A., E-mail: rtotah@u.washington.edu [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

  3. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    International Nuclear Information System (INIS)

    Kaspera, Rüdiger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

    2012-01-01

    Highlights: ► Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k cat ∼ 25 min −1 ). ► Reduction is a direct hydride transfer from R-NADP 2 H to the carbonyl moiety. ► P450 domain variants enhance reduction through potential allosteric/redox interactions. ► Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k cat of ∼25 min −1 was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP 2 H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP 2 H but not D 2 O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

  4. Inhibition of citric acid accumulation by manganese ions in Aspergillus niger mutants with reduced citrate control of phosphofructokinase

    Energy Technology Data Exchange (ETDEWEB)

    Schreferl, G.; Kubicek, C.P.; Roehr, M.

    1986-03-01

    Mutant strains of Aspergillus niger with reduced citrate control of carbohydrate catabolism (cic mutants) grow faster than the parent strain on media containing 5% (wt/vol) citrate. The mutants tolerated a higher intracellular citrate concentration than the parent strain. One mutant (cic-7/3) contained phosphofructokinase activity significantly less sensitive towards citrate than the enzyme from the parent strain. When this mutant was grown under citrate accumulating conditions, acidogenesis was far less sensitive to inhibition by Mn/sup 2 +/ than in the parent strain. Some of the cic mutants also showed altered citrate inhibition of NADP-specific isocitrate dehydrogenase.

  5. Optimizing Cofactor Specificity of Oxidoreductase Enzymes for the Generation of Microbial Production Strains—OptSwap

    DEFF Research Database (Denmark)

    King, Zachary A.; Feist, Adam

    2013-01-01

    Central oxidoreductase enzymes (eg, dehydrogenases, reductases) in microbial metabolism often have preferential binding specificity for one of the two major currency metabolites NAD(H) and NADP(H). These enzyme specificities result in a division of the metabolic functionality of the currency...... specificities of oxidoreductase enzyme and complementary reaction knockouts. Using the Escherichia coli genome-scale metabolic model iJO1366, OptSwap predicted eight growth-coupled production designs with significantly greater product yields or substrate-specific productivities than designs predicted with gene...

  6. Identification of differentially expressed proteins during human urinary bladder cancer progression

    DEFF Research Database (Denmark)

    Memon, Ashfaque Ahmed; chang, Jong. w; Oh, Bong R.

    2005-01-01

    and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder...... cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may...

  7. Hypomorphic Temperature-Sensitive Alleles of NSDHL Cause CK Syndrome

    OpenAIRE

    McLarren, Keith W.; Severson, Tesa M.; du Souich, Christèle; Stockton, David W.; Kratz, Lisa E.; Cunningham, David; Hendson, Glenda; Morin, Ryan D.; Wu, Diane; Paul, Jessica E.; An, Jianghong; Nelson, Tanya N.; Chou, Athena; DeBarber, Andrea E.; Merkens, Louise S.

    2010-01-01

    CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholestero...

  8. Reaction Engineering of Biocatalytic Enantioselective Reduction: A Case Study for Aliphatic Ketones

    DEFF Research Database (Denmark)

    Leuchs, Susanne; Lima-Ramos, Joana; Greiner, Lasse

    2013-01-01

    , 15, 167–176.). In the present work, the process metrics of the ketone reduction were calculated. A cost analysis revealed that the enzyme costs are negligible, but the cost for nicotinamide cofactor NADP+ is dominating the overall cost of the chemical raw material followed by the ionic liquid (TEGO...... IL K5) used as solubiliser and the buffer. The overall cost of chemicals was €148/kgproduct. To assess the environmental impact of the process, the E-factor (kgwaste/kgproduct) 132 and the process mass intensity 133 (PMI, kgsubstrate/kgproduct) were calculated. A process model based on initial rate...

  9. JST Thesaurus Headwords and Synonyms: NADPトランスヒドロゲナーゼ [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term NADPトランスヒドロゲナーゼ 名詞 一般 * * * * NAD...(P)トランスヒドロゲナーゼ NAD(P)トランスヒドロゲナーゼ エヌエイディー(ピー)トランスヒドロゲナーゼ Thesaurus2015 200906007955537034 C LS38 UNKNOWN_2 NADP トランスヒドロゲナーゼ

  10. JST Thesaurus Headwords and Synonyms: NADPグリコヒドロラーゼ [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term NADPグリコヒドロラーゼ 名詞 一般 * * * * NADヌク...レオシダーゼ NADヌクレオシダーゼ エヌエイディーヌクレオシダーゼ Thesaurus2015 200906060754826613 C LS38 UNKNOWN_2 NADP グリコヒドロラーゼ

  11. JST Thesaurus Headwords and Synonyms: NADPトランスヒドロゲナーゼ類 [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term NADPトランスヒドロゲナーゼ類 名詞 一般 * * * * NA...D(P)トランスヒドロゲナーゼ NAD(P)トランスヒドロゲナーゼ エヌエイディー(ピー)トランスヒドロゲナーゼ Thesaurus2015 200906007955537034 C LS38 UNKNOWN_2 NADP トランスヒドロゲナーゼ 類

  12. A reductive aminase from Aspergillus oryzae

    OpenAIRE

    Grogan, Gideon James; Aleku, Godwin; France, Scott; Man, Henry Wing-Hong; Mangas-Sanchez, Juan; Sharma, Mahima; Montgomery, Sarah L; Leipold, Friedemann; Hussain, Shahed; Turner, Nicholas

    2017-01-01

    Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae (AspRedAm, Uniprot code Q2TW47) which can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to empl...

  13. Data mining of the transcriptome of Plasmodium falciparum: the pentose phosphate pathway and ancillary processes

    Directory of Open Access Journals (Sweden)

    Ginsburg Hagai

    2005-03-01

    Full Text Available Abstract The general paradigm that emerges from the analysis of the transcriptome of the malaria parasite Plasmodium falciparum is that the expression clusters of genes that code for enzymes engaged in the same cellular function is coordinated. Here the consistency of this perception is examined by analysing specific pathways that metabolically-linked. The pentose phosphate pathway (PPP is a fundamental element of cell biochemistry since it is the major pathway for the recycling of NADP+ to NADPH and for the production of ribose-5-phosphate that is needed for the synthesis of nucleotides. The function of PPP depends on the synthesis of NADP+ and thiamine pyrophosphate, a co-enzyme of the PPP enzyme transketolase. In this essay, the transcription of gene coding for enzymes involved in the PPP, thiamine and NAD(P+ syntheses are analysed. The genes coding for two essential enzymes in these pathways, transaldolase and NAD+ kinase could not be found in the genome of P. falciparum. It is found that the transcription of the genes of each pathway is not always coordinated and there is usually a gene whose transcription sets the latest time for the full deployment of the pathway's activity. The activity of PPP seems to involve only the oxidative arm of PPP that is geared for maximal NADP+ reduction and ribose-5-phosphate production during the early stages of parasite development. The synthesis of thiamine diphosphate is predicted to occur much later than the expression of transketolase. Later in the parasite cycle, the non-oxidative arm of PPP that can use fructose-6-phosphate and glyceraldehyde-3-phosphate supplied by glycolysis, becomes fully deployed allowing to maximize the production of ribose-5-phosphate. These discrepancies require direct biochemical investigations to test the activities of the various enzymes in the developing parasite. Notably, several transcripts of PPP enzyme-coding genes display biphasic pattern of transcription unlike most

  14. Spatial and temporal regulation of the metabolism of reactive oxygen and nitrogen species during the early development of pepper (Capsicum annuum) seedlings.

    Science.gov (United States)

    Airaki, Morad; Leterrier, Marina; Valderrama, Raquel; Chaki, Mounira; Begara-Morales, Juan C; Barroso, Juan B; del Río, Luis A; Palma, José M; Corpas, Francisco J

    2015-09-01

    The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development. The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate-glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide ((·)NO), superoxide radical (O2 (·-)) and peroxynitrite (ONOO(-)) was investigated using confocal laser scanning microscopy. The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme. There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H2O2 and NO have implications for signalling, and their temporal and spatial regulation contributes

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

    Science.gov (United States)

    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.

  16. Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

    DEFF Research Database (Denmark)

    Nissen, T.L.; Anderlund, M.; Nielsen, Jens

    2001-01-01

    The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/ADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can...... cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein...

  17. Las quinoproteínas alcohol deshidrogenasas en los sistemas bacterianos: distribución, clasificación, estructura y función

    OpenAIRE

    Saúl Gómez-Manzo; Roberto Arreguín-Espinosa; Martha Contreras-Zentella; Edgardo Escamilla-Marván

    2005-01-01

    Existe una gran diversidad de alcohol deshidrogenasas (ADHs) microbianas; las cuales son divididas en tres grandes grupos: (a) Las que son dependientes de las coenzimas NAD o NADP, (b) Las que son independientes de estas coenzimas; sin embargo, utilizan pirroloquinolina quinona (PQQ) y hemo tipo C como grupo prostético y (c) Las oxidasas dependientes de FAD que catalizan la reacción irreversible de alcoholes. Las ADHs que utilizan el PQQ, se encuentran a su vez divididas en tres t...

  18. Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

    Science.gov (United States)

    Ding, Wei; Si, Meiru; Zhang, Weipeng; Zhang, Yaoling; Chen, Can; Zhang, Lei; Lu, Zhiqiang; Chen, Shaolin; Shen, Xihui

    2015-01-01

    Vanillin dehydrogenase (VDH) is a crucial enzyme involved in the degradation of lignin-derived aromatic compounds. Herein, the VDH from Corynebacterium glutamicum was characterized. The relative molecular mass (Mr) determined by SDS-PAGE was ~51kDa, whereas the apparent native Mr values revealed by gel filtration chromatography were 49.5, 92.3, 159.0 and 199.2kDa, indicating the presence of dimeric, trimeric and tetrameric forms. Moreover, the enzyme showed its highest level of activity toward vanillin at pH 7.0 and 30C, and interestingly, it could utilize NAD+ and NADP+ as coenzymes with similar efficiency and showed no obvious difference toward NAD+ and NADP+. In addition to vanillin, this enzyme exhibited catalytic activity toward a broad range of substrates, including p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, o-phthaldialdehyde, cinnamaldehyde, syringaldehyde and benzaldehyde. Conserved catalytic residues or putative cofactor interactive sites were identified based on sequence alignment and comparison with previous studies, and the function of selected residues were verified by site-directed mutagenesis analysis. Finally, the vdh deletion mutant partially lost its ability to grow on vanillin, indicating the presence of alternative VDH(s) in Corynebacterium glutamicum. Taken together, this study contributes to understanding the VDH diversity from bacteria and the aromatic metabolism pathways in C. glutamicum. PMID:25622822

  19. Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis.

    Science.gov (United States)

    Wang, Guodong; Pichersky, Eran

    2007-03-01

    Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which is derived from NAD, have important roles as a redox carriers in metabolism. A combination of de novo and salvage pathways contribute to the biosynthesis of NAD in all organisms. The pathways and enzymes of the NAD salvage pathway in yeast and animals, which diverge at nicotinamide, have been extensively studied. Yeast cells convert nicotinamide to nicotinic acid, while mammals lack the enzyme nicotinamidase and instead convert nicotinamide to nicotinamide mononucleotide. Here we show that Arabidopsis thaliana gene At2g22570 encodes a nicotinamidase, which is expressed in all tissues, with the highest levels observed in roots and stems. The 244-residue protein, designated AtNIC1, converts nicotinamide to nicotinic acid and has a Km value of 118 +/- 17 microM and a Kcat value of 0.93 +/- 0.13 sec(-1). Plants homozygous for a null AtNIC1 allele, nic1-1, have lower levels of NAD and NADP under normal growth conditions, indicating that AtNIC1 participates in a yeast-type NAD salvage pathway. Mutant plants also exhibit hypersensitivity to treatments of abscisic acid and NaCl, which is correlated with their inability to increase the cellular levels of NAD(H) under these growth conditions, as occurs in wild-type plants. We also show that the growth of the roots of wild-type but not nic1-1 mutant plants is inhibited and distorted by nicotinamide.

  20. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    International Nuclear Information System (INIS)

    Ciulli, Alessio; Lobley, Carina M. C.; Tuck, Kellie L.; Smith, Alison G.; Blundell, Tom L.; Abell, Chris

    2007-01-01

    A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP + that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP + , with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes

  1. Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Bhumika S., E-mail: bhumika.shah@mq.edu.au; Tetu, Sasha G. [Macquarie University, Research Park Drive, Sydney, NSW 2109 (Australia); Harrop, Stephen J. [University of New South Wales, Sydney, NSW 2052 (Australia); Paulsen, Ian T.; Mabbutt, Bridget C. [Macquarie University, Research Park Drive, Sydney, NSW 2109 (Australia)

    2014-09-25

    The structure of a short-chain dehydrogenase encoded within genomic islands of A. baumannii strains has been solved to 2.4 Å resolution. This classical SDR incorporates a flexible helical subdomain. The NADP-binding site and catalytic side chains are identified. Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access.

  2. Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii

    International Nuclear Information System (INIS)

    Shah, Bhumika S.; Tetu, Sasha G.; Harrop, Stephen J.; Paulsen, Ian T.; Mabbutt, Bridget C.

    2014-01-01

    The structure of a short-chain dehydrogenase encoded within genomic islands of A. baumannii strains has been solved to 2.4 Å resolution. This classical SDR incorporates a flexible helical subdomain. The NADP-binding site and catalytic side chains are identified. Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access

  3. Improved daily precipitation nitrate and ammonium concentration models for the Chesapeake Bay Watershed

    International Nuclear Information System (INIS)

    Grimm, J.W.; Lynch, J.A.

    2005-01-01

    Daily precipitation nitrate and ammonium concentration models were developed for the Chesapeake Bay Watershed (USA) using a linear least-squares regression approach and precipitation chemistry data from 29 National Atmospheric Deposition Program/National Trends Network (NADP/NTN) sites. Only weekly samples that comprised a single precipitation event were used in model development. The most significant variables in both ammonium and nitrate models included: precipitation volume, the number of days since the last event, a measure of seasonality, latitude, and the proportion of land within 8 km covered by forest or devoted to industry and transportation. Additional variables included in the nitrate model were the proportion of land within 0.8 km covered by water and/or forest. Local and regional ammonia and nitrogen oxide emissions were not as well correlated as land cover. Modeled concentrations compared very well with event chemistry data collected at six NADP/AirMoN sites within the Chesapeake Bay Watershed. Wet deposition estimates were also consistent with observed deposition at selected sites. Accurately describing the spatial distribution of precipitation volume throughout the watershed is important in providing critical estimates of wet-fall deposition of ammonium and nitrate. - A linear least-squares regression approach was used to develop daily precipitation nitrate and ammonium concentration models for the Chesapeake Bay Watershed

  4. Preprotein import into chloroplasts via the Toc and Tic complexes is regulated by redox signals in Pisum sativum.

    Science.gov (United States)

    Stengel, Anna; Benz, J Philipp; Buchanan, Bob B; Soll, Jürgen; Bölter, Bettina

    2009-11-01

    The import of nuclear-encoded preproteins is necessary to maintain chloroplast function. The recognition and transfer of most precursor proteins across the chloroplast envelopes are facilitated by two membrane-inserted protein complexes, the translocons of the chloroplast outer and inner envelope (Toc and Tic complexes, respectively). Several signals have been invoked to regulate the import of preproteins. In our study, we were interested in redox-based import regulation mediated by two signals: regulation based on thiols and on the metabolic NADP+/NADPH ratio. We sought to identify the proteins participating in the regulation of these transport pathways and to characterize the preprotein subgroups whose import is redox-dependent. Our results provide evidence that the formation and reduction of disulfide bridges in the Toc receptors and Toc translocation channel have a strong influence on import yield of all tested preproteins that depend on the Toc complex for translocation. Furthermore, the metabolic NADP+/NADPH ratio influences not only the composition of the Tic complex, but also the import efficiency of most, but not all, preproteins tested. Thus, several Tic subcomplexes appear to participate in the translocation of different preprotein subgroups, and the redox-active components of these complexes likely play a role in regulating transport.

  5. Alternative photosynthetic electron transport pathways during anaerobiosis in the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Hemschemeier, Anja; Happe, Thomas

    2011-08-01

    Oxygenic photosynthesis uses light as energy source to generate an oxidant powerful enough to oxidize water into oxygen, electrons and protons. Upon linear electron transport, electrons extracted from water are used to reduce NADP(+) to NADPH. The oxygen molecule has been integrated into the cellular metabolism, both as the most efficient electron acceptor during respiratory electron transport and as oxidant and/or "substrate" in a number of biosynthetic pathways. Though photosynthesis of higher plants, algae and cyanobacteria produces oxygen, there are conditions under which this type of photosynthesis operates under hypoxic or anaerobic conditions. In the unicellular green alga Chlamydomonas reinhardtii, this condition is induced by sulfur deficiency, and it results in the production of molecular hydrogen. Research on this biotechnologically relevant phenomenon has contributed largely to new insights into additional pathways of photosynthetic electron transport, which extend the former concept of linear electron flow by far. This review summarizes the recent knowledge about various electron sources and sinks of oxygenic photosynthesis besides water and NADP(+) in the context of their contribution to hydrogen photoproduction by C. reinhardtii. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    Science.gov (United States)

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  7. Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances.

    Directory of Open Access Journals (Sweden)

    Du Toit W P Schabort

    Full Text Available The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production.

  8. pH-tuneable binding of 2′-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Ciulli, Alessio [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Lobley, Carina M. C. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Tuck, Kellie L. [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom); Smith, Alison G. [Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA (United Kingdom); Blundell, Tom L. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Abell, Chris, E-mail: ca26@cam.ac.uk [University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

    2007-02-01

    A combined crystallographic, calorimetric and mutagenic study has been used to show how changes in pH give rise to two distinct binding modes of 2′-phospho-ADP-ribose to ketopantoate reductase. The crystal structure of Escherichia coli ketopantoate reductase in complex with 2′-monophosphoadenosine 5′-diphosphoribose, a fragment of NADP{sup +} that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP{sup +}, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ‘reversed binding mode’ observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.

  9. A novel lung slice system with compromised antioxidant defenses

    Energy Technology Data Exchange (ETDEWEB)

    Hardwick, S.J.; Adam, A.; Cohen, G.M. (Univ. of London (England)); Smith, L.L. (Imperial Chemical Industries PLC, Cheshire (England))

    1990-04-01

    In order to facilitate the study of oxidative stress in lung tissue, rat lung slices with impaired antioxidant defenses were prepared and used. Incubation of lung slices with the antineoplastic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 {mu}M) in an amino acid-rich medium for 45 min produced a near-maximal (approximately 85%), irreversible inhibition of glutathione reductase, accompanied by only a modest (approximately 15%) decrease in pulmonary nonprotein sulfhydryls (NPSH) and no alteration in intracellular ATP, NADP{sup +}, and NADPH levels. The amounts of NADP(H), ATP, and NPSH were stable over a 4-hr incubation period following the removal from BCNU. The viability of the system was further evaluated by measuring the rate of evolution of {sup 14}CO{sub 2} from D-({sup 14}C(U))-glucose. The rates of evolution were almost identical in the compromised system when compared with control slices over a 4-hr time period. By using slices with compromised oxidative defenses, preliminary results have been obtained with paraquat, nitrofurantoin, and 2,3-dimethoxy-1,4-naphthoquinone.

  10. Near-UV radiation acts as a beneficial factor for physiological responses in cucumber plants.

    Science.gov (United States)

    Mitani-Sano, Makiko; Tezuka, Takafumi

    2013-11-05

    Effects of near-UV radiation on the growth and physiological activity of cucumber plants were investigated morphologically, physiologically and biochemically using 3-week-old seedlings grown under polyvinyl chloride films featuring transmission either above 290 nm or above 400 nm in growth chambers. The hypocotyl length and leaf area of cucumber seedlings were reduced but the thickness of leaves was enhanced by near-UV radiation, due to increased upper/lower epidermis thickness, palisade parenchyma thickness and volume of palisade parenchyma cells. Photosynthetic and respiratory activities were also promoted by near-UV radiation, associated with general enhancement of physiological/biochemical responses. Particularly, metabolic activities in the photosynthetic system of chloroplasts and the respiratory system of mitochondria were analyzed under the conditions of visible light with and without near-UV radiation. For example, the activities of NAD(P)-dependent enzymes such as glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in chloroplasts and isocitrate dehydrogenase (ICDH) in mitochondria were elevated, along with levels of pyridine nucleotides (nicotinamide coenzymes) [NAD(H) and NADP(H)] and activity of NAD kinase (NADP forming enzyme). Taken together, these data suggest that promotion of cucumber plant growth by near-UV radiation involves activation of carbon and nitrogen metabolism in plants. The findings of this research showed that near-UV radiation reaching the Earth's surface is a beneficial factor for plant growth. Copyright © 2013. Published by Elsevier B.V.

  11. Immobilization of malate dehydrogenase on carbon nanotubes for development of malate biosensor.

    Science.gov (United States)

    Ruhal, A; Rana, J S; Kumar, S; Kumar, A

    2012-12-22

    An amperometric malic acid biosensor was developed by immobilizing malate dehydrogenase on multi-walled carbon nanotubes (MWCNT) coated on screen printed carbon electrode. The screen printed carbon electrode is made up of three electrodes viz., carbon as working, platinum as counter and silver as reference electrode. Detection of L-malic acid concentration provides important information about the ripening and shelf life of the fruits. The NADP specific malate dehydrogenase was immobilized on carboxylated multiwalled carbon nanotubes using cross linker EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide] on screen printed carbon electrode. An amperometric current was measured by differential pulse voltammetry (DPV) which increases with increasing concentrations of malic acid at fixed concentration of NADP. Enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. The detection limit of malic acid by the sensor was 60 - 120 μM and sensitivity of the sensor was 60 μM with a response time of 60s. The usual detection methods of malic acid are nonspecific, time consuming and less sensitive. However, an amperometric malic acid nanosensor is quick, specific and more sensitive for detection of malic acid in test samples.

  12. Attributes for MRB_E2RF1 Catchments by Major River Basins in the Conterminous United States: Normalized Atmospheric Deposition for 2002, Nitrate (NO3)

    Science.gov (United States)

    Wieczorek, Michael; LaMotte, Andrew E.

    2010-01-01

    This tabular data set represents the average normalized (wet) deposition, in kilograms per square kilometer multiplied by 100, of Nitrate (NO3) for the year 2002 compiled for every MRB_E2RF1 catchment of the Major River Basins (MRBs, Crawford and others, 2006). Estimates of NO3 deposition are based on National Atmospheric Deposition Program (NADP) measurements (B. Larsen, U.S. Geological Survey, written. commun., 2007). De-trending methods applied to the year 2002 are described in Alexander and others, 2001. NADP site selection met the following criteria: stations must have records from 1995 to 2002 and have a minimum of 30 observations. The MRB_E2RF1 catchments are based on a modified version of the U.S. Environmental Protection Agency's (USEPA) ERF1_2 and include enhancements to support national and regional-scale surface-water quality modeling (Nolan and others, 2002; Brakebill and others, 2011). Data were compiled for every MRB_E2RF1 catchment for the conterminous United States covering New England and Mid-Atlantic (MRB1), South Atlantic-Gulf and Tennessee (MRB2), the Great Lakes, Ohio, Upper Mississippi, and Souris-Red-Rainy (MRB3), the Missouri (MRB4), the Lower Mississippi, Arkansas-White-Red, and Texas-Gulf (MRB5), the Rio Grande, Colorado, and the Great basin (MRB6), the Pacific Northwest (MRB7) river basins, and California (MRB8).

  13. Attributes for MRB_E2RF1 Catchments by Major River Basins in the Conterminous United States: Normalized Atmospheric Deposition for 2002, Total Inorganic Nitrogen

    Science.gov (United States)

    Wieczorek, Michael; LaMotte, Andrew E.

    2010-01-01

    This tabular data set represents the average normalized atmospheric (wet) deposition, in kilograms per square kilometer multiplied by 100, of Total Inorganic Nitrogen for the year 2002 compiled for every MRB_E2RF1 catchment of selected Major River Basins (MRBs, Crawford and others, 2006). Estimates of Total Inorganic Nitrogen deposition are based on National Atmospheric Deposition Program (NADP) measurements (B. Larsen, U.S. Geological Survey, written. commun., 2007). De-trending methods applied to the year 2002 are described in Alexander and others, 2001. NADP site selection met the following criteria: stations must have records from 1995 to 2002 and have a minimum of 30 observations. The MRB_E2RF1 catchments are based on a modified version of the U.S. Environmental Protection Agency's (USEPA) ERF1_2 and include enhancements to support national and regional-scale surface-water quality modeling (Nolan and others, 2002; Brakebill and others, 2011). Data were compiled for every MRB_E2RF1 catchment for the conterminous United States covering New England and Mid-Atlantic (MRB1), South Atlantic-Gulf and Tennessee (MRB2), the Great Lakes, Ohio, Upper Mississippi, and Souris-Red-Rainy (MRB3), the Missouri (MRB4), the Lower Mississippi, Arkansas-White-Red, and Texas-Gulf (MRB5), the Rio Grande, Colorado, and the Great basin (MRB6), the Pacific Northwest (MRB7) river basins, and California (MRB8).

  14. Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis.

    Science.gov (United States)

    Li, Zhi; Sun, Hanxiao; Mo, Xuemei; Li, Xiuying; Xu, Bo; Tian, Peng

    2013-06-01

    The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP(+) dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C16:0, C18:0, C16:1, C18:1, and C18:2. Our results indicated that heterologous expression of NADP(+)-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis.

  15. A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization.

    Science.gov (United States)

    Kim, Tae-Su; Patel, Sanjay K S; Selvaraj, Chandrabose; Jung, Woo-Suk; Pan, Cheol-Ho; Kang, Yun Chan; Lee, Jung-Kul

    2016-09-16

    A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

  16. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation

    KAUST Repository

    Rose, Nicholas D.; Regan, John M.

    2015-01-01

    © 2015 Elsevier B.V. Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD+, respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP+, respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  17. The Mechanisms of Oxygen Reduction in the Terminal Reducing Segment of the Chloroplast Photosynthetic Electron Transport Chain.

    Science.gov (United States)

    Kozuleva, Marina A; Ivanov, Boris N

    2016-07-01

    The review is dedicated to ascertainment of the roles of the electron transfer cofactors of the pigment-protein complex of PSI, ferredoxin (Fd) and ferredoxin-NADP reductase in oxygen reduction in the photosynthetic electron transport chain (PETC) in the light. The data regarding oxygen reduction in other segments of the PETC are briefly analyzed, and it is concluded that their participation in the overall process in the PETC under unstressful conditions should be insignificant. Data concerning the contribution of Fd to the oxygen reduction in the PETC are examined. A set of collateral evidence as well as results of direct measurements of the involvement of Fd in this process in the presence of isolated thylakoids led to the inference that this contribution in vivo is negligible. The increase in oxygen reduction rate in the isolated thylakoids in the presence of either Fd or Fd plus NADP + under increasing light intensity was attributed to the increase in oxygen reduction executed by the membrane-bound oxygen reductants. Data are presented which imply that a main reductant of the O 2 molecule in the terminal reducing segment of the PETC is the electron transfer cofactor of PSI, phylloquinone. The physiological significance of characteristic properties of oxygen reductants in this segment of the PETC is discussed. © 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.

  18. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    Energy Technology Data Exchange (ETDEWEB)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-07-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

  19. Conversion of ethanol to acetaldehyde by human placental homogenates and villi in vitro

    International Nuclear Information System (INIS)

    Blomquist, C.H.; Lindemann, N.J.; Hakanson, E.Y.

    1986-01-01

    The authors have previously reported that placental villi in vitro metabolize acetaldehyde (Ach), and that Ach forms adducts with placental subcellular fractions. In the experiments reported here the authors have investigated the capacity of placental homogenates and villi to generate Ach from ethanol (EtOH). When placental homogenates (0.5 g wet weight) prepared in 50 mM Tris. pH 7.5, were incubated with 20 μM [1- 14 C]ethanol and an NADP- generating system, Ach was formed at the rate of 0.18 nmol/h/g wet weight of tissue, based on counts trappable with semicarbazide. NAD was as effective as NADP. Omission of cofactor resulted in a 69% decrease in activity. The addition of a human serum ultrafiltrate (25,000 m.w. cut-off) to 20% had no effect on Ach formation, whole serum at 20% reduced reaction by 60%. Sodium azide at 40 mM completely abolished Ach formation, 1,10-phenanthroline at 0.4 mM inhibited approximately 50%. In contrast, no Ach formation was detected when 1.0-g fragments of villous tissue were incubated with 20 μM [1- 14 C]EtOH. The data suggest that villous tissue is capable of Ach formation by a catalase-like activity, but the capacity of intact villi for EtOH oxidation is low

  20. Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic bacterium isolated from a sterilization drying oven

    Directory of Open Access Journals (Sweden)

    Maximiliano J. Amenábar

    2012-02-01

    Full Text Available Glutamate dehydrogenase from axenic bacterial cultures of anew microorganism, called GWE1, isolated from the interior ofa sterilization drying oven, was purified by anion-exchange andmolecular-exclusion liquid chromatography. The apparent molecularmass of the native enzyme was 250.5 kDa and wasshown to be an hexamer with similar subunits of molecularmass 40.5 kDa. For glutamate oxidation, the enzyme showedan optimal pH and temperature of 8.0 and 70oC, respectively.In contrast to other glutamate dehydrogenases isolated frombacteria, the enzyme isolated in this study can use both NAD+and NADP+ as electron acceptors, displaying more affinity forNADP+ than for NAD+. No activity was detected with NADHor NADPH, 2-oxoglutarate and ammonia. The enzyme was exceptionallythermostable, maintaining more than 70% of activityafter incubating at 100oC for more than five hours suggestingbeing one of the most thermoestable enzymes reported inthe family of dehydrogenases. [BMB reports 2012; 45(2: 91-95

  1. Attributes for MRB_E2RF1 Catchments by Major River Basins in the Conterminous United States: Normalized Atmospheric Deposition for 2002, Ammonium (NH4)

    Science.gov (United States)

    Wieczorek, Michael; LaMotte, Andrew E.

    2010-01-01

    This tabular data set represents the average normalized (wet) deposition, in kilograms per square kilometer multiplied by 100, of ammonium (NH4) for the year 2002 compiled for every MRB_E2RF1 catchment of the Major River Basins (MRBs, Crawford and others, 2006). Estimates of NH4 deposition are based on National Atmospheric Deposition Program (NADP) measurements (B. Larsen, U.S. Geological Survey, written. commun., 2007). De-trending methods applied to the year 2002 are described in Alexander and others, 2001. NADP site selection met the following criteria: stations must have records from 1995 to 2002 and have a minimum of 30 observations. The MRB_E2RF1 catchments are based on a modified version of the U.S. Environmental Protection Agency's (USEPA) ERF1_2 and include enhancements to support national and regional-scale surface-water quality modeling (Nolan and others, 2002; Brakebill and others, 2011). Data were compiled for every MRB_E2RF1 catchment for the conterminous United States covering New England and Mid-Atlantic (MRB1), South Atlantic-Gulf and Tennessee (MRB2), the Great Lakes, Ohio, Upper Mississippi, and Souris-Red-Rainy (MRB3), the Missouri (MRB4), the Lower Mississippi, Arkansas-White-Red, and Texas-Gulf (MRB5), the Rio Grande, Colorado, and the Great basin (MRB6), the Pacific Northwest (MRB7) river basins, and California (MRB8).

  2. [Characterization of a malic enzyme isoform V from Mucor circinelloides].

    Science.gov (United States)

    Zhang, Yingtong; Chen, Haiqin; Song, Yuanda; Zhang, Hao; Chen, Yongquan; Chen, Wei

    2016-02-04

    We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides. me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently. The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it. A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

  3. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

    Directory of Open Access Journals (Sweden)

    Stephen A Brose

    2016-11-01

    Full Text Available Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA biosynthesis (FAS followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FAS maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FAS inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FAS inhibitors, TOFA (inhibits Acetyl-CoA carboxylase and cerulenin (inhibits FA synthase, increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FAS inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

  4. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia.

    Science.gov (United States)

    Brose, Stephen A; Golovko, Svetlana A; Golovko, Mikhail Y

    2016-01-01

    Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased [Formula: see text]/NAD + and [Formula: see text]/NADP + ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke.

  5. Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

    Science.gov (United States)

    Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

    1983-07-01

    Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

  6. A novel lung slice system with compromised antioxidant defenses

    International Nuclear Information System (INIS)

    Hardwick, S.J.; Adam, A.; Cohen, G.M.; Smith, L.L.

    1990-01-01

    In order to facilitate the study of oxidative stress in lung tissue, rat lung slices with impaired antioxidant defenses were prepared and used. Incubation of lung slices with the antineoplastic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 μM) in an amino acid-rich medium for 45 min produced a near-maximal (approximately 85%), irreversible inhibition of glutathione reductase, accompanied by only a modest (approximately 15%) decrease in pulmonary nonprotein sulfhydryls (NPSH) and no alteration in intracellular ATP, NADP + , and NADPH levels. The amounts of NADP(H), ATP, and NPSH were stable over a 4-hr incubation period following the removal from BCNU. The viability of the system was further evaluated by measuring the rate of evolution of 14 CO 2 from D-[ 14 C(U)]-glucose. The rates of evolution were almost identical in the compromised system when compared with control slices over a 4-hr time period. By using slices with compromised oxidative defenses, preliminary results have been obtained with paraquat, nitrofurantoin, and 2,3-dimethoxy-1,4-naphthoquinone

  7. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase.

    Science.gov (United States)

    Liang, Bo; Zhang, Shu; Lang, Qiaolin; Song, Jianxia; Han, Lihui; Liu, Aihua

    2015-07-16

    A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP(+)-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP(+) involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current-time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM-1 mM and 2-10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N=3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Peculiarities of glucose and glycerol metabolism in Nocardia vaccinii IMB B-7405

    Directory of Open Access Journals (Sweden)

    T. P. Pirog

    2015-04-01

    Full Text Available It has been established that in cells of Nocardia vaccinii IMB B-7405 (surfactant producer glucose catabolism is performed through pentose phosphate cycle as well as through gluconate (activi­ty of NAD+-dependent glucose-6- phosphate dehydrogenase and FAD+-dependent glucose dehydrogenase 835 ± 41 and 698 ± 35 nmol∙min-1∙mg-1 of protein respectively. 6-Phosphogluconate formed in the gluconokinase reaction is involved in the pentose phosphate cycle (activity of constitutive NADP+-dependent 6-phosphogluconate dehydrogenase 357 ± 17 nmol∙min-1∙mg-1 of protein. Glyce­rol catabolism to dihydroxyacetonephosphate (the intermediate of glycolysis may be performed in two ways: through glycerol-3-phosphate (glycerol kinase activity 244 ± 12 nmol∙min-1∙mg-1 of protein and through dihydroxyacetone. Replenishment of the C4-dicarboxylic acids pool in N. vaccinii IMV B-7405 grown on glucose and glycerol occurs in the phosphoenolpyruvate(PEPcarboxylase reaction (714–803 nmol∙min-1∙mg-1 of protein. 2-Oxoglutara­te was involved in tricarboxylic acid cycle by alternate pathway with the participation of 2-oxoglutarate synthase. The observed activity of both key enzymes of gluconeogenesis (PEP- carboxykinase and PEP-synthase, trehalose phosphate synthase and NADP+-dependent glutamate dehydrogenase confirmed the ability of IMV B-7405 strain to the synthesis of surface active glyco- and aminolipids, respectively.

  9. Improved daily precipitation nitrate and ammonium concentration models for the Chesapeake Bay Watershed.

    Science.gov (United States)

    Grimm, J W; Lynch, J A

    2005-06-01

    Daily precipitation nitrate and ammonium concentration models were developed for the Chesapeake Bay Watershed (USA) using a linear least-squares regression approach and precipitation chemistry data from 29 National Atmospheric Deposition Program/National Trends Network (NADP/NTN) sites. Only weekly samples that comprised a single precipitation event were used in model development. The most significant variables in both ammonium and nitrate models included: precipitation volume, the number of days since the last event, a measure of seasonality, latitude, and the proportion of land within 8km covered by forest or devoted to industry and transportation. Additional variables included in the nitrate model were the proportion of land within 0.8km covered by water and/or forest. Local and regional ammonia and nitrogen oxide emissions were not as well correlated as land cover. Modeled concentrations compared very well with event chemistry data collected at six NADP/AirMoN sites within the Chesapeake Bay Watershed. Wet deposition estimates were also consistent with observed deposition at selected sites. Accurately describing the spatial distribution of precipitation volume throughout the watershed is important in providing critical estimates of wet-fall deposition of ammonium and nitrate.

  10. Reproductive strategies and genetic variability in tropical freshwater fish

    Directory of Open Access Journals (Sweden)

    Maria Dolores Peres Lassala

    2007-01-01

    Full Text Available We estimated the genetic variability of nine fish species from the Brazilian upper Paraná River floodplain (Astyanax altiparanae, Hoplias malabaricus, Leporinus lacustris, Loricariichthys platymetopon, Parauchenipterus galeatus, Pimelodus maculatus, Rhaphiodon vulpinus, Roeboides paranensis and Serrasalmus marginatus based on data for 36 putative allozyme loci obtained using corn starch gel electrophoresis of 13 enzymatic systems: aspartate aminotransferase (EC 2.6.1.1, acid phosphatase (EC 3.1.3.2, esterase (EC 3.1.1.1, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8, glucose-6-phosphate dehydrogenase (EC 1.1.1.49, glucose-6-phosphate isomerase (EC 5.3.1.9, Iditol dehydrogenase (EC 1.1.1.14, isocitrate dehydrogenase - NADP+ (EC 1.1.1.42, L-lactate dehydrogenase (EC 1.1.1.27, malate dehydrogenase (EC 1.1.1.37, malate dehydrogenase-NADP+ (EC 1.1.1.40, phosphoglucomutase (EC 5.4.2.2 and superoxide dismutase, (EC 1.15.1.1. The mean expected heterozygosity varied from zero to 0.147. When data from the literature for 75 species of tropical fish were added to the nine species of this study, the heterozygosity values differed significantly among the groups of different reproductive strategies. The highest mean heterozygosity was for the non-migratory without parental care, followed by the long-distance migratory, and the lowest mean was for the non-migratory with parental care or internal fecundation.

  11. Oxygen and hydrogen isotope fractionation during cellulose metabolism in Lemna gibba L

    International Nuclear Information System (INIS)

    Yakir, D.; DeNiro, M.J.

    1990-01-01

    Lemna gibba L. B3 was grown under heterotrophic, photoheterotrophic, and autotrophic conditions in water having a variety of hydrogen and oxygen isotopic compositions. The slopes of the linear regression lines between the isotopic composition of water and leaf cellulose indicated that under the three growth conditions about 40, 70, and 100% of oxygens and carbon-bound hydrogens of cellulose exchanged with those of water prior to cellulose formation. Using the equations of the linear relationships, we estimated the overall fractionation factors between water and the exchanged oxygen and carbon bound-hydrogen of cellulose. At least two very different isotope effects must determine the hydrogen isotopic composition of Lemna cellulose. One reflects the photosynthetic reduction of NADP, while the second reflects exchange reactions that occur subsequent to NADP reduction. Oxygen isotopic composition of cellulose apparently is determined by a single type of exchange reaction with water. Under different growth conditions, variations in metabolic fluxes affect the hydrogen isotopic composition of cellulose by influencing the extent to which the two isotope effects mentioned above are recorded. The oxygen isotopic composition of cellulose is not affected by such changes in growth conditions

  12. [Concentration of monoamines and activity of several enzymes in the arcuate nucleus of the hypothalamus in young and aging rats during the estrous cycle].

    Science.gov (United States)

    Grantyn', V A

    1976-07-01

    The arcuate nucleus (AN) and the median eminence (ME) of the hypothalamus were investigated in young and ageing female rats. During the estral cycle (EC) the monoamine (MA) content, the monoaminoxidase (MAO), NADP and NAD-diaphorase activities were determined in the AN, and the MA content and the activity of alkaline phosphatase (AP) -- in the ME. In young rats in the proestrus-estrus there was an increase in the activity of the NADP and NAD-diaphorase and of the MA content, but a decrease of the MAO activity. This indicated an intensified function of the nucleus at these stages of the EC. Accumulation of the MA in the ME was noted in the diestrus, while in the proestrus their concentration sharply fell; on the other hand, the activity of the AP was considerably increased. In the ageing rats the dynamics of the indices under study during the EC were largely unchanged. However, the functional activity of the AN proved to increase, and in the ME and elevation of the MA concentration and disturbance of its release from the nerve terminals was seen.

  13. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation

    KAUST Repository

    Rose, Nicholas D.

    2015-12-01

    © 2015 Elsevier B.V. Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD+, respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP+, respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  14. Thermodynamics of the oxidation-reduction reaction {2 glutathionered(aq) + NADPox(aq)=glutathioneox(aq) + NADPred(aq)}

    International Nuclear Information System (INIS)

    Tewari, Yadu B.; Goldberg, Robert N.

    2003-01-01

    Microcalorimetry, spectrophotometry, and high-performance liquid chromatography (h.p.l.c.) have been used to conduct a thermodynamic investigation of the glutathione reductase catalyzed reaction {2 glutathione red (aq) + NADP ox (aq)=glutathione ox (aq) + NADP red (aq)}. The reaction involves the breaking of a disulfide bond and is of particular importance because of the role glutathione red plays in the repair of enzymes. The measured values of the apparent equilibrium constant K ' for this reaction ranged from 0.5 to 69 and were measured over a range of temperature (288.15 K to 303.15 K), pH (6.58 to 8.68), and ionic strength I m (0.091 mol · kg -1 to 0.90 mol · kg -1 ). The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations led to values of thermodynamic quantities at T=298.15 K and I m =0 for a chemical reference reaction that involves specific ionic forms. Thus, for the reaction {2 glutathione red - (aq) + NADP ox 3- (aq)=glutathione ox 2- (aq) + NADP red 4- (aq) + H + (aq)}, the equilibrium constant K=(6.5±4.4)·10 -11 , the standard molar enthalpy of reaction Δ r H o m =(6.9±3.0) kJ · mol -1 , the standard molar Gibbs free energy change Δ r G o m =(58.1±1.7) kJ · mol -1 , and the standard molar entropy change Δ r S o m =-(172±12) J · K -1 · mol -1 . Under approximately physiological conditions (T=311.15 K, pH=7.0, and I m =0.25 mol · kg -1 the apparent equilibrium constant K ' ∼0.013. The results of the several studies of this reaction from the literature have also been examined and analyzed using the chemical equilibrium model. It was found that much of the literature is in agreement with the results of this study. Use of our results together with a value from the literature for the standard electromotive force E o for the NADP redox reaction leads to E o =0.166 V (T=298.15 K and I

  15. Human 3α-hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT reverse binding and enzyme down-regulation decreasing MCF7 cell growth.

    Science.gov (United States)

    Zhang, Bo; Hu, Xiao-Jian; Wang, Xiao-Qiang; Thériault, Jean-François; Zhu, Dao-Wei; Shang, Peng; Labrie, Fernand; Lin, Sheng-Xiang

    2016-04-15

    Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP(+) Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  16. Atypical protein disulfide isomerases (PDI: Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A.

    Directory of Open Access Journals (Sweden)

    Benjamin Selles

    Full Text Available Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b'-a' and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH, peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors of PDI which contributes to understand why this protein family expanded along evolution.

  17. Quantifying particulate matter deposition in Niwot Ridge, Colorado: Collection of dry deposition using marble inserts and particle imaging using the FlowCAM

    Science.gov (United States)

    Goss, Natasha R.; Mladenov, Natalie; Seibold, Christine M.; Chowanski, Kurt; Seitz, Leslie; Wellemeyer, T. Barret; Williams, Mark W.

    2013-12-01

    Atmospheric wet and dry deposition are important sources of carbon for remote alpine lakes and soils. The carbon inputs from dry deposition in alpine National Atmospheric Deposition Program (NADP) collectors, including aeolian dust and biological material, are not well constrained due to difficulties in retaining particulate matter in the collectors. Here, we developed and tested a marble insert for dry deposition collection at the Niwot Ridge Long Term Ecological Research Station (NWT LTER) Soddie site (3345 m) between 24 May and 8 November 2011. We conducted laboratory tests of the insert's effect on particulate matter (PM) mass and non-purgeable organic carbon (DOC) and found that the insert did not significantly change either measurement. Thus, the insert may enable dry deposition collection of PM and DOC at NADP sites. We then developed a method for enumerating the collected wet and dry deposition with the Flow Cytometer and Microscope (FlowCAM), a dynamic-image particle analysis tool. The FlowCAM has the potential to establish morphology, which affects particle settling and retention, through particle diameter and aspect ratio. Particle images were used to track the abundance of pollen grains over time. Qualitative image examination revealed that most particles were biological in nature, such as intact algal cells and pollen. Dry deposition loading to the Soddie site as determined by FlowCAM measurements was highly variable, ranging from 100 to >230 g ha-1 d-1 in June-August 2011 and peaking in late June. No significant difference in diameter or aspect ratio was found between wet and dry deposition, suggesting fundamental similarities between those deposition types. Although FlowCAM statistics and identification of particle types proved insightful, our total-particle enumeration method had a high variance and underestimated the total number of particles when compared to imaging of relatively large volumes (60-125 mL) from a single sample. We recommend use of

  18. Ferredoxin-linked chloreplast enzymes. Progress report, August 15, 1990--August 14, 1993

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-01-01

    Progress has clearly been made on all of the goals set forth in the original proposal. Although the monoclonal antibodies raised against FNR turned out no to be useful for mapping the FNR/ferredoxin or FNR/NADP+ interaction domains, good progress has been made on mapping the FNR/ferredoxin interaction domains by an alternative technique, differential chemical modification. Furthermore, the techniques developed for differential chemical modifications of these two proteins - taurine modification of aspartate and glutamate residues and biotin modification of lysine residues - should be useful for mapping the interaction domains of many proteins that associate through electrostatic interactions. Finally, progress has also been made with respect to another ferredoxin-dependent enzyme involved in the earliest steps of plant nitrogen metabolism - nitrite reductase. Questions concerning the subunit composition and heme content of the enzyme have been resolved and evidence demonstrating the involvement of lysine and arginine residues in binding ferredoxin has been obtained for the first time.

  19. Biosynthesis of NAD from nicotinic acid and nicotinamide by resting cells of Arthrobacter globiformis

    International Nuclear Information System (INIS)

    Kuwahara, Masaaki

    1978-01-01

    Isotopically labeled nicotinic acid and nicotinamide were incorporated into the metabolites of nicotinic acid-dependent pathway (Preiss-Handler pathway) of the NAD biosynthesis by resting cells of Arthrobacter globiformis. Azaserine and adenosine markedly stimulated the accumulation of NAD in the cells. Radioactive nicotinic acid and nicotinamide were also incorporated into an unknown compound when the cells were incubated in the presence of azaserine. Cell-free extract of the organism showed the NAD synthetase activity, which required ammonium ion and ATP for the amidation of deamido-NAD. Adenosine inhibited the enzyme activity. The organism possessed nicotinamidase, suggesting deamidation is the first step in the biosynthesis of NAD from nicotinamide. The activity was inhibited by NAD, NADP and NMN. (auth.)

  20. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    Science.gov (United States)

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-09-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

  1. Prospects for improving CO2 fixation in C3-crops through understanding C4-Rubisco biogenesis and catalytic diversity.

    Science.gov (United States)

    Sharwood, Robert E; Ghannoum, Oula; Whitney, Spencer M

    2016-06-01

    By operating a CO2 concentrating mechanism, C4-photosynthesis offers highly successful solutions to remedy the inefficiency of the CO2-fixing enzyme Rubisco. C4-plant Rubisco has characteristically evolved faster carboxylation rates with low CO2 affinity. Owing to high CO2 concentrations in bundle sheath chloroplasts, faster Rubisco enhances resource use efficiency in C4 plants by reducing the energy and carbon costs associated with photorespiration and lowering the nitrogen investment in Rubisco. Here, we show that C4-Rubisco from some NADP-ME species, such as maize, are also of potential benefit to C3-photosynthesis under current and future atmospheric CO2 pressures. Realizing this bioengineering endeavour necessitates improved understanding of the biogenesis requirements and catalytic variability of C4-Rubisco, as well as the development of transformation capabilities to engineer Rubisco in a wider variety of food and fibre crops. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Action of sulphite on plant malate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Ziegler, I.

    1974-01-01

    SO/sub 3//sup 2 -/ acts on NAD- and NADP-dependent malate dehydrogenase in several ways. Firstly, SO/sub 3//sup 2 -/ favours the appearance of low MW species (65000 and 39000 daltons) in Sephadex gel chromatography. Secondly, the enzyme from which is obtained by gel chromatography with dithioerythritol plus nucleotide cofactor is changed in the presence of SO/sub 3//sup 2 -/. This is indicated by the appearance of a linear reaction (instead of curvilinear), and by the abolition of the biphasic sigmoidal kinetics on varying substrate and cofactor concentrations. Thus the inhibition of initial velocity at high substrate or cofactor concentrations is even more marked than at lower ones. Thirdly, SO/sub 3//sup 2 -/ strongly reduces the activity in substrate saturating conditions.

  3. Biochemical characteristics of glucose-6-phosphate dehydrogenase variants among the Malays of Singapore with report of a new non-deficient (GdSingapore) and three deficient variants.

    Science.gov (United States)

    Saha, N; Hong, S H; Wong, H A; Jeyaseelan, K; Tay, J S

    1991-12-01

    Biochemical characteristics of one non-deficient fast G6PD variant (GdSingapore) and six different deficient variants (three new, two Mahidol, one each of Indonesian and Mediterranean) were studied among the Malays of Singapore. The GdSingapore variant had normal enzyme activity (82%) and fast electrophoretic mobilities (140% in TEB buffer, 160% in phosphate and 140% in Tris-HCl buffer systems respectively). This variant is further characterized by normal Km for G6P; utilization of analogues (Gal6P, 2dG6P; dAmNADP), heat stability and pH optimum. The other six deficient G6PD variants had normal electrophoretic mobility in TEB buffer with enzyme activities ranging from 1 to 12% of GdB+. The biochemical characteristics identity them to be 2 Mahidol, 1 Indonesian and 1 Mediterranean variants and three new deficient variants.

  4. ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

    Science.gov (United States)

    Sehgal, Pankaj; Olesen, Claus; Møller, Jesper V

    2016-01-01

    Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

  5. JST Thesaurus Headwords and Synonyms: フェレドキシンNADPレダクターゼ [MeCab user dictionary for science technology term[Archive

    Lifescience Database Archive (English)

    Full Text Available MeCab user dictionary for science technology term フェレドキシンNADPレダクターゼ 名詞 一般 * * * * フ...ェレドキシンNADPレダクターゼ フェレドキシンNADPレダクターゼ フェレドキシンエヌエイディーピーレダクターゼ Thesaurus2015 200906031562191487 C LS38 UNKNOWN_2 フェレドキシン NADP レダクターゼ

  6. Pyridine metabolism and trigonelline synthesis in leaves of the mangrove legume trees Derris indica (Millettia pinnata) and Caesalpinia crista.

    Science.gov (United States)

    Yin, Yuling; Sasamoto, Hamako; Ashihara, Hiroshi

    2011-12-01

    The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (= Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 microg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.

  7. Purification and characterization of the amine dehydrogenase from a facultative methylotroph.

    Science.gov (United States)

    Coleman, J P; Perry, J J

    1984-01-01

    Strain RA-6 is a pink-pigmented organism which can grow on a variety of substrates including methylamine. It can utilize methylamine as sole source of carbon via an isocitrate lyase negative serine pathway. Methylamine grown cells contain an inducible primary amine dehydrogenase [primary amine: (acceptor) oxidoreductase (deaminating)] which is not present in succinate grown cells. The amine dehydrogenase was purified to over 90% homogeneity. It is an acidic protein (isoelectric point of 5.37) with a molecular weight of 118,000 containing subunits with approximate molecular weights of 16,500 and 46,000. It is active on an array of primary terminal amines and is strongly inhibited by carbonyl reagents. Cytochrome c or artificial electron acceptors are required for activity; neither NAD nor NADP can serve as primary electron acceptor.

  8. Redox Homeostasis in Plants under Abiotic Stress: Role of electron carriers, energy metabolism mediators and proteinaceous thiols

    Directory of Open Access Journals (Sweden)

    Dhriti Kapoor

    2015-03-01

    Full Text Available Contemporaneous presence of both oxidized and reduced forms of electron carriers is mandatory in efficient flux by plant electron transport cascades. This requirement is considered as redox poising that involves the movement of electron from multiple sites in respiratory and photosynthetic electron transport chains to molecular oxygen. This flux triggers the formation of superoxide, consequently give rise to other reactive oxygen species (ROS under adverse environmental conditions like drought, high or low temperature, heavy metal stress etc. that plants owing during their life span. Plant cells synthesize ascorbate, an additional hydrophilic redox buffer, which protect the plants against oxidative challenge. Large pools of antioxidants also preside over the redox homeostasis. Besides, tocopherol is a liposoluble redox buffer, which efficiently scavenges the ROS like singlet oxygen. In addition, proteinaceous thiol members such as thioredoxin, peroxiredoxin and glutaredoxin, electron carriers and energy metabolism mediators phosphorylated (NADP and non-phosphorylated (NAD+ coenzyme forms interact with ROS, metabolize and maintain redox homeostasis.

  9. Statistical methodology for exploring elevational differences in precipitation chemistry

    International Nuclear Information System (INIS)

    Warren, W.G.; Boehm, M.; Link, D.

    1992-01-01

    A statistical methodology for exploring the relationships between elevation and precipitation chemistry is outlined and illustrated. The methodology utilizes maximum likelihood estimates and likelihood ratio tests with contour ellipses of assumed bivariate lognormal distributions used to assist in interpretation. The approach was illustrated using 12 NADP/NTN sites located in six study areas in the Wyoming and Colorado Rockies. These sites are part of the Rocky Mountain Deposition Monitoring Project (RMDMP), which was initiated in 1986 to investigate the relationships between elevation and the chemistry of precipitation. The results indicate differences in sulfate concentrations between airsheds, between snow and rain, and between higher and lower elevations. In general, sulfate concentrations in snow are greater at lower elevations and this difference is independent of concentration. A similar relationship for rain was not well established. In addition there is evidence that, overall, the sulfate concentrations differed between the six study areas, although pairwise differences were not always significant

  10. [Effects of melaxen and valdoxan on the activity of glutathione antioxidant system and NADPH-producing enzymes in rat heart under experimental hyperthyroidism conditions].

    Science.gov (United States)

    Gorbenko, M V; Popova, T N; Shul'gin, K K; Popov, S S

    2013-01-01

    The effects of melaxen and valdoxan on the activity of glutathione antioxidant system and some NADPH-producing enzymes have been studied under conditions of experimental hyperthyroidism in rat heart. Under the action of these drugs, reduced glutathione (GSH) content increased as compared to values observed under the conditions of pathology. It has been established that the activities of glutathione reductase (GR), glutathione peroxidase (GP), glucose-6-phosphate dehydrogenase, and NADP isocitrate dehydrogenase (increased under pathological conditions) change toward the intact control values upon the introduction of both drugs. The influence of melaxen and valdoxan, capable of producing antioxidant effect, leads apparently to the inhibition of free-radical oxidation processes and, as a consequence, the reduction of mobilization degree of the glutathione antioxidant system.

  11. [Quantitative histoenzymatic analysis of the adenohypophysis and adrenal cortex during the early stages of involution].

    Science.gov (United States)

    Prochukhanov, R A; Rostovtseva, T I

    1977-11-01

    A method of quantitative histenzymatic analysis was applied for determination of the involution changes of the neuroendocrine system. The activity of NAD- and NADP-reductases, acid and alkaline phosphatases, glucose-6-phosphoric dehydrogenase, 3-OH-steroid-dehydrogenase, 11-hydroxysteroid dehydrogenases was investigated in the adenohypophysis and in the adrenal cortex of rats aged 4 and 12 months. There were revealed peculiarities attending the structural-metabolic provision of physiological reconstructions of the neuro-endocrine system under conditions of the estral cycle at the early involution stages. An initial reduction of the cell ular-vascular transport with the retention of the functional activity of the intracellular organoids was demonstrated in ageing animals.

  12. Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

    DEFF Research Database (Denmark)

    Mark, Christina; Zor, Kinga; Heiskanen, Arto

    such as redox activity, O2 and H2O2 concentration, pH, cell viability and release of target enzymes such as α-amylase. We have optimised an intracellular, whole-cell redox activity assay[3] that detects changes in redox activity in barley aleurone layer during PCD. The assay uses a double mediator......This project focuses on developing and applying a tissue culture system with electrochemical and optical detection techniques for tissue culture of barley aleurone layer to increase understanding of the underlying mechanisms of programmed cell death (PCD) in plants. The major advantage......-system to electrochemically measure redox activity via changes in the NADP:NADPH ratio. Experiments show that redox activity changes depend on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD. We have also successfully detected PCD induced by phytohormones in barley aleurone layer using...

  13. Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses

    Directory of Open Access Journals (Sweden)

    Rajib Saha

    2016-05-01

    Full Text Available Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H, and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP+ showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium.

  14. Cytosolic Calcium, hydrogen peroxide, and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: Parabolic flight data

    Science.gov (United States)

    Hampp, Ruediger; Hausmann, Niklas; Neef, Maren; Fengler, Svenja

    Callus cell cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights in order to assess molecular short-term responses to altered gravity fields. Using transgenic cell lines, hydrogen peroxide and cytosolic Ca2+ were continuously monitored. In parallel, the metabolism of samples was chemically quenched (RNAlater, Ambion, for RNA; acid/base for NADPH, NADP) at typical stages of a parabola (1g before pull up; end of pull up (1.8 g), end of microgravity (µg, 20 sec), and end of pull out (1.8 g)). Cells exhibited an increase of both Ca2+ and hydrogen peroxide with the onset of µg, and a decline thereafter. This behaviour was accompanied by a decrease of the NADPH/NADP redox ratio, indicating a Ca2+-dependent activation of a NADPH oxidase. Microarray analyses revealed concomitant expression profiles. At the end of the microgravity phase, 396 transcripts were specifically up-, while 485 were down-regulated. Up-regulation was dominated by Ca2+- and ROS(reactive oxygen species)-related gene products. The same material was also used for the analysis of phosphopeptides by 2D SDS PAGE. Relevant spots were identified by liquid chromatography-MS. With the exception of a chaperone (HSP 70-3), hypergravity (1.8 g) and microgravity modified different sets of proteins. These are partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of reactive oxygen species. Taken together, these data show that both gene expression and protein modulation jointly respond within seconds to alterations in the gravity field, with a focus on metabolic adaptation, signalling and control of ROS.

  15. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China); Zhang, Shu [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Lang, Qiaolin [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Song, Jianxia; Han, Lihui [Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Liu, Aihua, E-mail: liuah@qibebt.ac.cn [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China)

    2015-07-16

    Highlights: • E. coli surface-dispalyed Gldh exhibiting excellent enzyme activity and stability. • Sensitive amperometric biosensor for glutamate using Gldh-bacteria and MWNTs. • The glutamate biosensor exhibited high specificity and stability. - Abstract: A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP{sup +}-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP{sup +} involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.

  16. Purine Turnover Metabolites and Selected Antioxidants in Blood of Goats during the Periparturient Period

    Directory of Open Access Journals (Sweden)

    Ewa Skotnicka

    2010-01-01

    Full Text Available The aim of this study was to examine the relationships between the erythrocyte concentrations of ATP, ADP, AMP, NAD+, NADP+ and adenylate energy charge (AEC and their metabolites: inosine (Ino, hypoxanthine (Hyp, uric acid (UA, selected blood antioxidants: superoxide dismutase (SOD, glutathione reductase (GR of goats during the periparturient period. The study was conducted on 12 clinically healthy pregnant goats (Capra hircus - 75% genotype of the Polish noble white aged between 2-3 years. Blood was taken from the external jugular vein early in the morning before feeding and obtained twice (4 weeks and 1 week before delivery and twice (2 and 3 weeks after delivery. The ATP concentration did not change significantly. One week before delivery ADP, AMP and Ino concentrations were significantly higher and AEC value significantly lower compared to values in the third week after delivery (p ≤ 0.02, p ≤ 0.05, p ≤ 0.03 and p ≤ 0.01, respectively. One week before and two weeks after delivery we observed a significant increase in SOD and GR activities and an increase in NAD+ and NADP+ concentrations but a significant decrease in Hyp and UA concentrations. Constant ATP concentration and the changes in the dynamics and tendency of Hyp and UA concentrations show that in the periparturient period in goats, purine metabolism undergoes adaptive changes. The balance between resynthetic processes and adenine nucleotide degradation is retained in order to level the oxygen and energy lacks. The results of our study show that the maintenance of prooxidative homeostasis in goats of peripartal period depends mainly on enzymatic mechanisms.

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

    Science.gov (United States)

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

    2017-07-01

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

  18. Homocysteine, antioxidant vitamins and lipids as biomarkers of neurodegeneration in Alzheimer's disease versus non-Alzheimer's dementia.

    Science.gov (United States)

    Raszewski, Grzegorz; Chwedorowicz, Roman; Chwedorowicz, Agnieszka; Gustaw Rothenberg, Katarzyna

    2016-01-01

    Evidence for the benefit of antioxidants' based therapeutic intervention in dementia are inconsistent. Parallel studies in disease forms of dementia different than Alzheimer's are even less conclusive. In this study, the role of serum levels of homocysteine (tHcy), lipids and antioxidants in predicting the risk of cognitive decline in Alzheimer's disease (AD) versus non-Alzheimer's dementias (n-AD). The objective was to add to the ongoing cumulative research to establish the biochemical baseline for potential nutri-therapeutic intervention in different forms of dementia. 65 participants with dementia (DP-s) were divided into two groups: ADP--patients with Alzheimer's disease and n-ADP--patients with dementia of a different etiology than primary neurodegenerative dementia in the course of Alzheimer's disease. Cognitive function was assessed by Mini-Mental State Examination (MMSE) and related to plasma levels of tHcy, folate, vitamins B-6, B-12, lipids and vitamins A and E for both groups. Also examined were associations between cognitive impairment and several variables (age, education, duration of dementia) that might confound nutrition-cognition associations. A significant reduction in serum vitamin A levels and elevation of total cholesterol levels were shown for the DP-s group compared to those in the control group. Moreover, significant differences were found in MMSE data and serum vitamin E and tHcy levels between patients with ADP and n-ADP. The scores for MMSE showed a correlation with the vitamin E levels and duration of dementia in the ADP group and/or correlation with tHcy, levels of vitamins A and/or E, and duration of dementia in the n-ADP group. The results obtained suggest that elevated serum tHcy and decreased levels of vitamins A and E are associated with an increased risk of non-Alzheimer's dementias, although further studies involving a larger cohort are now needed to verify these results.

  19. Glucose availability controls adipogenesis in mouse 3T3-L1 adipocytes via up-regulation of nicotinamide metabolism.

    Science.gov (United States)

    Jackson, Robert M; Griesel, Beth A; Gurley, Jami M; Szweda, Luke I; Olson, Ann Louise

    2017-11-10

    Expansion of adipose tissue in response to a positive energy balance underlies obesity and occurs through both hypertrophy of existing cells and increased differentiation of adipocyte precursors (hyperplasia). To better understand the nutrient signals that promote adipocyte differentiation, we investigated the role of glucose availability in regulating adipocyte differentiation and maturation. 3T3-L1 preadipocytes were grown and differentiated in medium containing a standard differentiation hormone mixture and either 4 or 25 mm glucose. Adipocyte maturation at day 9 post-differentiation was determined by key adipocyte markers, including glucose transporter 4 (GLUT4) and adiponectin expression and Oil Red O staining of neutral lipids. We found that adipocyte differentiation and maturation required a pulse of 25 mm glucose only during the first 3 days of differentiation. Importantly, fatty acids were unable to substitute for the 25 mm glucose pulse during this period. The 25 mm glucose pulse increased adiponectin and GLUT4 expression and accumulation of neutral lipids via distinct mechanisms. Adiponectin expression and other early markers of differentiation required an increase in the intracellular pool of total NAD/P. In contrast, GLUT4 protein expression was only partially restored by increased NAD/P levels. Furthermore, GLUT4 mRNA expression was mediated by glucose-dependent activation of GLUT4 gene transcription through the cis-acting GLUT4-liver X receptor element (LXRE) promoter element. In summary, this study supports the conclusion that high glucose promotes adipocyte differentiation via distinct metabolic pathways and independently of fatty acids. This may partly explain the mechanism underlying adipocyte hyperplasia that occurs much later than adipocyte hypertrophy in the development of obesity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Gene Expression Patterns during Light and Dark Infection of Prochlorococcus by Cyanophage.

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    Luke R Thompson

    Full Text Available Cyanophage infecting the marine cyanobacteria Prochlorococcus and Synechococcus require light and host photosystem activity for optimal reproduction. Many cyanophages encode multiple photosynthetic electron transport (PET proteins, which are presumed to maintain electron flow and produce ATP and NADPH for nucleotide biosynthesis and phage genome replication. However, evidence suggests phage augment NADPH production via the pentose phosphate pathway (PPP, thus calling into question the need for NADPH production by PET. Genes implicated in cyclic PET have since been identified in cyanophage genomes. It remains an open question which mode of PET, cyclic or linear, predominates in infected cyanobacteria, and thus whether the balance is towards producing ATP or NADPH. We sequenced transcriptomes of a cyanophage (P-HM2 and its host (Prochlorococcus MED4 throughout infection in the light or in the dark, and analyzed these data in the context of phage replication and metabolite measurements. Infection was robust in the light, but phage were not produced in the dark. Host gene transcripts encoding high-light inducible proteins and two terminal oxidases (plastoquinol terminal oxidase and cytochrome c oxidase-implicated in protecting the photosynthetic membrane from light stress-were the most enriched in light but not dark infection. Among the most diminished transcripts in both light and dark infection was ferredoxin-NADP+ reductase (FNR, which uses the electron acceptor NADP+ to generate NADPH in linear photosynthesis. The phage gene for CP12, which putatively inhibits the Calvin cycle enzyme that receives NADPH from FNR, was highly expressed in light infection. Therefore, both PET production of NADPH and its consumption by carbon fixation are putatively repressed during phage infection in light. Transcriptomic evidence is thus consistent with cyclic photophosphorylation using oxygen as the terminal electron acceptor as the dominant mode of PET under

  1. Antidiabetic Effect of Fresh Nopal (Opuntia ficus-indica in Low-Dose Streptozotocin-Induced Diabetic Rats Fed a High-Fat Diet

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    Seung Hwan Hwang

    2017-01-01

    Full Text Available The objective of the present study was to evaluate α-glucosidase inhibitory and antidiabetic effects of Nopal water extract (NPWE and Nopal dry power (NADP in low-dose streptozotocin- (STZ- induced diabetic rats fed a high-fat diet (HFD. The type 2 diabetic rat model was induced by HFD and low-dose STZ. The rats were divided into four groups as follows: (1 nondiabetic rats fed a regular diet (RD-Control; (2 low-dose STZ-induced diabetic rats fed HFD (HF-STZ-Control; (3 low-dose STZ-induced diabetic rats fed HFD and supplemented with NPWE (100 mg/kg body weight, HF-STZ-NPWE; and (4 low-dose STZ-induced diabetic rats fed HFD and supplemented with comparison medication (rosiglitazone, 10 mg/kg, body weight, HF-STZ-Rosiglitazone. In results, NPWE and NADP had IC50 values of 67.33 and 86.68 μg/mL, both of which exhibit inhibitory activities but lower than that of acarbose (38.05 μg/mL while NPWE group significantly decreased blood glucose levels compared to control and NPDP group on glucose tolerance in the high-fat diet fed rats model (P<0.05. Also, the blood glucose levels of HR-STZ-NPWE group were significantly lower (P<0.05 than HR-STZ-Control group on low-dose STZ-induced diabetic rats fed HFD. Based on these findings, we suggested that NPWE could be considered for the prevention and/or treatment of blood glucose and a potential use as a dietary supplement.

  2. Antidiabetic Effect of Fresh Nopal (Opuntia ficus-indica) in Low-Dose Streptozotocin-Induced Diabetic Rats Fed a High-Fat Diet.

    Science.gov (United States)

    Hwang, Seung Hwan; Kang, Il-Jun; Lim, Soon Sung

    2017-01-01

    The objective of the present study was to evaluate α -glucosidase inhibitory and antidiabetic effects of Nopal water extract (NPWE) and Nopal dry power (NADP) in low-dose streptozotocin- (STZ-) induced diabetic rats fed a high-fat diet (HFD). The type 2 diabetic rat model was induced by HFD and low-dose STZ. The rats were divided into four groups as follows: (1) nondiabetic rats fed a regular diet (RD-Control); (2) low-dose STZ-induced diabetic rats fed HFD (HF-STZ-Control); (3) low-dose STZ-induced diabetic rats fed HFD and supplemented with NPWE (100 mg/kg body weight, HF-STZ-NPWE); and (4) low-dose STZ-induced diabetic rats fed HFD and supplemented with comparison medication (rosiglitazone, 10 mg/kg, body weight, HF-STZ-Rosiglitazone). In results, NPWE and NADP had IC 50 values of 67.33 and 86.68  μ g/mL, both of which exhibit inhibitory activities but lower than that of acarbose (38.05  μ g/mL) while NPWE group significantly decreased blood glucose levels compared to control and NPDP group on glucose tolerance in the high-fat diet fed rats model ( P < 0.05). Also, the blood glucose levels of HR-STZ-NPWE group were significantly lower ( P < 0.05) than HR-STZ-Control group on low-dose STZ-induced diabetic rats fed HFD. Based on these findings, we suggested that NPWE could be considered for the prevention and/or treatment of blood glucose and a potential use as a dietary supplement.

  3. Functional properties and structural characterization of rice δ1-pyrroline-5-carboxylate reductase

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    Giuseppe eForlani

    2015-07-01

    Full Text Available The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L. for δ1-pyrroline-5-carboxylate (P5C reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in E. coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human and bacterial enzymes.

  4. Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

    Science.gov (United States)

    Küssau, Tanja; Flipo, Marion; Van Wyk, Niel; Viljoen, Albertus; Olieric, Vincent; Kremer, Laurent; Blaise, Mickaël

    2018-05-01

    In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP + -bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP + -bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.

  5. Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves.

    Science.gov (United States)

    Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

    2002-05-01

    In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

  6. Photophosphorylation capacity of stable spheroplast preparations of anabaena.

    Science.gov (United States)

    Spiller, H

    1980-09-01

    Spheroplasts from Anabaena 7119 (formerly designated Nostoc muscorum) were prepared in the presence of serum albumin in 0.5 molar sucrose. Electron transport and photophosphorylation were preserved (> 70% of the maximum rate for 1 week). The pH profile of electron transport and photophosphorylation in the reactions H(2)O --> NADP, H(2)O --> methyl viologen, and H(2)O --> ferricyanide shows that uncoupling by ammonia is small throughout and increases slightly with higher pH. ADP + Pi increased NADP reduction from H(2)O by 2.5-fold. The ratios of ATP formed per electron pair transported ranged from 0.9 to 1.5. Effects of catalase and superoxide dismutase on the overall O(2) balance implicate pseudocyclic electron transport and phosphorylation. The quenching of 9-aminoacridine fluorescence indicates the formation of a Delta pH from 2 to 2.6 during illumination. This pH gradient is abolished by uncouplers; however, complete uncoupling is achieved only by 3-chlorocarbonyl cyanide phenylhydrazone or valinomycin + NH(4) (+). In the presence of NH(4) (+) alone, the membrane potential may act as the driving force for photophosphorylation.Increasing amounts of bovine serum albumin protected phosphorylation from uncoupling by silicomolybdic acid. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea-insensitive water oxidation by silicomolybdic acid provides evidence that the site for 3-(3,4-dichlorophenyl)-1, 1-dimethylurea action is on the acceptor and not donor side of photosystem II in the procaryote Anabaena. It is concluded that stable spheroplasts retain coupled electron transport approaching in vivo rates.

  7. Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system.

    Science.gov (United States)

    Lopert, Pamela; Patel, Manisha

    2014-05-30

    Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H2O2) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H2O2 catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP(+), provides the link between mitochondrial respiration and H2O2 detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H2O2 in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H2O2, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H2O2 catabolism, (b) decreased NADPH and increased NADP(+) levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H2O2 and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H2O2 antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

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    Pascal eREY

    2013-10-01

    Full Text Available Plants display a remarkable diversity of thioredoxins (Trxs, reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast.

  9. Nitrate uptake and utilization is modulated by exogenous gamma-aminobutyric acid in Arabidopsis thaliana seedlings.

    Science.gov (United States)

    Barbosa, Jose M; Singh, Narendra K; Cherry, Joe H; Locy, Robert D

    2010-06-01

    Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO(3)(-)) in the growth medium. At NO(3)(-) concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO(3)(-), the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO(3)(-) uptake at low NO(3)(-), while GABA inhibited NO(3)(-) uptake at high NO(3)(-). Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.

  10. Fructose Alters Intermediary Metabolism of Glucose in Human Adipocytes and Diverts Glucose to Serine Oxidation in the One–Carbon Cycle Energy Producing Pathway

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    Vijayalakshmi Varma

    2015-06-01

    Full Text Available Increased consumption of sugar and fructose as sweeteners has resulted in the utilization of fructose as an alternative metabolic fuel that may compete with glucose and alter its metabolism. To explore this, human Simpson-Golabi-Behmel Syndrome (SGBS preadipocytes were differentiated to adipocytes in the presence of 0, 1, 2.5, 5 or 10 mM of fructose added to a medium containing 5 mM of glucose representing the normal blood glucose concentration. Targeted tracer [1,2-13C2]-d-glucose fate association approach was employed to examine the influence of fructose on the intermediary metabolism of glucose. Increasing concentrations of fructose robustly increased the oxidation of [1,2-13C2]-d-glucose to 13CO2 (p < 0.000001. However, glucose-derived 13CO2 negatively correlated with 13C labeled glutamate, 13C palmitate, and M+1 labeled lactate. These are strong markers of limited tricarboxylic acid (TCA cycle, fatty acid synthesis, pentose cycle fluxes, substrate turnover and NAD+/NADP+ or ATP production from glucose via complete oxidation, indicating diminished mitochondrial energy metabolism. Contrarily, a positive correlation was observed between glucose-derived 13CO2 formed and 13C oleate and doses of fructose which indicate the elongation and desaturation of palmitate to oleate for storage. Collectively, these results suggest that fructose preferentially drives glucose through serine oxidation glycine cleavage (SOGC pathway one-carbon cycle for NAD+/NADP+ production that is utilized in fructose-induced lipogenesis and storage in adipocytes.

  11. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

    Science.gov (United States)

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

    2012-01-01

    NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

  12. Investigations about the in vitro import of nucleus encoded cyanellar ploypeptides

    International Nuclear Information System (INIS)

    Brandtner, M.

    1991-05-01

    Cyanelles are the plastids of the eukaryotic alga Cyanophora paradoxa. They share several features with cyanobacteria: a cell wall made of peptidoglycan, concentric thylakoid membranes and phycobilisomes as light harvesting complexes. Therefore cyanelles were regarded as 'missing link' between the chloroplasts and the prokaryotic ancestor according to the endosymbiotic hypothesis. The genome of the cyanelles resembles that of plastids concerning size and organization. So cyanelles have to import 90% of their proteins. In order to study the import of proteins into cyanelles, it was necessary to isolate a nucleus-encoded gene from a cDNA library. The gene could be expressed in vitro in a suitable plasmid. The radioactive labelled precursor will be incubated with isolated cyanelles. After their lysis their proteins will be run on a gel and the fate of the precursor -whether it was imported and processed respectively or not - will be detected. A method for mRNA isolation was adapted with respect to Cyanophora paradoxa. Based on the mRNA cDNA libraries were established. In parallel the mRNA was in vitro translated. Import experiments with the translation products were performed into cyanelles and pea chloroplasts. Additionally it was tried to import four precursors of higher plants into cyanelles. The isolated cyanelles were not competent for protein uptake. The gene for ferredoxin-NADP-reductase was found in a lambda ZAP cDNA library by screening with homologous antibodies. The transit peptide for the import into cyanelles shows the same general features as chloroplast transit peptides do, but differs from the three known transit peptides of ferredoxin-NADP-reductase from higher plants. (author)

  13. Disabled infectious single cycle herpes simplex virus (DISC-HSV) is a candidate vector system for gene delivery/expression of GM-CSF in human prostate cancer therapy.

    Science.gov (United States)

    Parkinson, Richard J; Mian, Shahid; Bishop, Michael C; Gray, Trevor; Li, Geng; McArdle, Stephanie E B; Ali, Selman; Rees, Robert C

    2003-06-15

    DISC-HSV is a replication incompetent herpes simplex virus that is a highly efficient vector for the transduction of genes in vivo and in vitro. We examine the ability of DISC-HSV to infect human prostate cancer cell-lines and xenograft tumor models, and induce expression of reporter and therapeutic cytokine genes. Infection was confirmed by cellular staining for the beta-galactosidase reporter gene product, and by EM. Human GM-CSF production following DISC-hGMCSF infection was measured using ELISA. The metabolic activity of infected cells was determined by NADP/NADPH assay. Cell death was estimated by cell-cycle analysis using flow cytometry with propidium iodide staining. Infection of DU145, PC3 and LNCaP cells with DISC-HSV was dose dependent. Cells infected with DISC-hGM-CSF released significant levels of hGM-CSF for 3 days. NADP/NADPH assay suggested that infected cells continued to be metabolically active for 3 days post-infection, which was consistent with flow cytometry findings that cell death did not occur within 7 days of infection. Tumor xenografts injected with DISC-HSV expressed beta-galactosidase, and intracellular viral particles were demonstrated using EM. We have previously reported the rejection of established tumors following intra-tumoral injection of DISC-GMCSF. This study demonstrates the ability of DISC-HSV to infect prostate cancer and express GMCSF at significant levels. We suggest that prostate cancer is a potential target for therapy using DISC-HSV containing GM-CSF. Copyright 2003 Wiley-Liss, Inc.

  14. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  15. A Model of Oxidative Stress Management: Moderation of Carbohydrate Metabolizing Enzymes in SOD1-Null Drosophila melanogaster

    Science.gov (United States)

    Bernard, Kristine E.; Parkes, Tony L.; Merritt, Thomas J. S.

    2011-01-01

    The response to oxidative stress involves numerous genes and mutations in these genes often manifest in pleiotropic ways that presumably reflect perturbations in ROS-mediated physiology. The Drosophila melanogaster SOD1-null allele (cSODn108) is proposed to result in oxidative stress by preventing superoxide breakdown. In SOD1-null flies, oxidative stress management is thought to be reliant on the glutathione-dependent antioxidants that utilize NADPH to cycle between reduced and oxidized form. Previous studies suggest that SOD1-null Drosophila rely on lipid catabolism for energy rather than carbohydrate metabolism. We tested these connections by comparing the activity of carbohydrate metabolizing enzymes, lipid and triglyceride concentration, and steady state NADPH:NADP+ in SOD1-null and control transgenic rescue flies. We find a negative shift in the activity of carbohydrate metabolizing enzymes in SOD1-nulls and the NADP+-reducing enzymes were found to have significantly lower activity than the other enzymes assayed. Little evidence for the catabolism of lipids as preferential energy source was found, as the concentration of lipids and triglycerides were not significantly lower in SOD1-nulls compared with controls. Using a starvation assay to impact lipids and triglycerides, we found that lipids were indeed depleted in both genotypes when under starvation stress, suggesting that oxidative damage was not preventing the catabolism of lipids in SOD1-null flies. Remarkably, SOD1-nulls were also found to be relatively resistant to starvation. Age profiles of enzyme activity, triglyceride and lipid concentration indicates that the trends observed are consistent over the average lifespan of the SOD1-nulls. Based on our results, we propose a model of physiological response in which organisms under oxidative stress limit the production of ROS through the down-regulation of carbohydrate metabolism in order to moderate the products exiting the electron transport chain. PMID

  16. Atmospheric nitrogen inputs to the Delaware Inland Bays: the role of ammonia

    International Nuclear Information System (INIS)

    Scudlark, Joseph R.; Jennings, Jennifer A.; Roadman, Megan J.; Savidge, Karen B.; Ullman, William J.

    2005-01-01

    + wet deposition within the Inland Bays watershed. In a pilot study, precipitation composition at the Lewes NADP-AIRMoN site (DE 02) was compared with that at a satellite site established at Riverdale on the Indian River Estuary, approximately 21 km southwest. While the volume-weighted mean precipitation NO 3 - concentrations did not differ significantly between sites, the NH 4 + concentration observed at Riverdale (26.3 μmoles L -1 ) was 73% greater than at Lewes (15.2 μmoles L -1 ). More recently, a NADP site was established at Trap Pond, DE (DE 99), which was intentionally located within the region of intense poultry production. A comparison of the initial two years (6/2001-5/2003) of precipitation chemistry data from Trap Pond with other nearby NADP-AIRMoN sites (Lewes and Smith Island) reveals fairly homogeneous NO 3 - wet deposition, but significant spatial differences (∼60%) in the NH 4 + wet flux. Overall, these results suggest that local emissions and below-cloud scavenging provide a significant contribution to regional atmospheric N deposition. - Local emissions from poultry production appear to significantly contribute to wet and dry atmospheric NH x loading to the Delaware Inland Bays

  17. The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes

    International Nuclear Information System (INIS)

    Chiu, Hsiu-Ju; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes. Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P) + -dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD + has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP + and NAD + . The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from

  18. Overexpression of E3 Ubiquitin Ligase Gene AdBiL Contributes to Resistance against Chilling Stress and Leaf Mold Disease in Tomato

    Directory of Open Access Journals (Sweden)

    Shuangchen Chen

    2017-06-01

    Full Text Available Ubiquitination is a common regulatory mechanism, playing a critical role in diverse cellular and developmental processes in eukaryotes. However, a few reports on the functional correlation between E3 ubiquitin ligases and reactive oxygen species (ROS or reactive nitrogen species (RNS metabolism in response to stress are currently available in plants. In the present study, the E3 ubiquitin ligase gene AdBiL (Adi3 Binding E3 Ligase was introduced into tomato line Ailsa Craig via Agrobacterium-mediated method. Transgenic lines were confirmed for integration into the tomato genome using PCR. Transcription of AdBiL in various transgenic lines was determined using real-time PCR. Evaluation of stress tolerance showed that T1 generation of transgenic tomato lines showed only mild symptoms of chilling injury as evident by higher biomass accumulation and chlorophyll content than those of non-transformed plants. Compared with wild-type plants, the contents of AsA, AsA/DHA, GSH and the activity of GaILDH, γ-GCS and GSNOR were increased, while H2O2, O2.−, MDA, NO, SNOs, and GSNO accumulations were significantly decreased in AdBiL overexpressing plants in response to chilling stress. Furthermore, transgenic tomato plants overexpressing AdBiL showed higher activities of enzymes such as G6PDH, 6PGDH, NADP-ICDH, and NADP-ME involved in pentose phosphate pathway (PPP. The transgenic tomato plants also exhibited an enhanced tolerance against the necrotrophic fungus Cladosporium fulvum. Tyrosine nitration protein was activated in the plants infected with leaf mold disease, while the inhibition could be recovered in AdBiL gene overexpressing lines. Taken together, our results revealed a possible physiological role of AdBiL in the activation of the key enzymes of AsA–GSH cycle, PPP and down-regulation of GSNO reductase, thereby reducing oxidative and nitrosative stress in plants. This study demonstrates an optimized transgenic strategy using AdBiL gene for crop

  19. Activation of Signaling Cascades by Weak Extremely Low Frequency Electromagnetic Fields

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    Einat Kapri-Pardes

    2017-10-01

    Full Text Available Background/Aims: Results from recent studies suggest that extremely low frequency magnetic fields (ELF-MF interfere with intracellular signaling pathways related to proliferative control. The mitogen-activated protein kinases (MAPKs, central signaling components that regulate essentially all stimulated cellular processes, include the extracellular signal-regulated kinases 1/2 (ERK1/2 that are extremely sensitive to extracellular cues. Anti-phospho-ERK antibodies serve as a readout for ERK1/2 activation and are able to detect minute changes in ERK stimulation. The objective of this study was to explore whether activation of ERK1/2 and other signaling cascades can be used as a readout for responses of a variety of cell types, both transformed and non-transformed, to ELF-MF. Methods: We applied ELF-MF at various field strengths and time periods to eight different cell types with an exposure system housed in a tissue culture incubator and followed the phosphorylation of MAPKs and Akt by western blotting. Results: We found that the phosphorylation of ERK1/2 is increased in response to ELF-MF. However, the phosphorylation of ERK1/2 is likely too low to induce ELF-MF-dependent proliferation or oncogenic transformation. The p38 MAPK was very slightly phosphorylated, but JNK or Akt were not. The effect on ERK1/2 was detected for exposures to ELF-MF strengths as low as 0.15 µT and was maximal at ∼10 µT. We also show that ERK1/2 phosphorylation is blocked by the flavoprotein inhibitor diphenyleneiodonium, indicating that the response to ELF-MF may be exerted via NADP oxidase similar to the phosphorylation of ERK1/2 in response to microwave radiation. Conclusions: Our results further indicate that cells are responsive to ELF-MF at field strengths much lower than previously suspected and that the effect may be mediated by NADP oxidase. However, the small increase in ERK1/2 phosphorylation is probably insufficient to affect proliferation and oncogenic

  20. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    Energy Technology Data Exchange (ETDEWEB)

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    This project used the cyanobacterial species Synechocystis PCC 6803 to pursue two lines of inquiry, with each line addressing one of the two main factors affecting hydrogen (H2) production in Synechocystis PCC 6803: NADPH availability and O2 sensitivity. H2 production in Synechocystis PCC 6803 requires a very high NADPH:NADP+ ratio, that is, the NADP pool must be highly reduced, which can be problematic because several metabolic pathways potentially can act to raise or lower NADPH levels. Also, though the [NiFe]-hydrogenase in PCC 6803 is constitutively expressed, it is reversibly inactivated at very low O2 concentrations. Largely because of this O2 sensitivity and the requirement for high NADPH levels, a major portion of overall H2 production occurs under anoxic conditions in the dark, supported by breakdown of glycogen or other organic substrates accumulated during photosynthesis. Also, other factors, such as N or S limitation, pH changes, presence of other substances, or deletion of particular respiratory components, can affect light or dark H2 production. Therefore, in the first line of inquiry, under a number of culture conditions with wild type (WT) Synechocystis PCC 6803 cells and a mutant with impaired type I NADPH-dehydrogenase (NDH-1) function, we used H2 production profiling and metabolic flux analysis, with and without specific inhibitors, to examine systematically the pathways involved in light and dark H2 production. Results from this work provided rational bases for metabolic engineering to maximize photobiological H2 production on a 24-hour basis. In the second line of inquiry, we used site-directed mutagenesis to create mutants with hydrogenase enzymes exhibiting greater O2 tolerance. The research addressed the following four tasks: 1. Evaluate the effects of various culture conditions (N, S, or P limitation; light/dark; pH; exogenous organic carbon) on H2 production profiles of WT cells and an NDH-1 mutant; 2. Conduct metabolic flux analyses for

  1. Alternative oxidase pathway optimizes photosynthesis during osmotic and temperature stress by regulating cellular ROS, malate valve and antioxidative systems

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    DINAKAR eCHALLABATHULA

    2016-02-01

    Full Text Available The present study reveals the importance of alternative oxidase (AOX pathway in optimizing photosynthesis under osmotic and temperature stress conditions in the mesophyll protoplasts of Pisum sativum. The responses of photosynthesis and respiration were monitored at saturating light intensity of 1000 µmoles m-2 s-1 at 25 oC under a range of sorbitol concentrations from 0.4 M to 1.0M to induce hyper-osmotic stress and by varying the temperature of the thermo-jacketed pre-incubation chamber from 25 oC to 10 oC to impose sub-optimal temperature stress. Compared to controls (0.4 M sorbitol and 25 OC, the mesophyll protoplasts showed remarkable decrease in NaHCO3-dependent O2 evolution (indicator of photosynthetic carbon assimilation, under both hyper-osmotic (1.0 M sorbitol and sub-optimal temperature stress conditions (10 OC, while the decrease in rates of respiratory O2 uptake were marginal. The capacity of AOX pathway increased significantly in parallel to increase in intracellular pyruvate and reactive oxygen species (ROS levels under both hyper-osmotic stress and sub-optimal temperature stress under the background of saturating light. The ratio of redox couple (Malate/OAA related to malate valve increased in contrast to the ratio of redox couple (GSH/GSSG related to antioxidative system during hyper-osmotic stress. Nevertheless, the ratio of GSH/GSSG decreased in the presence of sub-optimal temperature, while the ratio of Malate/OAA showed no visible changes. Also, the redox ratios of pyridine nucleotides increased under hyper-osmotic (NADH/NAD and sub-optimal temperature (NADPH/NADP stresses, respectively. However, upon restriction of AOX pathway by using salicylhydroxamic acid (SHAM, the observed changes in NaHCO3 dependent O2 evolution, cellular ROS, redox ratios of Malate/OAA, NAD(PH/NAD(P and GSH/GSSG were further aggravated under stress conditions with concomitant modulations in NADP-MDH and antioxidant enzymes. Taken together, the

  2. Implications of terminal oxidase function in regulation of salicylic acid on soybean seedling photosynthetic performance under water stress.

    Science.gov (United States)

    Tang, Yanping; Sun, Xin; Wen, Tao; Liu, Mingjie; Yang, Mingyan; Chen, Xuefei

    2017-03-01

    The aim of this study is to investigate whether exogenous application of salicylic acid (SA) could modulate the photosynthetic capacity of soybean seedlings in water stress tolerance, and to clarify the potential functions of terminal oxidase (plastid terminal oxidase (PTOX) and alternative oxidase (AOX)) in SA' s regulation on photosynthesis. The effects of SA and water stress on gas exchange, pigment contents, chlorophyll fluorescence, enzymes (guaiacol peroxidase (POD; EC 1.11.1.7), superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11) and NADP-malate dehydrogenase (NADP-MDH; EC1.1.1.82)) activity and transcript levels of PTOX, AOX1, AOX2a, AOX2b were examined in a hydroponic cultivation system. Results indicate that water stress significantly decreased the photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (E), pigment contents (Chla + b, Chla/b, Car), maximum quantum yield of PSⅡphotochemistry (Fv/Fm), efficiency of excitation capture of open PSⅡcenter (Fv'/Fm'), quantum efficiency of PSⅡphotochemistry (ΦPSⅡ), photochemical quenching (qP), and increased malondialdehyde (MDA) content and the activity of all the enzymes. SA pretreatment led to significant decreases in Ci and MDA content, and increases in Pn, Gs, E, pigment contents, Fv/Fm, Fv'/Fm', ΦPSⅡ, qP, and the activity of all the enzymes. SA treatment and water stress alone significantly up-regulated the expression of PTOX, AOX1 and AOX2b. SA pretreatment further increased the transcript levels of PTOX and AOX2b of soybean seedling under water stress. These results indicate that SA application alleviates the water stress-induced decrease in photosynthesis may mainly through maintaining a lower reactive oxygen species (ROS) level, a greater PSⅡefficiency, and an enhanced alternative respiration and chlororespiration. PTOX and AOX may play important roles in SA-mediated resistance to water stress. Copyright © 2016

  3. Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir.

    Science.gov (United States)

    He, Xiu-Juan; Chen, Shao-Yun; Wu, Jian-Ping; Yang, Li-Rong; Xu, Gang

    2015-11-01

    tert-Butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) is a valuable chiral synthon, which is used for the synthesis of the cholesterol-lowering drugs atorvastatin and rosuvastatin. To date, only the alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefir (LkADH) have demonstrated catalytic activity toward the asymmetric reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (CDOH) to (S)-CHOH. Herein, a tetrad mutant of LkADH (LkTADH), A94T/F147L/L199H/A202L, was screened to be more efficient in this bioreduction process, exhibiting a 3.7- and 42-fold improvement in specific activity toward CDOH (1.27 U/mg) over LbADH (0.34 U/mg) and wild-type LkADH (0.03 U/mg), respectively. The molecular basis for the improved catalytic activity of LkTADH toward CDOH was investigated using homology modeling and docking analysis. Two major issues had a significant impact on the biocatalytic efficiency of this process, including (i) the poor aqueous stability of the substrate and (ii) partial substrate inhibition. A fed-batch strategy was successfully developed to address these issues and maintain a suitably low substrate concentration throughout the entire process. Several other parameters were also optimized, including the pH, temperature, NADP(+) concentration and cell loading. A final CDOH concentration of 427 mM (100 g/L) gave (S)-CHOH in 94 % yield and 99.5 % e.e. after a reaction time of 38 h with whole cells expressing LkTADH. The space-time yield and turnover number of NADP(+) in this process were 10.6 mmol/L/h and 16,060 mol/mol, respectively, which were the highest values ever reported. This new approach therefore represents a promising alternative for the efficient synthesis of (S)-CHOH.

  4. Redox balancing in recombinant strains of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Anderlund, M

    1998-09-01

    transhydrogenation between the two coenzyme systems NADP(H) and NAD(H) during anaerobic growth, S. cerevisiae was transformed with a plasma membrane bound AB-transhydrogenase from E. coli and with a cytoplasmic BB-transhydrogenase from A. vinelandii. Expression of both types changed the intracellular nucleotide levels. The NADPH/NADP{sup +} ratio was reduced while the NADH/NAD{sup +} ratio was almost constant. An increased formation of 2-oxoglutarate, glycerol and acetate was observed during anaerobic glucose fermentation 206 refs, 8 figs, 3 tabs

  5. Functional proteomic analysis of Ankaferd® Blood Stopper

    Directory of Open Access Journals (Sweden)

    Duygu Özel Demiralp

    2010-06-01

    Full Text Available Objective: Ankaferd® Blood Stopper (ABS comprises a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. The basic mechanism of action for ABS is the formation of an encapsulated protein network that provides focal points for vital erythrocyte aggregation. ABS–induced protein network formation with blood cells, particularly erythrocytes, covers the primary and secondary hemostatic system without disturbing individual coagulation factors. Materials and Methods: To understand the effect mechanisms of ABS on hemostasis, a proteomic analysis using 2D gel electrophoresis and mass spectrometer was performed. Results: Proteins of plant origin in Ankaferd® were NADP-dependent-malic enzyme, ribulose bisphosphate-carboxylase-large chain, maturase K, ATP synthase subunit-beta, ATP synthase subunit-alpha, chalcone-flavanone isomerase-1, chalcone-flavanone isomerase-2, and actin-depolymerizing factor. Furthermore, functional proteomic studies revealed that proteins resembling human peptides have been detected within Ankaferd®, including ATP synthase, mucin-16 (CD164 sialomucin-like 2 protein, coiled-coil domain containing 141 hypothetical protein LOC283638 isoform 1, hypothetical protein LOC283638 isoform 2, dynactin 5, complex I intermediate-associated protein 30, mitochondrial, NADH dehydrogenase (ubiquinone 1 alpha subcomplex, TP synthase, H+ transporting, mitochondrial actin binding 1 isoform, LIM domain and actin binding 1 isoform a, LIM domain and actin binding 1 isoform b, spectrin alpha non erythrocytic 1, prolactin releasing hormone receptor, utrophin, tet oncogene family member 2 isoform b, protein phosphatase 1 regulatory subunit 12A, NIMA (never in mitosis gene a-related kinase, ATP-binding cassette protein C12, Homo sapiens malic enzyme 1, mitochondrial NADP(+-dependent malic enzyme 3, ME2 protein, nuclear factor 1 B-type, abhydrolase domain-containing protein 12B, E

  6. Tree ring proxies show physiological responses of eastern red cedar to increased CO2 and SO4 concentrations over the 20th century

    Science.gov (United States)

    Thomas, R. B.; Spal, S.; Maxwell, S.; Nippert, J. B.

    2011-12-01

    We examined the relationships between tree growth during the past century and the ratio of internal carbon dioxide concentration to atmospheric CO2 concentration (ci/ca) and instantaneous water-use efficiency (iWUE) by analyzing δ13C in tree rings of Juniperus virginiana growing on a limestone outcrop in West Virginia, US. Tree rings from years 1909 to 2008 from five Juniperus virginiana trees that ranged from 116 years to over 300 years in age were measured for basal area growth and used for isotopic analysis. Instantaneous WUE increased from approximately 75 to 112μmol mmol-1 over the past century, representing a 49% increase. In addition, we found a positive relationship between iWUE and the basal area increase over this time period, suggesting the increase in WUE translated into greater growth of the Juniperus trees. Typically, we might expect that increased growth of these trees reflects increased photosynthetic rates and decreased stomatal conductance rates resulting from increased atmospheric CO2 concentrations. However, this area of the central Appalachian Mountains has historically received some of the highest rates of acid deposition in the nation resulting from being downwind from an abundance of coal-fired power plants in the Ohio River valley. Our results show that ci/ca declined 14% between 1909 and 1980, but increased 9.6% between 1980 and 2009. We hypothesize that the directional change in ci/ca that occurred around 1980 was due to a reduction in sulfur emissions imposed by the Clean Air Act, environmental legislation enacted in 1970 and amended in 1990. Sulfur deposition measured by the National Atmospheric Deposition Program (NADP) in West Virginia near our Juniperus site shows a 53% decline between 1979 and 2009 and these NADP data show a highly significant negative correlation with ci/ca of Juniperus over this time period. Previously, experimental studies have shown that acidic sulfur mist leaches calcium from leaves causing a reduction in

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

    Science.gov (United States)

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

    2015-03-01

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

  8. Trends in mercury wet deposition and mercury air concentrations across the U.S. and Canada

    Science.gov (United States)

    Weiss-Penzias, Peter S.; Gay, David A.; Brigham, Mark E.; Parsons, Matthew T.; Gustin, Mae S.; ter Shure, Arnout

    2016-01-01

    This study examined the spatial and temporal trends of mercury (Hg) in wet deposition and air concentrations in the United States (U.S.) and Canada between 1997 and 2013. Data were obtained from the National Atmospheric Deposition Program (NADP) and Environment Canada monitoring networks, and other sources. Of the 19 sites with data records from 1997–2013, 53% had significant negative trends in Hg concentration in wet deposition, while no sites had significant positive trends, which is in general agreement with earlier studies that considered NADP data up until about 2010. However, for the time period 2007–2013 (71 sites), 17% and 13% of the sites had significant positive and negative trends, respectively, and for the time period 2008–2013 (81 sites) 30% and 6% of the sites had significant positive and negative trends, respectively. Non-significant positive tendencies were also widespread. Regional trend analyses revealed significant positive trends in Hg concentration in the Rocky Mountains, Plains, and Upper Midwest regions for the recent time periods in addition to significant positive trends in Hg deposition for the continent as a whole. Sulfate concentration trends in wet deposition were negative in all regions, suggesting a lower importance of local Hg sources. The trend in gaseous elemental Hg from short-term datasets merged as one continuous record was broadly consistent with trends in Hg concentration in wet deposition, with the early time period (1998–2007) producing a significantly negative trend (− 1.5 ± 0.2% year− 1) and the recent time period (2008–2013) displaying a flat slope (− 0.3 ± 0.1% year− 1, not significant). The observed shift to more positive or less negative trends in Hg wet deposition primarily seen in the Central-Western regions is consistent with the effects of rising Hg emissions from regions outside the U.S. and Canada and the influence of long-range transport in the free troposphere.

  9. Atmospheric mercury deposition to forests in the eastern USA.

    Science.gov (United States)

    Risch, Martin R; DeWild, John F; Gay, David A; Zhang, Leiming; Boyer, Elizabeth W; Krabbenhoft, David P

    2017-09-01

    Atmospheric mercury (Hg) deposition to forests is important because half of the land cover in the eastern USA is forest. Mercury was measured in autumn litterfall and weekly precipitation samples at a total of 27 National Atmospheric Deposition Program (NADP) monitoring sites in deciduous and mixed deciduous-coniferous forests in 16 states in the eastern USA during 2007-2014. These simultaneous, uniform, repeated, annual measurements of forest Hg include the broadest area and longest time frame to date. The autumn litterfall-Hg concentrations and litterfall mass at the study sites each year were combined with annual precipitation-Hg data. Rates of litterfall-Hg deposition were higher than or equal to precipitation-Hg deposition rates in 70% of the annual data, which indicates a substantial contribution from litterfall to total atmospheric-Hg deposition. Annual litterfall-Hg deposition in this study had a median of 11.7 μg per square meter per year (μg/m 2 /yr) and ranged from 2.2 to 23.4 μg/m 2 /yr. It closely matched modeled dry-Hg deposition, based on land cover at selected NADP Hg-monitoring sites. Mean annual atmospheric-Hg deposition at forest study sites exhibited a spatial pattern partly explained by statistical differences among five forest-cover types and related to the mapped density of Hg emissions. Forest canopies apparently recorded changes in atmospheric-Hg concentrations over time because litterfall-Hg concentrations decreased year to year and litterfall-Hg concentrations were significantly higher in 2007-2009 than in 2012-2014. These findings reinforce reported decreases in Hg emissions and atmospheric elemental-Hg concentrations during this same time period. Methylmercury (MeHg) was detected in all litterfall samples at all sites, compared with MeHg detections in less than half the precipitation samples at selected sites during the study. These results indicate MeHg in litterfall is a pathway into the terrestrial food web where it can

  10. Atmospheric mercury deposition to forests in the eastern USA

    International Nuclear Information System (INIS)

    Risch, Martin R.; DeWild, John F.; Gay, David A.; Zhang, Leiming; Boyer, Elizabeth W.; Krabbenhoft, David P.

    2017-01-01

    Atmospheric mercury (Hg) deposition to forests is important because half of the land cover in the eastern USA is forest. Mercury was measured in autumn litterfall and weekly precipitation samples at a total of 27 National Atmospheric Deposition Program (NADP) monitoring sites in deciduous and mixed deciduous-coniferous forests in 16 states in the eastern USA during 2007–2014. These simultaneous, uniform, repeated, annual measurements of forest Hg include the broadest area and longest time frame to date. The autumn litterfall-Hg concentrations and litterfall mass at the study sites each year were combined with annual precipitation-Hg data. Rates of litterfall-Hg deposition were higher than or equal to precipitation-Hg deposition rates in 70% of the annual data, which indicates a substantial contribution from litterfall to total atmospheric-Hg deposition. Annual litterfall-Hg deposition in this study had a median of 11.7 μg per square meter per year (μg/m 2 /yr) and ranged from 2.2 to 23.4 μg/m 2 /yr. It closely matched modeled dry-Hg deposition, based on land cover at selected NADP Hg-monitoring sites. Mean annual atmospheric-Hg deposition at forest study sites exhibited a spatial pattern partly explained by statistical differences among five forest-cover types and related to the mapped density of Hg emissions. Forest canopies apparently recorded changes in atmospheric-Hg concentrations over time because litterfall-Hg concentrations decreased year to year and litterfall-Hg concentrations were significantly higher in 2007–2009 than in 2012–2014. These findings reinforce reported decreases in Hg emissions and atmospheric elemental-Hg concentrations during this same time period. Methylmercury (MeHg) was detected in all litterfall samples at all sites, compared with MeHg detections in less than half the precipitation samples at selected sites during the study. These results indicate MeHg in litterfall is a pathway into the terrestrial food web where it can

  11. Methodology and significance of studies of atmospheric deposition in highway runoff

    Science.gov (United States)

    Colman, John A.; Rice, Karen C.; Willoughby, Timothy C.

    2001-01-01

    Atmospheric deposition and the processes that are involved in causing and altering atmospheric deposition in relation to highway surfaces and runoff were evaluated nationwide. Wet deposition is more easily monitored than dry deposition, and data on wet deposition are available for major elements and water properties (constituents affecting acid deposition) from the inter-agency National Atmospheric Deposition Program/ National Trends Network (NADP/NTN). Many trace constituents (metals and organic compounds) of interest in highway runoff loads, however, are not included in the NADP/NTN. Dry deposition, which constitutes a large part of total atmospheric deposition for many constituents in highway runoff loads, is difficult to monitor accurately. Dry-deposition rates are not widely available.Many of the highway-runoff investigations that have addressed atmospheric-deposition sources have had flawed investigative designs or problems with methodology. Some results may be incorrect because of reliance on time-aggregated data collected during a period of changing atmospheric emissions. None of the investigations used methods that could accurately quantify the part of highway runoff load that can be attributed to ambient atmospheric deposition. Lack of information about accurate ambient deposition rates and runoff loads was part of the problem. Samples collected to compute the rates and loads were collected without clean-sampling methods or sampler protocols, and without quality-assurance procedures that could validate the data. Massbudget calculations comparing deposition and runoff did not consider loss of deposited material during on-highway processing. Loss of deposited particles from highway travel lanes could be large, as has been determined in labeled particle studies, because of resuspension caused by turbulence from passing traffic. Although a cause of resuspension of large particles, traffic turbulence may increase the rate of deposition for small particles and

  12. Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6.

    Science.gov (United States)

    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

  13. Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

    Science.gov (United States)

    Gong, Ting; Xu, Xiaoqing; Che, You; Liu, Ruihua; Gao, Weixia; Zhao, Fengjie; Yu, Huilei; Liang, Jingnan; Xu, Ping; Song, Cunjiang; Yang, Chao

    2017-08-01

    An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP + ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

  14. Precipitation collector bias and its effects on temporal trends and spatial variability in National Atmospheric Deposition Program/National Trends Network data

    Science.gov (United States)

    Wetherbee, Gregory A.

    2017-01-01

    Precipitation samples have been collected by the National Atmospheric Deposition Program's (NADP) National Trends Network (NTN) using the Aerochem Metrics Model 301 (ACM) collector since 1978. Approximately one-third of the NTN ACM collectors have been replaced with N-CON Systems, Inc. Model ADS 00-120 (NCON) collectors. Concurrent data were collected over 6 years at 12 NTN sites using colocated ACM and NCON collectors in various precipitation regimes. Linear regression models of the colocated data were used to adjust for relative bias between the collectors. Replacement of ACM collectors with NCON collectors resulted in shifts in 10-year seasonal precipitation-weighted mean concentration (PWMC) trend slopes for: cations (−0.001 to −0.007 mgL−1yr−1), anions (−0.009 to −0.028 mgL−1yr−1), and hydrogen ion (+0.689 meqL-1yr−1). Larger shifts in NO3− and SO4−2 seasonal PWMC trend slopes were observed in the Midwest and Northeast US, where concentrations are generally higher than in other regions. Geospatial analysis of interpolated concentration rasters indicated regions of accentuated variability introduced by incorporation of NCON collectors into the NTN.

  15. Peroxisomal enzymes in the liver of rats with experimental diabetes mellitus type 2.

    Science.gov (United States)

    Turecký, L; Kupčová, V; Uhlíková, E; Mojto, V

    2014-01-01

    Diabetes mellitus is relatively frequently associated with fatty liver disease. Increased oxidative stress probably plays an important role in the development of this hepatopathy. One of possible sources of reactive oxygen species in liver is peroxisomal system. There are several reports about changes of peroxisomal enzymes in experimental diabetes, mainly enzymes of fatty acid oxidation. The aim of our study was to investigate the possible changes of activities of liver peroxisomal enzymes, other than enzymes of beta-oxidation, in experimental diabetes mellitus type 2. Biochemical changes in liver of experimental animals suggest the presence of liver steatosis. The changes of serum parameters in experimental group are similar to changes in serum of patients with non-alcoholic fatty liver disease. We have shown that diabetes mellitus influenced peroxisomal enzymes by the different way. Despite of well-known induction of peroxisomal beta-oxidation, the activities of catalase, aminoacid oxidase and NADH-cytochrome b(5) reductase were not significantly changed and the activities of glycolate oxidase and NADP-isocitrate dehydrogenase were significantly decreased. The effect of diabetes on liver peroxisomes is probably due to the increased supply of fatty acids to liver in diabetic state and also due to increased oxidative stress. The changes of metabolic activity of peroxisomal compartment may participate on the development of diabetic hepatopathy.

  16. Continuous Reusability using Immobilized HasApf in Chemoenzymatic Deracemization: A New Heterogeneous Enzyme Catalysis

    Directory of Open Access Journals (Sweden)

    Hiroyuki Nagaoka

    2016-10-01

    Full Text Available This study found that the calibration curve of heme acquisition system A (HasA, a new reactive active species immobilized by a porous ceramic particle (ImHApf; immobilized HasA from Pseudomonas fluorescens can be constructed in the range of 1750–1450 cm−1 using Fourier transform infrared spectroscopy (FTIR analysis, and evaluated its catalytic efficiency. In the asymmetric oxidation of rac-1-(6-methoxynaphthalen-2-ylethanol (rac-1: a naproxen precursor, a product ketone from the (R-isomer is desymmetrized using NaBH4 and continuously reused even if treated with an organic solvent in 50 mM glycine–NaOH buffer at 40 °C in the absence of nicotinamide adenine dinucleotide (NAD(P, leading to >99% enantiomeric excess and >90% chemical yield; the activity was calculated at 0.74 ± 0.03 mU/(mg·min and the turnover number was determined to be approximately 2 × 106. It was confirmed that the other sec-alcohols such as rac-1-(2-naphthylethanol (rac-2 and m- and p-substituted rac-1-phenyl ethanols (rac-3ab–6ab using ImHApf can also yield a single stereoisomer from a racemate. Therefore, HasA immobilization can be expected to become an important tool for building an environmentally friendly system that promotes industrial sustainability.

  17. Sunlight mediated synthesis of silver nanoparticles using redox phytoprotein and their application in catalysis and colorimetric mercury sensing.

    Science.gov (United States)

    Ahmed, Khan Behlol Ayaz; Senthilnathan, Rajendran; Megarajan, Sengan; Anbazhagan, Veerappan

    2015-10-01

    Owing to the benign nature, plant extracts mediated green synthesis of metal nanoparticles (NPs) is rapidly expanding. In this study, we demonstrated the successful green synthesis of silver nanoparticles (AgNPs) by utilizing natural sunlight and redox protein complex composed of ferredoxin-NADP(+) reductase (FNR) and ferredoxin (FD). The capping and stabilization of the AgNPs by the redox protein was confirmed by Fourier transform infrared spectroscopy. Light and redox protein is the prerequisite factor for the formation of AgNPs. The obtained result shows that the photo generated free radicals by the redox protein is responsible for the reduction of Ag(+) to Ag(0). Transmission electron microscopy revealed the formation of spherical AgNPs with size ranging from 10 to 15 nm. As-prepared AgNPs exhibit excellent catalytic activity toward the degradation of hazardous organic dyes, such as methylene blue, methyl orange and methyl red. These bio-inspired AgNPs is highly sensitive and selective in sensing hazardous mercury ions in the water at micromolar concentration. In addition, FNR/FD extract stabilized AgNPs showed good antimicrobial activity against gram positive and gram negative bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-01

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

  19. The utilization of aconate and itaconate by Micrococcus sp

    Science.gov (United States)

    Cooper, R. A.; Itiaba, K.; Kornberg, H. L.

    1965-01-01

    1. An organism, identified as Micrococcus sp., was isolated by elective culture on aconate; it also grew on itaconate. 2. Washed suspensions of the aconate-grown organism readily oxidized intermediates of the tricarboxylic acid cycle, aconate and succinic semialdehyde, but not itaconate. Itaconate-grown cells oxidized tricarboxylic acid-cycle intermediates, succinic semialdehyde and itaconate, but not aconate. Succinate-grown cells oxidized neither itaconate nor aconate. 3. Extracts of aconate-grown cells catalysed the formation of succinic semialdehyde and carbon dioxide, in equimolar amounts, from aconate. In the presence of NAD or NADP, succinic semialdehyde was oxidized to succinate with concomitant reduction of the coenzyme. 4. Extracts of itaconate-grown cells catalysed the formation of pyruvate and acetyl-CoA from itaconyl-CoA. 5. Key enzymes involved in the formation of succinate from aconate, and of pyruvate and acetyl-CoA from itaconate, were distinct and inducible: their formation preceded growth on the appropriate substrate. PMID:14342240

  20. Purification and characterization of vanillin dehydrogenases from alkaliphile Micrococcus sp. TA1 and neutrophile Burkholderia cepacia TM1.

    Science.gov (United States)

    Mitsui, Ryoji; Hirota, Mizuho; Tsuno, Takuo; Tanaka, Mitsuo

    2010-02-01

    Vanillin dehydrogenases (VDHs) were purified and characterized from two bacterial strains that have different pH dependencies for growth. The alkaliphile Micrococcus sp. TA1, isolated from an alkaline spa, can grow on several aromatic compounds such as ferulic acid, vanillin, vanillic acid, and protocatechuic acid under alkaline conditions. The neutrophile Burkholderia cepacia TM1, which was isolated previously, also grew on the above-mentioned compounds because they functioned as the sole carbon source under neutral conditions. Purified VDHs showed activities toward some aromatic aldehydes. These enzymes have the same subunit molecular mass of about 57 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differed in some of their observed properties. Native molecular masses also differed between the purified enzymes. These were 250 kDa for the enzyme from alkaliphilic strain TA1 and 110 kDa for that from neutrophilic strain TM1, as determined by gel filtration. The enzyme from strain TA1 required NADP(+) as a coenzyme for its activity, but that from strain TM1 required NAD(+). These results are important because this is the first report of an alkaliphilic bacterium consuming lignin monomers.

  1. The Role of Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Katarína Smolková

    2012-01-01

    Full Text Available Isocitrate dehydrogenase 2 (IDH2 is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP+-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD+ accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction, which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction to D-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations. D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factor α.

  2. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    Science.gov (United States)

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. 9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1.

    Science.gov (United States)

    Pace-Asciak, C R; Domazet, Z

    1984-11-14

    15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.

  4. Mechanism of mercuric chloride resistance in microorganisms. II. NADPH-dependent reduction of mercuric chloride and vaporization of mercury from mercuric chloride by a multiple drug resistant strain of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Komura, I; Funaba, T; Izaki, K

    1971-01-01

    The activity to vaporize a /sup 203/Hg compound from /sup 203/HgCl/sub 2/ was demonstrated in crude cell-free extracts of a strain of Escherichia coli W2252, which had acquired the multiple drug resistance. NADPH was essential for the vaporization, while NADH had only a slight stimulating effect and NADP/sup +/ had no effect. The oxidation of NADPH dependent on HgCl/sub 2/ was also demonstrated in the crude extracts, but the HgCl/sub 2/-dependent NADH oxidation could be demonstrated only when a partially purified enzyme preparation was used. The rate of NADH oxidation was much slower than that of NADPH oxidation. It was concluded that NADPH, and to a lesser extent NADH, act as electron donors for the enzymatic reduction of HgCl/sub 2/ and the vaporization occurs after this reduction. This reduction and subsequent vaporization seem to provide a mechanism of resistance to HgCl/sub 2/ in E. coli strains having the multiple drug resistance. 15 references, 4 figures, 4 tables.

  5. Characterization of phosphorylated isocitrate dehydrogenase and purification of the isocitrate dehydrogenase kinase/phosphatase of Escherichia coli

    International Nuclear Information System (INIS)

    Malloy, P.J.

    1985-01-01

    NADP + -specific isocitrate dehydrogenase (IDH; EC 1.1.1.42) was shown to be phosphorylated with ( 32 P)-orthophosphate in vivo in several strains of Escherichia coli. In strain KC 13, an adenylate cyclase deficient mutant, the specific activity of IDH decreased 70% when acetate was added to stationary phase cultures grown on glucose. The enzyme was immunoprecipitated from sonic extracts and shown to contain 32 P by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The results demonstrate that unlike many eukaryotic protein kinases, the protein kinase involved in the phosphorylation of IDH in E. coli does not require cyclic adenosine monophosphate for catalysis. Similarly, the phosphorylation of IDH was demonstrated in E. coli mutants deficient in either isocitrate lyase or malate synthase. The incorporation of 32 P in IDH was demonstrated following SDS-PAGE and autoradiography of the immunoprecipitated enzyme. These results suggest that the conditions required for the phosphorylation of IDH do not depend on the functioning of the glyoxylate shunt. Following in vivo 32 P-labeling of E. coli strain F143/KL259 in the presence of acetate, 32 P-labeled IDH was isolated from sonicated extracts of the cells. The 32 P-enzyme was carboxylmethylated and digested with trypsin. A single 32 P-labeled peptide was isolated from the tryptic digest. Amino acid analysis of the purified 32 P-labeled peptide showed that the peptide contains seven amino acids, including a single phosphorylated serine residue

  6. Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

    Science.gov (United States)

    Dirr, H W; Schabort, J C; Weitz, C

    1986-02-01

    Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

  7. Processes of malate catabolism during the anaerobic metabolism of grape berries

    International Nuclear Information System (INIS)

    Flanzy, C.; Andre, P.; Buret, M.; Chambroy, Y.; Garcia, P.

    1976-01-01

    In order to precise malate fate during the anaerobic metabolism of grape, malate- 3 - 14 C was injected into Carignan berries kept in darkness at 35 0 C under carbon dioxide atmosphere. The injection of labelled malate was effected in presence or not of non-labelled oxalate which inhibits malic enzyme (EC I.I.I.40). The analyses of the samples fixed after 3 and 7 days anaerobiosis concerned the titration of various substrates, organic acids, amino-acids and glycolysis products, and the measuring of the NADP + -malic enzyme (EC I.I.I.40) and malate dehydrogenase (EC I.I.I.40). Radioactivity is mainly observed in ethanol, amino-butyrate the non-separated group glycerate-shikimate and succinate. Malic enzyme acts in the first sequence of a process leading from malate to ethanol. Alanin synthesis seems to be stimulated in presence of oxalate. The results obtained and some hypotheses presented in the literature induce to suggest a utilization scheme for malate in the anaerobic metabolism of grape [fr

  8. Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant.

    Science.gov (United States)

    Ohta, Kazuhiro; Moriguchi, Ryo; Kanahama, Koki; Yamaki, Shohei; Kanayama, Yoshinori

    2005-12-01

    The enzyme NAD-dependent sorbitol dehydrogenase (SDH) is well characterized in the Rosaceae family of fruit trees, which synthesizes sorbitol as a translocatable photosynthate. Expressed sequence tags of SDH-like sequences have also been generated from various non-Rosaceae species that do not synthesize sorbitol as a primary photosynthetic product, but the physiological roles of the encoded proteins in non-Rosaceae plants are unknown. Therefore, we isolated an SDH-like cDNA (SDL) from tomato (Lycopersicon esculentum Mill.). Genomic Southern blot analysis suggested that SDL exists in the tomato genome as a single-copy gene. Northern blot analysis showed that SDL is ubiquitously expressed in tomato plants. Recombinant SDL protein was produced and purified for enzymatic characterization. SDL catalyzed the interconversion of sorbitol and fructose with NAD (H). SDL showed highest activity for sorbitol among the several substrates tested. SDL showed no activity with NADP+. Thus, SDL was identified as a SDH, although the Km values and substrate specificity of SDL were significantly different from those of SDH purified from the Japanese pear (Pyrus pyrifolia), a Rosaceae fruit tree. In addition, tomato was transformed with antisense SDL to evaluate the contribution of SDL to SDH activity in tomato. The transformation decreased SDH activity to approximately 50% on average. Taken together, these results provide molecular evidence of SDH in tomato, and SDL was renamed LeSDH.

  9. An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Joanna Griffin

    2011-01-01

    Full Text Available Sequence and structure comparisons of various glutamate dehydrogenases (GDH and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH. A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8 and slightly decreased activity with NADH. At position 242, “P6” of the “core fingerprint,” where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.

  10. Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

    Science.gov (United States)

    Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen

    2014-05-01

    Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

  11. Effect of trichloroethylene (TCE) toxicity on the enzymes of carbohydrate metabolism, brush border membrane and oxidative stress in kidney and other rat tissues.

    Science.gov (United States)

    Khan, Sheeba; Priyamvada, Shubha; Khan, Sara A; Khan, Wasim; Farooq, Neelam; Khan, Farah; Yusufi, A N K

    2009-07-01

    Trichloroethylene (TCE), an industrial solvent, is a major environmental contaminant. Histopathological examinations revealed that TCE caused liver and kidney toxicity and carcinogenicity. However, biochemical mechanism and tissue response to toxic insult are not completely elucidated. We hypothesized that TCE induces oxidative stress to various rat tissues and alters their metabolic functions. Male Wistar rats were given TCE (1000 mg/kg/day) in corn oil orally for 25 d. Blood and tissues were collected and analyzed for various biochemical and enzymatic parameters. TCE administration increased blood urea nitrogen, serum creatinine, cholesterol and alkaline phosphatase but decreased serum glucose, inorganic phosphate and phospholipids indicating kidney and liver toxicity. Activity of hexokinase, lactate dehydrogenase increased in the intestine and liver whereas decreased in renal tissues. Malate dehydrogenase and glucose-6-phosphatase and fructose-1, 6-bisphosphatase decreased in all tissues whereas increased in medulla. Glucose-6-phosphate dehydrogenase increased but NADP-malic enzyme decreased in all tissues except in medulla. The activity of BBM enzymes decreased but renal Na/Pi transport increased. Superoxide dismutase and catalase activities variably declined whereas lipid peroxidation significantly enhanced in all tissues. The present results indicate that TCE caused severe damage to kidney, intestine, liver and brain; altered carbohydrate metabolism and suppressed antioxidant defense system.

  12. Renal oncocytoma characterized by the defective complex I of the respiratory chain boosts the synthesis of the ROS scavenger glutathione.

    Science.gov (United States)

    Kürschner, Gerrit; Zhang, Qingzhou; Clima, Rosanna; Xiao, Yi; Busch, Jonas Felix; Kilic, Ergin; Jung, Klaus; Berndt, Nikolaus; Bulik, Sascha; Holzhütter, Hermann-Georg; Gasparre, Giuseppe; Attimonelli, Marcella; Babu, Mohan; Meierhofer, David

    2017-12-01

    Renal oncocytomas are rare benign tumors of the kidney and characterized by a deficient complex I (CI) enzyme activity of the oxidative phosphorylation (OXPHOS) system caused by mitochondrial DNA (mtDNA) mutations. Yet, little is known about the underlying molecular mechanisms and alterations of metabolic pathways in this tumor. We compared renal oncocytomas with adjacent matched normal kidney tissues on a global scale by multi-omics approaches, including whole exome sequencing (WES), proteomics, metabolomics, and metabolic pathway simulation. The abundance of proteins localized to mitochondria increased more than 2-fold, the only exception was a strong decrease in the abundance for CI subunits that revealed several pathogenic heteroplasmic mtDNA mutations by WES. We also observed renal oncocytomas to dysregulate main metabolic pathways, shunting away from gluconeogenesis and lipid metabolism. Nevertheless, the abundance of energy carrier molecules such as NAD + , NADH, NADP, ATP, and ADP were significantly higher in renal oncocytomas. Finally, a substantial 5000-fold increase of the reactive oxygen species scavenger glutathione can be regarded as a new hallmark of renal oncocytoma. Our findings demonstrate that renal oncocytomas undergo a metabolic switch to eliminate ATP consuming processes to ensure a sufficient energy supply for the tumor.

  13. Organophosphate pesticides-induced changes in the redox status of rat tissues and protective effects of antioxidant vitamins.

    Science.gov (United States)

    Mishra, Vibhuti; Srivastava, Nalini

    2015-04-01

    Organophosphates (OPs) pesticides are among the most toxic synthetic chemicals purposefully added in the environment. The common use of OP insecticides in public health and agriculture results in an environmental pollution and a number of acute and chronic poisoning events. Present study was aimed to evaluate the potential of monocrotophos and quinalphos to effect the redox status and glutathione (GSH) homeostasis in rat tissues and find out whether antioxidant vitamins have some protection on the pesticide-induced alterations. The results showed that these pesticides alone or in combination, caused decrease in the levels of GSH and the corresponding increase in the levels of GSSG, decreasing the GSH/GSSG ratio. The results also showed that NADPH/NADP(+) and NADH/NAD(+) ratios were decreased in the liver and brain of rats on exposure with mococrotophos, quinalphos, and their mixture. These pesticides, alone or in combination, caused alterations in the activities of GSH reductase and glucose-6-phosphate dehydrogenase in the rat tissues. However, the expression of the GSH recycling enzymes did not show significant alterations as compared to control. From the results, it can be concluded that these pesticides generate oxidative stress but their effects were not synergistic when given together and prior feeding of antioxidant vitamins tend to reduce the toxicities of these pesticides. Copyright © 2013 Wiley Periodicals, Inc.

  14. Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection.

    Science.gov (United States)

    Kolb, Alexander L; Corridon, Peter R; Zhang, Shijun; Xu, Weimin; Witzmann, Frank A; Collett, Jason A; Rhodes, George J; Winfree, Seth; Bready, Devin; Pfeffenberger, Zechariah J; Pomerantz, Jeremy M; Hato, Takashi; Nagami, Glenn T; Molitoris, Bruce A; Basile, David P; Atkinson, Simon J; Bacallao, Robert L

    2018-04-01

    Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine ( P <0.05) observed in controls and increased the mitochondria membrane potential ( P <0.05), maximal respiratory capacity ( P <0.05), and intracellular ATP levels ( P <0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning. Copyright © 2018 by the American Society of Nephrology.

  15. Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration

    Directory of Open Access Journals (Sweden)

    Jung Hyun Park

    2017-01-01

    Full Text Available Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants, such as reduced glutathione and protein thiols, and production of reactive oxygen species. Mitochondrial NADP+-dependent isocitrate dehydrogenase (idh2 regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of idh2 in acrolein-induced lung injury using idh2 short hairpin RNA- (shRNA- transfected Lewis lung carcinoma (LLC cells and idh2-deficient (idh2−/− mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In addition, acrolein-induced toxicity in idh2 shRNA-transfected LLC cells and in idh2 knockout mice was ameliorated by the antioxidant, N-acetylcysteine, through attenuation of oxidative stress resulting from idh2 deficiency. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2−/− mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

  16. Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration.

    Science.gov (United States)

    Park, Jung Hyun; Ku, Hyeong Jun; Lee, Jin Hyup; Park, Jeen-Woo

    2017-01-01

    Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants, such as reduced glutathione and protein thiols, and production of reactive oxygen species. Mitochondrial NADP + -dependent isocitrate dehydrogenase ( idh2 ) regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of idh2 in acrolein-induced lung injury using idh2 short hairpin RNA- (shRNA-) transfected Lewis lung carcinoma (LLC) cells and idh2 -deficient ( idh2 -/- ) mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In addition, acrolein-induced toxicity in idh2 shRNA-transfected LLC cells and in idh2 knockout mice was ameliorated by the antioxidant, N-acetylcysteine, through attenuation of oxidative stress resulting from idh2 deficiency. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2 -/- mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

  17. Functional Mitochondrial Complex I Is Required by Tobacco Leaves for Optimal Photosynthetic Performance in Photorespiratory Conditions and during Transients1

    Science.gov (United States)

    Dutilleul, Christelle; Driscoll, Simon; Cornic, Gabriel; De Paepe, Rosine; Foyer, Christine H.; Noctor, Graham

    2003-01-01

    The importance of the mitochondrial electron transport chain in photosynthesis was studied using the tobacco (Nicotiana sylvestris) mutant CMSII, which lacks functional complex I. Rubisco activities and oxygen evolution at saturating CO2 showed that photosynthetic capacity in the mutant was at least as high as in wild-type (WT) leaves. Despite this, steady-state photosynthesis in the mutant was reduced by 20% to 30% at atmospheric CO2 levels. The inhibition of photosynthesis was alleviated by high CO2 or low O2. The mutant showed a prolonged induction of photosynthesis, which was exacerbated in conditions favoring photorespiration and which was accompanied by increased extractable NADP-malate dehydrogenase activity. Feeding experiments with leaf discs demonstrated that CMSII had a lower capacity than the WT for glycine (Gly) oxidation in the dark. Analysis of the postillumination burst in CO2 evolution showed that this was not because of insufficient Gly decarboxylase capacity. Despite the lower rate of Gly metabolism in CMSII leaves in the dark, the Gly to Ser ratio in the light displayed a similar dependence on photosynthesis to the WT. It is concluded that: (a) Mitochondrial complex I is required for optimal photosynthetic performance, despite the operation of alternative dehydrogenases in CMSII; and (b) complex I is necessary to avoid redox disruption of photosynthesis in conditions where leaf mitochondria must oxidize both respiratory and photorespiratory substrates simultaneously. PMID:12529534

  18. Characterization of Developmental- and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf (Hibiscus cannabinus L.)

    Science.gov (United States)

    Lim, Hyoun-Sub; Park, Sang-Un; Bae, Hyeun-Jong; Natarajan, Savithiry

    2014-01-01

    Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments. PMID:24723816

  19. [Metabolism of thyroid gland cells as affected by prolactin and emotional-physical stress].

    Science.gov (United States)

    Strizhkov, V V

    1991-01-01

    A study was made of the role of prolactin (PRL) in the regulation of thyroid function in intact animals and in those exposed to stress (swimming was used as physical exercise). A single daily dose of 125 micrograms of PRL per 100 g of body mass was injected subcutaneously in 0.5 ml of saline solution during a week to male rats (control: intact rats; injection of 0.5 ml of saline solution subcutaneously). Redox enzymes; succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, NAD.H2 and NADP.H2, ATPase and monoamine oxidase, total protein, RNA and glycogen in glandular cells were investigated histochemically 24 h after the last injection of PRL or saline, 30 min., 1, 2, 3, 5 and 7 hours after swimming or right after complete fatigue (in the presence of experimental hyperprolactinemia). A conclusion has been made that one of the most important mechanisms of the adaptive effect of PRL is its ability to suppress thyroid function, thus decreasing the metabolism level, which results in reduction of oxygen consumption and improves body tolerance to stress.

  20. Anti-diabetic effects of DA-11004, a synthetic IDPc inhibitor in high fat high sucrose diet-fed C57BL/6J mice.

    Science.gov (United States)

    Shin, Chang Yell; Jung, Mi Young; Lee, In Ki; Son, Miwon; Kim, Dong Sung; Lim, Joong In; Kim, Soon Hoe; Yoo, Moohi; Huh, Tae Lin; Sohn, Young Taek; Kim, Won Bae

    2004-01-01

    DA-11004 is a synthetic, potent NADP-dependent isocitrate dehydrogenase (IDPc) inhibitor where IC50 for IDPc is 1.49 microM. The purpose of this study was to evaluate the effects of DA-11004 on the high fat high sucrose (HF)-induced obesity in male C57BL/6J mice. After completing a 8-week period of experimentation, the mice were sacrificed 1 hr after the last DA-11004 treatment and their blood, liver, and adipose tissues (epididymal and retroperitoneal fat) were collected. There was a significant difference in the pattern of increasing body weight between the HF control and the DA-11004 group. In the DA-11004 (100 mg/kg) treated group the increase in body weight significantly declined and a content of epididymal fat and retroperitoneal fat was also significantly decreased as opposed to the HF control. DA-11004 (100 mg/ kg) inhibited the IDPc activity, and thus, NADPH levels in plasma and the levels of free fatty acid (FFA) or glucose in plasma were less than the levels of the HF control group. In conclusion, DA-11004 inhibited the fatty acid synthesis in adipose tissues via IDPc inhibition, and it decreased the plasma glucose levels and FFA in HF diet-induced obesity of C57BL/6J mice.

  1. Identification of differentially expressed proteins during human urinary bladder cancer progression.

    Science.gov (United States)

    Memon, Ashfaque A; Chang, Jong W; Oh, Bong R; Yoo, Yung J

    2005-01-01

    Comparative proteome analysis was performed between RT4 (grade-1) and T24 (grade-3) bladder cancer cell lines, in an attempt to identify differentially expressed proteins during bladder cancer progression. Among those relatively abundant proteins, seven spots changed more than two-fold reproducibly and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may involve in tumor progression and metastasis. However, additional investigations are needed on large number of human samples to further verify these findings.

  2. Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry

    Science.gov (United States)

    Sharwood, Robert E.; Sonawane, Balasaheb V.; Ghannoum, Oula; Whitney, Spencer M.

    2016-01-01

    Plants operating C3 and C4 photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C3 and C4 plant soluble protein. We show that differences in plant extract preparation and assay conditions are required to measure NADP-malic enzyme and phosphoenolpyruvate carboxylase (pH 8, Mg2+, 22 °C) and phosphoenolpyruvate carboxykinase (pH 7, >2mM Mn2+, no Mg2+) maximal activities accurately. We validate how the omission of MgCl2 during leaf protein extraction, lengthy (>1min) centrifugation times, and the use of non-pure ribulose-1,5-bisphosphate (RuBP) significantly underestimate Rubisco activation status. We show how Rubisco activation status varies with leaf ontogeny and is generally lower in mature C4 monocot leaves (45–60% activation) relative to C3 monocots (55–90% activation). Consistent with their >3-fold lower Rubisco contents, full Rubisco activation in soluble protein from C4 leaves (<5min) was faster than in C3 plant samples (<10min), with addition of Rubisco activase not required for full activation. We conclude that Rubisco inactivation in illuminated leaves primarily stems from RuBP binding to non-carbamylated enzyme, a state readily reversible by dilution during cellular protein extraction. PMID:27122573

  3. A two-dimensional microscale model of gas exchange during photosynthesis in maize (Zea mays L.) leaves.

    Science.gov (United States)

    Retta, Moges; Ho, Quang Tri; Yin, Xinyou; Verboven, Pieter; Berghuijs, Herman N C; Struik, Paul C; Nicolaï, Bart M

    2016-05-01

    CO2 exchange in leaves of maize (Zea mays L.) was examined using a microscale model of combined gas diffusion and C4 photosynthesis kinetics at the leaf tissue level. Based on a generalized scheme of photosynthesis in NADP-malic enzyme type C4 plants, the model accounted for CO2 diffusion in a leaf tissue, CO2 hydration and assimilation in mesophyll cells, CO2 release from decarboxylation of C4 acids, CO2 fixation in bundle sheath cells and CO2 retro-diffusion from bundle sheath cells. The transport equations were solved over a realistic 2-D geometry of the Kranz anatomy obtained from light microscopy images. The predicted responses of photosynthesis rate to changes in ambient CO2 and irradiance compared well with those obtained from gas exchange measurements. A sensitivity analysis showed that the CO2 permeability of the mesophyll-bundle sheath and airspace-mesophyll interfaces strongly affected the rate of photosynthesis and bundle sheath conductance. Carbonic anhydrase influenced the rate of photosynthesis, especially at low intercellular CO2 levels. In addition, the suberin layer at the exposed surface of the bundle sheath cells was found beneficial in reducing the retro-diffusion. The model may serve as a tool to investigate CO2 diffusion further in relation to the Kranz anatomy in C4 plants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Photosynthetic plasticity in Flaveria brownii: Growth irradiance and the expression of C4 photosynthesis

    International Nuclear Information System (INIS)

    Cheng, Shuhua; Moore, B.D.; Wu, Jingrui; Edwards, G.E.; Ku, M.S.B.

    1989-01-01

    Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO 2 compensation point and the inhibition of photosynthesis by 21% O 2 were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C 4 cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO 2 compensation point and the degree of O 2 inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO 2 between Rubisco of the C 3 pathway and phosphoenolpyruvate carboxylase of the C 4 cycle was determined by exposing leaves to 14 CO 2 for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that ∼94% of the CO 2 was fixed by the C 4 cycle in high light grown plants, versus ∼78% in low light grown plants. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C 4 -like pattern of compartmentation. Pyruvate,Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants

  5. Hypomorphic temperature-sensitive alleles of NSDHL cause CK syndrome.

    Science.gov (United States)

    McLarren, Keith W; Severson, Tesa M; du Souich, Christèle; Stockton, David W; Kratz, Lisa E; Cunningham, David; Hendson, Glenda; Morin, Ryan D; Wu, Diane; Paul, Jessica E; An, Jianghong; Nelson, Tanya N; Chou, Athena; DeBarber, Andrea E; Merkens, Louise S; Michaud, Jacques L; Waters, Paula J; Yin, Jingyi; McGillivray, Barbara; Demos, Michelle; Rouleau, Guy A; Grzeschik, Karl-Heinz; Smith, Raffaella; Tarpey, Patrick S; Shears, Debbie; Schwartz, Charles E; Gecz, Jozef; Stratton, Michael R; Arbour, Laura; Hurlburt, Jane; Van Allen, Margot I; Herman, Gail E; Zhao, Yongjun; Moore, Richard; Kelley, Richard I; Jones, Steven J M; Steiner, Robert D; Raymond, F Lucy; Marra, Marco A; Boerkoel, Cornelius F

    2010-12-10

    CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development. Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. Novel inborn error of folate metabolism: identification by exome capture and sequencing of mutations in the MTHFD1 gene in a single proband.

    Science.gov (United States)

    Watkins, David; Schwartzentruber, Jeremy A; Ganesh, Jaya; Orange, Jordan S; Kaplan, Bernard S; Nunez, Laura Dempsey; Majewski, Jacek; Rosenblatt, David S

    2011-09-01

    An infant was investigated because of megaloblastic anaemia, atypical hemolytic uraemic syndrome, severe combined immune deficiency, elevated blood levels of homocysteine and methylmalonic acid, and a selective decreased synthesis of methylcobalamin in cultured fibroblasts. Exome sequencing was performed on patient genomic DNA. Two mutations were identified in the MTHFD1 gene, which encodes a protein that catalyses three reactions involved in cellular folate metabolism. This protein is essential for the generation of formyltetrahydrofolate and methylenetetrahydrofolate and important for nucleotide and homocysteine metabolism. One mutation (c.727+1G>A) affects the splice acceptor site of intron 8. The second mutation, c.517C>T (p.R173C), changes a critical arginine residue in the NADP-binding site of the protein. Mutations affecting this arginine have previously been shown to affect enzyme activity. Both parents carry a single mutation and an unaffected sibling carries neither mutation. The combination of two mutations in the MTHFRD1 gene, predicted to have severe consequences, in the patient and their absence in the unaffected sibling, supports causality. This patient represents the first case of an inborn error of folate metabolism affecting the trifunctional MTHFD1 protein. This report reinforces the power of exome capture and sequencing for the discovery of novel genes, even when only a single proband is available for study.

  7. Stereochemical diversity in lignan biosynthesis of Arctium lappa L.

    Science.gov (United States)

    Suzuki, Shiro; Umezawa, Toshiaki; Shimada, Mikio

    2002-06-01

    The stereochemistry of lignan biosynthesis in Arctium lappa L. is regulated organ-specifically. (+)-Secoisolariciresinol [81% enantiomeric excess (e.e.)] was isolated from A. lappa petioles. In sharp contrast, lignans whose predominant enantiomers have the opposite absolute configuration to that of (+)-secoisolariciresinol [i.e., (-)-matairesinol (>99% e.e.), (-)-arctigenin (>99% e.e.), and (-)-secoisolariciresinol (65% e.e.)] were isolated from seeds of the species. The stereochemical diversity of secoisolariciresinol was demonstrated with enzyme preparations from A. lappa petioles and seeds. Thus, a petiole enzyme preparation catalyzed the formation of (+)-pinoresinol (33% e.e.), (+)-lariciresinol (30% e.e.), and (+)-secoisolariciresinol (20% e.e.) from achiral coniferyl alcohol in the presence of NADPH and H202, whereas that from ripening seeds catalyzed the formation of (-)-pinoresinol (22% e.e.), (-)-lariciresinol (>99% e.e.), and (-)-secoisolariciresinol (38% e.e.) under the same conditions. In addition, the ripening seed enzyme preparation mediated the selective formation of the optically pure (>99% e.e.) (-)-enantiomer of matairesinol from racemic (+/-)-secoisolariciresinols in the presence of NADP. These results indicate that the stereochemical mechanism for lignan biosynthesis in A. lappa varies with organs, suggesting that multiple lignan-synthesizing isozymes are involved in the stereochemical control of lignan formation in A. lappa.

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

    Science.gov (United States)

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

    2015-08-01

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

  9. Detoxification of furfural in Corynebacterium glutamicum under aerobic and anaerobic conditions.

    Science.gov (United States)

    Tsuge, Yota; Hori, Yoshimi; Kudou, Motonori; Ishii, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2014-10-01

    The toxic fermentation inhibitors in lignocellulosic hydrolysates raise serious problems for the microbial production of fuels and chemicals. Furfural is considered to be one of the most toxic compounds among these inhibitors. Here, we describe the detoxification of furfural in Corynebacterium glutamicum ATCC13032 under both aerobic and anaerobic conditions. Under aerobic culture conditions, furfuryl alcohol and 2-furoic acid were produced as detoxification products of furfural. The ratio of the products varied depending on the initial furfural concentration. Neither furfuryl alcohol nor 2-furoic acid showed any toxic effect on cell growth, and both compounds were determined to be the end products of furfural degradation. Interestingly, unlike under aerobic conditions, most of the furfural was converted to furfuryl alcohol under anaerobic conditions, without affecting the glucose consumption rate. Both the NADH/NAD(+) and NADPH/NADP(+) ratio decreased in the accordance with furfural concentration under both aerobic and anaerobic conditions. These results indicate the presence of a single or multiple endogenous enzymes with broad and high affinity for furfural and co-factors in C. glutamicum ATCC13032.

  10. Mitochondrial metabolism of pyruvate is essential for regulating glucose-stimulated insulin secretion.

    Science.gov (United States)

    Patterson, Jessica N; Cousteils, Katelyn; Lou, Jennifer W; Manning Fox, Jocelyn E; MacDonald, Patrick E; Joseph, Jamie W

    2014-05-09

    It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in β-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that β-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 β-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-β-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.

  11. FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors.

    Science.gov (United States)

    Guan, Xiaoqian; Chen, Shuai; Voon, Chia Pao; Wong, Kam-Bo; Tikkanen, Mikko; Lim, Boon L

    2018-01-01

    Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP + oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

  12. Efficacy of turmeric on blood sugar and polyol pathway in diabetic albino rats.

    Science.gov (United States)

    Arun, N; Nalini, N

    2002-01-01

    In the traditional system of medicine, Ayurveda, several spices and herbs are thought to possess medicinal properties. Among the spices, turmeric rhizomes (Curcuma longa. Linn.) are used as flavoring and coloring agents in the Indian diet everyday. In this research, we studied the effect of turmeric and its active principle, curcumin, on diabetes mellitus in a rat model. Alloxan was used to induce diabetes. Administration of turmeric or curcumin to diabetic rats reduced the blood sugar, Hb and glycosylated hemoglobin levels significantly. Turmeric and curcumin supplementation also reduced the oxidative stress encountered by the diabetic rats. This was demonstrated by the lower levels of TBARS (thiobarbituric acid reactive substances), which may have been due to the decreased influx of glucose into the polyol pathway leading to an increased NADPH/NADP ratio and elevated activity of the potent antioxdiant enzyme GPx. Moreover, the activity of SDH (sorbitol dehydrogenase), which catalyzes the conversion of sorbitol to fructose, was lowered significantly on treatment with turmeric or curcumin. These results also appeared to reveal that curcumin was more effective in attenuating diabetes mellitus related changes than turmeric.

  13. Effect of light on respiration and development of photosynthetic cells. Progress report, September 1, 1975--June 1, 1976

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, M

    1976-06-01

    Adaptation and deadaptation of unicellular algal cells to a hydrogen metabolism may involve not only hydrogenase but also the site of entry of electrons from NADH. NADPH can also serve as an electron donor in a chloroplast preparation fortified with hydrogenase but the rate of photoevolution falls off rapidly perhaps due to competition with NADP for electrons. The evolution of H/sub 2/ with reduced pyridine nucleotide as electron donor is coupled to ATP formation. Evidence is presented that soluble ferredoxin may not be the immediate donor of H/sub 2/. Hydrogenases from Chlamydomonas and Scenedesmus have been highly purified and will be used to test the stoichiometry of NADH disappearance, ATP and H/sub 2/ formation and also whether H/sub 2/-photoevolution in contrast to dark evolution is ferredoxin dependent. Studies with intact Chlamydomonas provide evidence that a reduced carbon compound and not water is the source of H/sub 2/. The presence of acetate eliminates the CO/sub 2/ production. The ratio of CO/sub 2/:H/sub 2/ is 1:2 and evolution is blocked by inhibitors of glycolysis. Acetate has a strong effect on H/sub 2/O. Cells starved over a long period still retain photoreduction but will not evolve H/sub 2/ until glucose is added.

  14. Reductive Stress in Inflammation-Associated Diseases and the Pro-Oxidant Effect of Antioxidant Agents

    Directory of Open Access Journals (Sweden)

    Israel Pérez-Torres

    2017-10-01

    Full Text Available Abstract: Reductive stress (RS is the counterpart oxidative stress (OS, and can occur in response to conditions that shift the redox balance of important biological redox couples, such as the NAD+/NADH, NADP+/NADPH, and GSH/GSSG, to a more reducing state. Overexpression of antioxidant enzymatic systems leads to excess reducing equivalents that can deplete reactive oxidative species, driving the cells to RS. A feedback regulation is established in which chronic RS induces OS, which in turn, stimulates again RS. Excess reducing equivalents may regulate cellular signaling pathways, modify transcriptional activity, induce alterations in the formation of disulfide bonds in proteins, reduce mitochondrial function, decrease cellular metabolism, and thus, contribute to the development of some diseases in which NF-κB, a redox-sensitive transcription factor, participates. Here, we described the diseases in which an inflammatory condition is associated to RS, and where delayed folding, disordered transport, failed oxidation, and aggregation are found. Some of these diseases are aggregation protein cardiomyopathy, hypertrophic cardiomyopathy, muscular dystrophy, pulmonary hypertension, rheumatoid arthritis, Alzheimer’s disease, and metabolic syndrome, among others. Moreover, chronic consumption of antioxidant supplements, such as vitamins and/or flavonoids, may have pro-oxidant effects that may alter the redox cellular equilibrium and contribute to RS, even diminishing life expectancy.

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

    Science.gov (United States)

    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.

  16. An aldo-keto reductase, Bbakr1, is involved in stress response and detoxification of heavy metal chromium but not required for virulence in the insect fungal pathogen, Beauveria bassiana.

    Science.gov (United States)

    Wang, Huifang; He, Zhangjiang; Luo, Linli; Zhao, Xin; Lu, Zhuoyue; Luo, Tingying; Li, Min; Zhang, Yongjun

    2018-02-01

    The aldo-keto reductases (AKRs) belong to the NADP-dependent oxidoreductase superfamily, which play important roles in various physiological functions in prokaryotic and eukaryotic organisms. However, many AKR superfamily members remain uncharacterized. Here, a downstream target gene of the HOG1 MAPK pathways coding for an aldo-keto reductase, named Bbakr1, was characterized in the insect fungal pathogen, Beauveria bassiana. Bbakr1 expression increased in response to osmotic and salt stressors, and oxidative and heavy metal (chromium) stress. Deletion of Bbakr1 caused a reduction in conidiation, as well as delayed conidial germination. ΔBbakr1 displayed increased sensitivity to osmotic/high-salt stress with decreased compatible solute accumulation. In addition, the mutant was more sensitive to high concentrations of the heavy metal, chromium, and to oxidative stress than the wild type cells, with impaired ability to detoxify active aldehyde that might accumulate due to lipid peroxidation. However, over-expressing Bbakr1 in either the wild type strain or a ΔBbhog1 background did not cause any obvious changes in phenotypes as compared to their controls. Little effect on virulence was seen for either the ΔBbakr1 or overexpression strains in insect bioassays via cuticle infection or intrahemocoel injection assays, suggesting that Bbakr1 is not required for virulence. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Isolation and characterization of two different flavodoxins from the eukaryote Chlorella fusca.

    Science.gov (United States)

    Peleato, M L; Ayora, S; Inda, L A; Gómez-Moreno, C

    1994-01-01

    Two different molecular forms of flavodoxin from the green alga Chlorella fusca have been purified to homogeneity and their properties compared. The molecular masses are 22 kDa (flavodoxin I) and 20 kDa (flavodoxin II). Western blots of axenic crude extract show the two bands. Both are single polypeptide chains and their N-terminal sequences differ but are very similar. Each form contains 1 mol of FMN/mol of apoprotein, exhibits a typical flavodoxin u.v.-visible absorption spectrum and does not contain covalently bound phosphate. The oxidation-reduction properties of the FMN in the flavodoxins differ considerably. Redox potentials of flavodoxin I at pH8 are -240 mV for the oxidized/semiquinone couple and -350 mV for the semiquinone/hydroquinone couple. Flavodoxin II gives more electronegative values: -278 mV and -458 mV respectively. Flavodoxin II fulfils better the redox requirements for photosynthetic electron transport and, as expected, it is more efficient at mediating NADP+ photoreduction in the photosynthetic electron flow. A new h.p.l.c. method for flavodoxin purification is described, which is useful for the isolation of very similar anionic proteins. Images Figure 2 Figure 3 PMID:7945206

  18. The contribution of stored malate and citrate to the substrate requirements of metabolism of ripening peach (Prunus persica L. Batsch) flesh is negligible. Implications for the occurrence of phosphoenolpyruvate carboxykinase and gluconeogenesis.

    Science.gov (United States)

    Famiani, Franco; Farinelli, Daniela; Moscatello, Stefano; Battistelli, Alberto; Leegood, Richard C; Walker, Robert P

    2016-04-01

    The first aim of this study was to determine the contribution of stored malate and citrate to the substrate requirements of metabolism in the ripening flesh of the peach (Prunus persica L. Batsch) cultivar Adriatica. In the flesh, stored malate accumulated before ripening could contribute little or nothing to the net substrate requirements of metabolism. This was because there was synthesis and not dissimilation of malate throughout ripening. Stored citrate could potentially contribute a very small amount (about 5.8%) of the substrate required by metabolism when the whole ripening period was considered, and a maximum of about 7.5% over the latter part of ripening. The second aim of this study was to investigate why phosphoenolpyruvate carboxykinase (PEPCK) an enzyme utilised in gluconeogenesis from malate and citrate is present in peach flesh. The occurrence and localisation of enzymes utilised in the metabolism of malate, citrate and amino acids were determined in peach flesh throughout its development. Phosphoenolpyruvate carboxylase (essential for the synthesis of malate and citrate) was present in the same cells and at the same time as PEPCK and NADP-malic enzyme (both utilised in the dissimilation of malate and citrate). A hypothesis is presented to explain the presence of these enzymes and to account for the likely occurrence of gluconeogenesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  19. Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway

    Science.gov (United States)

    Mandal, Abhishek; Das, Sushmita; Roy, Saptarshi; Ghosh, Ayan Kumar; Sardar, Abul Hasan; Verma, Sudha; Saini, Savita; Singh, Ruby; Abhishek, Kumar; Kumar, Ajay; Mandal, Chitra; Das, Pradeep

    2016-01-01

    The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis. PMID:26808657

  20. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens

    International Nuclear Information System (INIS)

    Ahn, Jae-Woo; Lee, Shin Youp; Kim, Sangwoo; Cho, Sun Ja; Lee, Sun Bok; Kim, Kyung-Jin

    2011-01-01

    Recombinant glucuronic acid dehydrogenase from the halophilic bacterium Chromohalobacter salexigens has been crystallized and X-ray diffraction data collected to a maximum resolution of 2.1 Å. Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD + rather than NADP + as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 M calcium acetate, 0.1 M Tris–HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The GluUADH crystal belonged to space group P6 3 , with unit-cell parameters a = b = 122.58, c = 150.49 Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V M ) is 2.78 Å 3 Da −1 . The structure was solved by the single anomalous dispersion method and structure refinement is in progress

  1. Photosynthetic pathways and the geographical distribution of grasses in South West Africa/Namibia

    International Nuclear Information System (INIS)

    Ellis, R.P.; Vogel, J.C.; Fuls, A.

    1980-01-01

    Analysis of floristic lists for South West Africa/Namibia shows that, throughout the territory, more than 95% of the grass species occurring in any given area display the C 4 photosynthetic pathway. Exceptions are areas in the north-east and southwest where between 5% and 18% of the grass species are of the C 3 type. The south-western district of Luderitz falls within the winter rainfall area and it is only here that temperate C 3 genera are found. The C 3 species in the north-east belong to tropical groups. Most of the South West African C 3 grasses grow in specialized habitats and are either hydrophytes or sciophytes. Subdivision of the C 4 grasses into the three subtypes of the C 4 pathway reveals distinctive distributional trends. Malate formers or NADP-me species clearly become more abundant with increasing rainfall, whereas the aspartate formers show the opposite tendency. However, within the aspartate forming group the results show that it is specifically the NAD-me type of species which dominate in areas of very low precipitation, notably in the Namib and pre-Namib areas where rainfall is less than 200 mm/yr. The PEP-ck species form a group intermediate between the malate formers and the NAD-me grasses, especially as far as their water requirements are concerned [af

  2. A novel malic enzyme gene, Mime2, from Mortierella isabellina M6-22 contributes to lipid accumulation.

    Science.gov (United States)

    Li, Shan; Li, Lingyan; Xiong, Xiangfeng; Ji, Xiuling; Wei, Yunlin; Lin, Lianbing; Zhang, Qi

    2018-05-18

    This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation. Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as K m and V max for NADP + were determined. The effects of EDTA or metal ions (Mn 2+ , Mg 2+ , Co 2+ , Cu 2+ , Ca 2+ , or Zn 2+ ) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively. The act ivity of MIME2 was significantly increased by Mg 2+ , Ca 2+ , or Mn 2+ at 0.5 mM but inhibited by Cu 2+ or Zn 2+ (p M6-22 contributes to lipid accumulation in strain YM25235.

  3. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  4. Comparative Proteomic Analysis Reveals Proteins Putatively Involved in Toxin Biosynthesis in the Marine Dinoflagellate Alexandrium catenella

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    2013-01-01

    Full Text Available Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P < 0.05, and 53 proteins were identified using database searching. These proteins were involved in a variety of biological processes, i.e., protein modification and biosynthesis, metabolism, cell division, oxidative stress, transport, signal transduction, and translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to, alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  5. Comparative proteomic analysis reveals proteins putatively involved in toxin biosynthesis in the marine dinoflagellate Alexandrium catenella.

    Science.gov (United States)

    Wang, Da-Zhi; Gao, Yue; Lin, Lin; Hong, Hua-Sheng

    2013-01-22

    Alexandrium is a neurotoxin-producing dinoflagellate genus resulting in paralytic shellfish poisonings around the world. However, little is known about the toxin biosynthesis mechanism in Alexandrium. This study compared protein profiles of A. catenella collected at different toxin biosynthesis stages (non-toxin synthesis, initial toxin synthesis and toxin synthesizing) coupled with the cell cycle, and identified differentially expressed proteins using 2-DE and MALDI-TOF-TOF mass spectrometry. The results showed that toxin biosynthesis of A. catenella occurred within a defined time frame in the G1 phase of the cell cycle. Proteomic analysis indicated that 102 protein spots altered significantly in abundance (P translation. Among them, nine proteins with known functions in paralytic shellfish toxin-producing cyanobacteria, i.e., methionine S-adenosyltransferase, chloroplast ferredoxin-NADP+ reductase, S-adenosylhomocysteinase, adenosylhomocysteinase, ornithine carbamoyltransferase, inorganic pyrophosphatase, sulfotransferase (similar to), alcohol dehydrogenase and arginine deiminase, varied significantly at different toxin biosynthesis stages and formed an interaction network, indicating that they might be involved in toxin biosynthesis in A. catenella. This study is the first step in the dissection of the behavior of the A. catenella proteome during different toxin biosynthesis stages and provides new insights into toxin biosynthesis in dinoflagellates.

  6. Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

    DEFF Research Database (Denmark)

    Willemoes, Martin; Kilstrup, Mogens; Roepstorff, P.

    2002-01-01

    revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis...... was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced......The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has...

  7. Discovery, synthesis and in combo studies of a tetrazole analogue of clofibric acid as a potent hypoglycemic agent.

    Science.gov (United States)

    Navarrete-Vázquez, Gabriel; Alaniz-Palacios, Alfredo; Hidalgo-Figueroa, Sergio; González-Acevedo, Cristina; Ávila-Villarreal, Gabriela; Estrada-Soto, Samuel; Webster, Scott P; Medina-Franco, José L; López-Vallejo, Fabian; Guerrero-Álvarez, Jorge; Tlahuext, Hugo

    2013-06-01

    A tetrazole isosteric analogue of clofibric acid (1) was prepared using a short synthetic route and was characterized by elemental analysis, NMR ((1)H, (13)C) spectroscopy, and single-crystal X-ray diffraction. The in vitro inhibitory activity of 1 against 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) was evaluated, showing a moderate inhibitory enzyme activity (51.17% of inhibition at 10 μM), being more active than clofibrate and clofibric acid. The antidiabetic activity of compound 1 was determined at 50 mg/Kg single dose using a non insulin dependent diabetes mellitus rat model. The results indicated a significant decrease of plasma glucose levels, during the 7h post-administration. Additionally, we performed a molecular docking of 1 into the ligand binding pocket of one subunit of human 11β-HSD1. In this model, compound 1 binds into the catalytic site of 11β-HSD1 in two different orientations. Both of them, show important short contacts with the catalytic residues Ser 170, Tyr 183, Asp 259 and also with the nicotinamide ring of NADP(+). Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Genética molecular de las hemoglobinas bacterianas: estructura, regulación y función

    Directory of Open Access Journals (Sweden)

    Elizabeth Hernández Urzúa

    2004-01-01

    Full Text Available Las hemoglobinas se definen como aquellas hemoproteínas que unen reversiblemente el oxígeno. Estas proteínas se encuentran distribuidas en plantas, protozoarios, hongos y bacterias. Las hemoglobinas presentes en los microorganismos se han dividido en tres grupos. En el primer grupo se encuentran aquellas hemoproteínas que presentan un solo dominio con el grupo hemo, por ejemplo la hemoglobina de Vitreoscilla. En el segundo se encuentran aquellas hemoglobinas que presentan dos diferentes dominios, el primer dominio con el grupo hemo el cual es homólogo a la hemoglobina de Vitreoscilla y un segundo dominio con actividad de reductasa el cual es homólogo a la familia de las proteínas ferrodoxín NADP- reductasas (FNR, a éstas hemoglobinas se les conoce como flavohemoglobinas, un ejemplo es Hmp, la flavohemoglobina de Escherichia coli, ésta fue la primera flavohemoglobina estudiada a nivel molecular. En el tercer grupo se encuentran las hemoglobinas truncadas, éstas son proteínas pequeñas de 20 a 30 aminoácidos en donde se encuentra el dominio hemo. Poco se sabe acerca de la función de estas hemoglobinas, pero se ha reportado su participación en la protección y destoxificación del óxido nítrico.

  9. Loch Vale Watershed Long-Term Ecological Research and Monitoring Program: Quality Assurance Report, 2003-09

    Science.gov (United States)

    Richer, Eric E.; Baron, Jill S.

    2011-01-01

    The Loch Vale watershed project is a long-term research and monitoring program located in Rocky Mountain National Park that addresses watershed-scale ecosystem processes, particularly as they respond to atmospheric deposition and climate variability. Measurements of precipitation depth, precipitation chemistry, discharge, and surface-water quality are made within the watershed and elsewhere in Rocky Mountain National Park. As data collected for the program are used by resource managers, scientists, policy makers, and students, it is important that all data collected in Loch Vale watershed meet high standards of quality. In this report, data quality was evaluated for precipitation, discharge, and surface-water chemistry measurements collected during 2003-09. Equipment upgrades were made at the Loch Vale National Atmospheric Deposition Program monitoring site to improve precipitation measurements and evaluate variability in precipitation depth and chemistry. Additional solar panels and batteries have been installed to improve the power supply, and data completeness, at the NADP site. As a result of equipment malfunction, discharge data for the Loch Outlet were estimated from October 18, 2005, to August 17, 2006. Quality-assurance results indicate that more than 98 percent of all surface-water chemistry measurements were accurate and precise. Records that did not meet quality criteria were removed from the database. Measurements of precipitation depth, precipitation chemistry, discharge, and surface-water quality were all sufficiently complete and consistent to support project data needs.

  10. Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Shanshan Sun

    2017-04-01

    Full Text Available Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD, a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen–glucose-deprivation/reperfusion (OGD/R model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XFe24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP+ ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance.

  11. Investigation on the Metabolic Regulation of pgi gene knockout Escherichia coli by Enzyme Activities and Intracellular Metabolite Concentrations

    Directory of Open Access Journals (Sweden)

    Nor ‘Aini, A. R.

    2006-01-01

    Full Text Available An integrated analysis of the cell growth characteristics, enzyme activities, intracellular metabolite concentrations was made to investigate the metabolic regulation of pgi gene knockout Escherichia coli based on batch culture and continuous culture which was performed at the dilution rate of 0.2h-1. The enzymatic study identified that pathways of pentose phosphate, ED pathway and glyoxylate shunt were all active in pgi mutant. The glycolysis enzymes i.e glyceraldehyde-3-phosphate dehydrogenase, fructose diphosphatase, pyruvate kinase, triose phosphate isomerase were down regulated implying that the inactivation of pgi gene reduced the carbon flux through glycolytic pathway. Meanwhile, the pentose phosphate pathway was active as a major route for intermediary carbohydrate metabolism instead of glycolysis. The pentose phosphate pathway generates most of the major reducing co-factor NADPH as shown by the increased of NADPH/NADP+ ratio in the mutant when compared with the parent strain. The fermentative enzymes such as acetate kinase and lactate dehydrogenase were down regulated in the mutant. Knockout of pgi gene results in the significant increase in the intracellular concentration of glucose-6-phosphate and decrease in the concentration of oxaloacetate. The slow growth rate of the mutant was assumed to be affected by the accumulation of glucose-6-phosphate and imbalance of NADPH reoxidation.

  12. Diagnosis and epidemiology of red blood cell enzyme disorders

    Directory of Open Access Journals (Sweden)

    Richard Van Wijk

    2013-03-01

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

  13. A tight association in two genetically unlinked dispersal related traits in sympatric and allopatric salt marsh beetle populations.

    Science.gov (United States)

    Van Belleghem, Steven M; Hendrickx, Frederik

    2014-02-01

    Local adaptation likely involves selection on multiple, genetically unlinked traits to increase fitness in divergent habitats. Conversely, recombination is expected to counteract local adaptation under gene flow by breaking down adaptive gene combinations. Western European populations of the salt marsh beetle Pogonus chalceus are characterized by large interpopulation variation at various geographical ranges in two traits related to dispersal ability, i.e. wing size and different allozymes of the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (mtIdh) gene. In this study, we tested whether variation in wing length was as strongly genetically determined in locally adapted populations in a sympatric mosaic compared to allopatric populations, and if variation in mtIDH and wing size was genetically unlinked. We demonstrate that the genetic determination of wing size is very high (h (2) = 0.90) in sympatry and of comparable magnitude as geographically separated populations. Second, we show that, although frequencies of mtIDH allozymes are tightly associated with mean population wing size across Western European populations, the correlation is strongly reduced within some of the populations. These findings demonstrate that the divergence involves at least two traits under independent genetic control and that the genetically distinct ecotypes are retained at geographical distances with ample opportunity for gene flow.

  14. Biochemical characterization of an isoform of GDP-D-mannose-4,6-dehydratase from Mortierella alpina.

    Science.gov (United States)

    Wang, Hongchao; Zhang, Chen; Chen, Haiqin; Yang, Qin; Zhou, Xin; Gu, Zhennan; Zhang, Hao; Chen, Wei; Chen, Yong Q

    2016-10-01

    To clarify the molecular mechanism of GDP-L-fucose biosynthesis in Mortierella alpina. Analysis of the M. alpina genome suggests that there were two isofunctional GDP-D-mannose-4,6-dehydratase genes (GMD1 and GMD2) that have never been found in a microorganism before. GMD2 was expressed heterologously in Escherichia coli and purified to homogeneity. The addition of exogenous NAD(+) or NADP(+) was not essential for GMD2 activity. GMD2 may have considerable importance for GDP-L-fucose biosynthesis under nitrogen starvation. The transcriptional regulation of GMD1 may be more susceptible to GDP and GTP than that of GMD2. Significant changes were observed in the concentration of GDP-L-fucose (30 and 36 % inhibition respectively) and total fatty acids (18 and 12 % inhibition respectively) in M. alpina grown on GMD inhibitors medium, which suggests that GDP-L-fucose is functionally significant in lipid metabolism. This is the first time that an isofunctional GDP-D-mannose-4,6-dehydratase has been characterized in a microorganism.

  15. Metaproteomics analysis of the functional insights into microbial communities of combined hydrogen and methane production by anaerobic fermentation from reed straw.

    Directory of Open Access Journals (Sweden)

    Xuan Jia

    Full Text Available A metaproteomic approach was used to analyse the proteins expressed and provide functional evidence of key metabolic pathways in the combined production of hydrogen and methane by anaerobic fermentation (CHMP-AF for reed straw utilisation. The functions and structures of bacteria and archaea populations show significant succession in the CHMP-AF process. There are many kinds of bacterial functional proteins, mainly belonging to phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, that are involved in carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Ferredoxin-NADP reductase, present in bacteria in genus Azotobacter, is an important enzyme for NADH/NAD+ equilibrium regulation in hydrogen production. The archaeal functional proteins are mainly involved in methane metabolism in energy metabolism, such as acetyl-CoA decarboxylase, and methyl-coenzyme M reductase, and the acetic acid pathway exhibited the highest proportion of the total. The archaea of genus Methanosarcina in phylum Euryarchaeota can produce methane under the effect of multi-functional proteins through acetic acid, CO2 reduction, and methyl nutrient pathways. The study demonstrates metaproteomics as a new way of uncovering community functional and metabolic activity. The combined information was used to identify the metabolic pathways and organisms crucial for lignocellulosic biomass degradation and biogas production. This also regulates the process from its protein levels and improves the efficiency of biogas production using reed straw biomass.

  16. In vitro metabolic engineering for the salvage synthesis of NAD(.).

    Science.gov (United States)

    Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

    2016-05-01

    Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  17. Structure of a NAD kinase from Thermotoga maritima at 2.3 Å resolution

    International Nuclear Information System (INIS)

    Oganesyan, Vaheh; Huang, Candice; Adams, Paul D.; Jancarik, Jaru; Yokota, Hisao A.; Kim, Rosalind; Kim, Sung-Hou

    2005-01-01

    The expression, purification, crystallization, and structure determination of NAD-kinase from T. maritima are reported. Similarity to other NAD-kinases as well as homo-oligomrization state of the enzyme from T. maritima are discussed. NAD kinase is the only known enzyme that catalyzes the formation of NADP, a coenzyme involved in most anabolic reactions and in the antioxidant defense system. Despite its importance, very little is known regarding the mechanism of catalysis and only recently have several NAD kinase structures been deposited in the PDB. Here, an independent investigation of the crystal structure of inorganic polyphosphate/ATP-NAD kinase, PPNK-THEMA, a protein from Thermotoga maritima, is reported at a resolution of 2.3 Å. The crystal structure was solved using single-wavelength anomalous diffraction (SAD) data collected at the Se absorption-peak wavelength in a state in which no cofactors or substrates were bound. It revealed that the 258-amino-acid protein is folded into two distinct domains, similar to recently reported NAD kinases. The N-terminal α/β-domain spans the first 100 amino acids and the last 30 amino acids of the polypeptide and has several topological matches in the PDB, whereas the other domain, which spans the middle 130 residues, adopts a unique β-sandwich architecture and only appreciably matches the recently deposited PDB structures of NAD kinases

  18. Increased Rate of NAD Metabolism Shortens Plant Longevity by Accelerating Developmental Senescence in Arabidopsis.

    Science.gov (United States)

    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.

  19. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    International Nuclear Information System (INIS)

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-01-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating [ 3 H]thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase

  20. Lipid droplets accumulation and other biochemical changes induced in the fungal pathogen Ustilago maydis under nitrogen-starvation.

    Science.gov (United States)

    Aguilar, Lucero Romero; Pardo, Juan Pablo; Lomelí, Mónica Montero; Bocardo, Oscar Ivan Luqueño; Juárez Oropeza, Marco A; Guerra Sánchez, Guadalupe

    2017-10-01

    In many organisms, the growth under nitrogen-deprivation or a poor nitrogen source impacts on the carbon flow distribution and causes accumulation of neutral lipids, which are stored as lipid droplets (LDs). Efforts are in progress to find the mechanism of LDs synthesis and degradation, and new organisms capable of accumulating large amounts of lipids for biotechnological applications. In this context, when Ustilago maydis was cultured in the absence of a nitrogen source, there was a large accumulation of lipid bodies containing mainly triacylglycerols. The most abundant fatty acids in lipid bodies at the stationary phase were palmitic, linoleic, and oleic acids, and they were synthesized de novo by the fatty-acid synthase. In regard to the production of NADPH for the synthesis of fatty acids, the cytosolic NADP + -dependent isocitrate dehydrogenase and the glucose-6-phosphate and 6-phosphogluconate dehydrogenases couple showed the highest specific activities, with a lower activity of the malic enzyme. The ATP-citrate lyase activity was not detected in any of the culture conditions, which points to a different mechanism for the transfer of acetyl-CoA into the cytosol. Protein and RNA contents decreased when U. maydis was grown without a nitrogen source. Due to the significant accumulation of triacylglycerols and the particular composition of fatty acids, U. maydis can be considered an alternative model for biotechnological applications.

  1. Construction and engineering of a thermostable self-sufficient cytochrome P450

    Energy Technology Data Exchange (ETDEWEB)

    Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  2. Mangifera indica L. extract (Vimang) and its main polyphenol mangiferin prevent mitochondrial oxidative stress in atherosclerosis-prone hypercholesterolemic mouse.

    Science.gov (United States)

    Pardo-Andreu, Gilberto L; Paim, Bruno A; Castilho, Roger F; Velho, Jesus A; Delgado, René; Vercesi, Anibal E; Oliveira, Helena C F

    2008-05-01

    Atherosclerosis is linked to a number of oxidative events ranging from low-density lipoprotein (LDL) oxidation to the increased production of intracellular reactive oxygen species (ROS). We have recently demonstrated that liver mitochondria isolated from the atherosclerosis-prone hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice have lower content of NADP(H)-linked substrates than the controls and, as consequence, higher sensitivity to oxidative stress and mitochondrial membrane permeability transition (MPT). In the present work, we show that oral supplementation with the antioxidants Mangifera indica L. extract (Vimang) or its main polyphenol mangiferin shifted the sensitivity of LDLr(-/-) liver mitochondria to MPT to control levels. These in vivo treatments with Vimang and mangiferin also significantly reduced ROS generation by both isolated LDLr(-/-) liver mitochondria and spleen lymphocytes. In addition, these antioxidant treatments prevented mitochondrial NAD(P)H-linked substrates depletion and NADPH spontaneous oxidation. In summary, Vimang and mangiferin spared the endogenous reducing equivalents (NADPH) in LDLr(-/-) mice mitochondria correcting their lower antioxidant capacity and restoring the organelle redox homeostasis. The effective bioavailability of these compounds makes them suitable antioxidants with potential use in atherosclerosis susceptible conditions.

  3. Identification of Multiple Soluble Fe(III Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

    Directory of Open Access Journals (Sweden)

    Subrata Pal

    2014-01-01

    Full Text Available Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III and Cr(VI anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs, thioredoxin reductase (Trx, NADP(H-nitrite reductase (Ntr, and thioredoxin disulfide reductase (Tdr were determined to be responsible for Fe(III reductase activity. Amino acid sequence and three-dimensional (3D structural similarity analyses of the T. indiensis Fe(III reductases were carried out with Cr(VI reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI reduction as well.

  4. Characterization and Evaluation of Acid Rain in East Central Florida from 1978 to 1995 and Evaluation of Some Chromatographic/Spectroscopic Results from Leachate Samples from CELSS

    Science.gov (United States)

    Madsen, Brooks C.

    1996-01-01

    The results of monitoring the chemical composition of rain in east-central Florida have shown that the rain is moderately acid. The measured acidity of rain is less than that observed in other regions of the U. S., however, it does suggest that the level of acidity is substantial. The annual chemical composition of rain at UCF and at KSC has shown moderate variability. Extreme daily and monthly variations are observed, however these variations are not addressed here. The total ionic composition of rain collected at FL99 is greater than that for rain collected at UCF, however this can be accounted for by site proximity to the ocean with the accompanying marine influence. Difference in acidity data collected from the UCF and FL99 sites which are separated by 50 km may be due in part to the differences that have been observed between laboratory and field pH measurements. Trend assessment for precipitation composition requires evaluation of data that covers some minimum time period. In fact, the subdivision of the multi-year UCF record into individual 10 year records as described above can lead to the conclusion that a significant increase, a significant decrease or no trend exists for acidity depending upon the time period chosen for evaluation. Trend evaluation has also been accomplished by linear and nonlinear regression analysis using monthly volume weighted average concentrations and deposition using the UCF data set and some of the Florida NADP data set.

  5. Effect of aluminum on metabolism of organic acids and chemical forms of aluminum in root tips of Eucalyptus camaldulensis Dehnh.

    Science.gov (United States)

    Ikka, Takashi; Ogawa, Tsuyoshi; Li, Donghua; Hiradate, Syuntaro; Morita, Akio

    2013-10-01

    Eucalyptus (Eucalyptus camaldulensis) has relatively high resistance to aluminum (Al) toxicity than the various herbaceous plants and model plant species. To investigate Al-tolerance mechanism, the metabolism of organic acids and the chemical forms of Al in the target site (root tips) in Eucalyptus was investigated. To do this, 2-year old rooted cuttings of E. camaldulensis were cultivated in half-strength Hoagland solution (pH 4.0) containing Al (0, 0.25, 0.5, 1.0, 2.5 and 5.0mM) salts for 5weeks; growth was not affected at concentrations up to 2.5mM even with Al concentration reaching 6000μgg(-1) DW. In roots, the citrate content also increased with increasing Al application. Concurrently, the activities of aconitase and NADP(+)-isocitrate dehydrogenase, which catalyze the decomposition of citrate, decreased. On the other hand, the activity of citrate synthase was not affected at concentrations up to 2.5mM Al. (27)Al-NMR spectroscopic analyses were carried out where it was found that Al-citrate complexes were a major chemical form present in cell sap of root tips. These findings suggested that E. camaldulensis detoxifies Al by forming Al-citrate complexes, and that this is achieved through Al-induced citrate accumulation in root tips via suppression of the citrate decomposition pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Nicotinamide-NAD sequence: redox process and related behavior, behavior and properties of intermediate and final products

    International Nuclear Information System (INIS)

    Elving, P.J.; Schmakel, C.O.; Santhanam, K.S.V.

    1976-01-01

    Illustrations of the application of analytical chemical techniques to the study of chemical phenomena are given. In particular, electrochemical techniques and methodology and, to a lesser extent, spectrophotometry were used to investigate the solution behavior, adsorption, redox processes including coupled chemical reactions, and allied aspects of biologically significant compounds and of their intermediate and final redox products, e.g., the behavior of the free radicals produced by initial one-electron processes. This approach is illustrated by the consideration of the behavior in aqueous and nonaqueous media of a sequence of compounds ranging from nicotinamide (3-carbamoylpyridine) to NAD + and NADP + ; the latter compounds function as coenzymes for the pyridinoproteins which are principal components in the Krebs citric acid cycle and in the electron transport chain in biological redox reactions. The discussion is presented under the following section headings: interpretation of electrochemical behavior; mechanistic patterns; kinetic aspects of charge-transfer and chemical reactions; correlation with theoretically calculated parameters; and, mechanisms of biological oxidation-reduction reactions. The use of pulse radiolysis, chronopotentiometric, and cyclic voltammetric methods in studies on free radical dimerization rates is reviewed in the discussion of the kinetic aspects of charge-transfer and chemical reactions. (188 references)

  7. IDH1/2 mutations target a key hallmark of cancer by deregulating cellular metabolism in glioma.

    Science.gov (United States)

    Zhang, Chunzhi; Moore, Lynette M; Li, Xia; Yung, W K Alfred; Zhang, Wei

    2013-09-01

    Isocitrate dehydrogenase (IDH) enzymes have recently become a focal point for research aimed at understanding the biology of glioma. IDH1 and IDH2 are mutated in 50%-80% of astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas but are seldom mutated in primary glioblastomas. Gliomas with IDH1/2 mutations always harbor other molecular aberrations, such as TP53 mutation or 1p/19q loss. IDH1 and IDH2 mutations may serve as prognostic factors because patients with an IDH-mutated glioma survive significantly longer than those with an IDH-wild-type tumor. However, the molecular pathogenic role of IDH1/2 mutations in the development of gliomas is unclear. The production of 2-hydroxyglutarate and enhanced NADP+ levels in tumor cells with mutant IDH1/2 suggest mechanisms through which these mutations contribute to tumorigenesis. Elucidating the pathogenesis of IDH mutations will improve understanding of the molecular mechanisms of gliomagenesis and may lead to development of a new molecular classification system and novel therapies.

  8. Caloric restriction counteracts age-related changes in the activities of sorbitol metabolizing enzymes from mouse liver

    Science.gov (United States)

    Hagopian, Kevork; Ramsey, Jon J.; Weindruch, Richard

    2009-01-01

    The influence of caloric restriction (CR) on hepatic sorbitol-metabolizing enzyme activities was investigated in young and old mice. Aldose reductase and sorbitol dehydrogenase activities were significantly lower in old CR mice than in old controls. Young CR mice showed decreased aldose reductase activity and a trend towards decreased sorbitol dehydrogenase when compared to controls. Metabolites of the pathway, namely sorbitol, glucose and fructose were decreased by CR in young and old mice. Pyruvate levels were decreased by CR in both young and old mice, while lactate decreased only in old CR. Malate levels increased in old CR but remained unchanged in young CR, when compared with controls. Accordingly, the lactae/pyruvate and malate/pyruvate ratios in young and old CR mice were increased, indicating increased NADH/NAD and NADPH/NADP redox couples, respectively. The results indicate that decreased glucose levels under CR conditions lead to decreased sorbitol pathway enzyme activities and metabolite levels, and could contribute to the beneficial effects of long-term CR through decreased sorbitol levels and NADPH sparing. PMID:18953666

  9. Genetic variability in Oligosarcus paranensis (Teleostei: Characiformes from the São Francisco river, Ivaí river basin – Paraná State, Brazil - doi: 10.4025/actascibiolsci.v35i3.14179

    Directory of Open Access Journals (Sweden)

    Michele Rocha dos Santos

    2013-08-01

    Full Text Available The genetic variability of Oligosarcus paranensis was estimated from a population collected in São Francisco river, Prudentópolis county in Paraná State (Brazil using the electrophoresis in starch gel technique. Eleven enzymatic systems were analyzed: Aspartate aminotransaminase (AAT; E. C. 2.6.1, Alcohol dehydrogenase (ADH; E. C. 1.1.1.1, Esterase (EST; E. C. 3.1.1.1, Glucose-6-phosphate isomerase (GPI; E. C. 5.3.1.9, Glycerol-3-Phosphate dehydrogenase (G3PDH; E. C. 1.1.1, Isocitrate dehydrogenase (IDH; E. C. 1.1.1.42, L-lactate dehydrogenase (LDH; E. C. 1.1.1.27, Malate dehydrogenase (MDH; E. C. 1.1.1.37 , Malate dehydrogenase NADP (ME; E. C. 1.1.1.40, Phosphoglucomutase (PGM; E. C. 5.4.2.2 and Sorbitol dehydrogenase (SORB; E.C. 1.1.1.14. Twenty loci were identified through 15% corn starch gel electrophoresis of which nine (45% were polymorphic. The average expected heterozygosity was estimated as 0.1229 ± 0.1728, and the observed was 0.0586 ± 0.1069, indicating high genetic variability. The average value of FIS = 0.5145 indicates homozygote excess.  

  10. Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

    Energy Technology Data Exchange (ETDEWEB)

    Pérez-Dorado, Inmaculada [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Bortolotti, Ana; Cortez, Néstor [Instituto de Biología Molecular y Celular de Rosario (Universidad Nacional de Rosario y CONICET), Suipacha 531, S2002LRK Rosario (Argentina); Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain)

    2008-05-01

    The flavodoxin NifF from R. capsulatus, a candidate for nitrogenase reduction during nitrogen fixation, has been crystallized using the hanging-drop vapour-diffusion method. Preliminary X-ray data processing at 2.17 Å resolution allowed determination of the crystal system and unit-cell parameters. Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution.

  11. Preliminary study of cell metabolism, by use of NBT test, determination the intensity of lipid peroxidation and antioxidant activity

    Directory of Open Access Journals (Sweden)

    Diana BEI

    2009-05-01

    Full Text Available Otto Warburg, in the early part of the 20th century, originated a hypothesis, that the cause of cancer is primarily a defect in energy metabolism.A decrease in the capacity of mitochondria to reduce NAD(P, together with a decline in the NAD(PH/NAD(P redox couple, uncouples oxidative phosphorylation, lead to depletion of ATP and decrease the cell viability.Nitro-bleu tetrazolium have been used to assay cell proliferation and viability. The method to measure cell proliferation is based on enzymatic cleavage of the tetrazolium salts to a water-soluble formazan dye.Succinate-tetrazolium reductase, is an enzymatic sistem, which belongs to the respiratory chain of the mitochondria and it is active only in viable cells. The reagent diffuses into the cells and it is cleaved to formazan. The absorption change is measured and analysed.Free radicals such as superoxide, can cause a damage in cellular components, but several antioxidants inhibiting the lipid peroxidation and limiting the level of free radicals in cells.In the present study we had in view the proliferation and viability of leukemia cells during antineoplastic treatment along with the alteration of the serum level of malondialdehyde (MDA and ceruloplasmin (CP. With serum level of malondialdehyde we monitored the presence of the lipid peroxidation by the reactive oxygen species, and with the oxidized ceruloplasmin level in blood serum we evidenced the activity of antioxidant system in blood.

  12. Expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni

    International Nuclear Information System (INIS)

    Tourigny, David S.; Elliott, Paul R.; Edgell, Louise J.; Hudson, Gregg M.; Moody, Peter C. E.

    2010-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of C. jejuni glyceraldehyde-3-phosphate dehydrogenase is reported. The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde-3-phosphate dehydrogenase that can utilize either NADP + or NAD + as coenzymes for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of both the wild type and an active-site mutant of the enzyme are presented. Preliminary X-ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4 1 22, with unit-cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme

  13. Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems.

    Science.gov (United States)

    Gurpilhares, Daniela B; Pessoa, Adalberto; Roberto, Inês C

    2015-07-01

    Remaining cells of Candida guilliermondii cultivated in hemicellulose-based fermentation medium were used as intracellular protein source. Recovery of glucose-6-phosphate dehydrogenase (G6PD) was attained in conventional aqueous two-phase systems (ATPS) was compared with integrated process involving mechanical disruption of cells followed by ATPS. Influences of polyethylene glycol molar mass (M PEG) and tie line lengths (TLL) on purification factor (PF), yields in top (Y T ) and bottom (Y B ) phases and partition coefficient (K) were evaluated. First scheme resulted in 65.9 % enzyme yield and PF of 2.16 in salt-enriched phase with clarified homogenate (M PEG 1500 g mol(-1), TLL 40 %); Y B of 75.2 % and PF B of 2.9 with unclarified homogenate (M PEG 1000 g mol(-1), TLL 35 %). The highest PF value of integrated process was 2.26 in bottom phase (M PEG 1500 g mol(-1), TLL 40 %). In order to optimize this response, a quadratic model was predicted for the response PFB for process integration. Maximum response achieved was PFB = 3.3 (M PEG 1500 g mol(-1), TLL 40 %). Enzyme characterization showed G6P Michaelis-Menten constant (K M ) equal 0.07-0.05, NADP(+) K M 0.02-1.98 and optimum temperature 70 °C, before and after recovery. Overall, our data confirmed feasibility of disruption/extraction integration for single-step purification of intracellular proteins from remaining yeast cells.

  14. Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

    Science.gov (United States)

    Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

    2004-10-01

    In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

  15. Characterization of Developmental- and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf (Hibiscus cannabinus L.

    Directory of Open Access Journals (Sweden)

    Ritesh Ghosh

    2014-01-01

    Full Text Available Cinnamoyl-CoA reductase (CCR is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L., which contains a 1,020-bp open reading frame (ORF, encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI of 6.27 (JX524276, HcCCR2. BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P binding domain (VTGAGGFIASWMVKLLLEKGY at N-terminal and probable catalytic domain (NWYCYGK. According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094 and Populus trichocarpa (CAC07424. HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments.

  16. Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    Nan Wang

    2011-01-01

    Full Text Available Alcohol dehydrogenases (ADH are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD or nicotinamide adenine dinucleotide phosphate (NADP, as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+. The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3, and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH.

  17. The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

    DEFF Research Database (Denmark)

    Zaganas, Ioannis; Pajecka, Kamilla; Nielsen, Camilla Wendel

    2013-01-01

    Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two...... human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.......0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced...

  18. Behaviour of glutathione in the cornea epithelium of cattle and rabbits under the influence of infrared radiation

    International Nuclear Information System (INIS)

    Scharsich, G.

    1978-01-01

    After creating a keratitis photoelectrica by means of a mercury/quartz analysis lamp, the concentration of GSH (glutathion, reduced) and GSSG (glutathion, oxidised) in the cornea epithelium of cattle and of rabbits at different times after irradiation was determined. The question of whether ultraviolet radiation considerably influences the GSH/GSSG quotient before the keratitis photoelectrica is fully formed morphologically, could be answered as follows: There is no considerable change in the GSH/GSSG quotient during the first 7-10 hours after the irradiation. Then, when there is, also morphologically, a keratitis photoelectrica, the GSH/GSSG quotient is doubled. The linkage of the GSH/GSSG system and hexosephosphatshunt via the NADP/NADPH system leads us to the conclusion that irradiation does not cause any direct changes in the GSH/GSSG system. Only when the cornea epithelium visably disintegrates an increased reduction of the glutathion occurs, like in several other damaging influences. Thus the found increase of the GSH/GSSG quotient 7-10 hours after ultraviolet irradiation might be unspecific. (orig./AJ) [de

  19. Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Kazumichi Nagasawa

    2013-08-01

    The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control and a low environmental temperature (5°C, cold exposure. Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

  20. Screening of traditional Chinese medicines with therapeutic potential on chronic obstructive pulmonary disease through inhibiting oxidative stress and inflammatory response.

    Science.gov (United States)

    Zhou, Ming-Xing; Wei, Xuan; Li, Ai-Ling; Wang, Ai-Min; Lu, Ling-Zi; Yang, Yue; Ren, Dong-Mei; Wang, Xiao-Ning; Wen, Xue-Sen; Lou, Hong-Xiang; Shen, Tao

    2016-09-13

    Chronic obstructive pulmonary disease (COPD) is a major public health problem and gives arise to severe chronic morbidity and mortality in the world. Inflammatory response and oxidative stress play dominant roles in the pathological mechanism of COPD, and have been regarded to be two important targets for the COPD therapy. Traditional Chinese medicines (TCMs) possess satisfying curative effects on COPD under guidance of the TCM theory in China, and merit in-depth investigations as a resource of lead compounds. One hundred ninety-six of TCMs were collected, and extracted to establish a TCM extract library, and then further evaluated for their potency on inhibitions of oxidative stress and inflammatory response using NADP(H):quinone oxidoreductase (QR) assay and nitric oxide (NO) production assay, respectively. Our investigation observed that 38 of the tested TCM extracts induced QR activity in hepa 1c1c7 murine hepatoma cells, and 55 of them inhibited NO production in RAW 264.7 murine macrophages at the tested concentrations. Noteworthily, 20 of TCM extracts simultaneously inhibited oxidative stress and inflammatory responses. The observed bioactive TCMs, particularly these 20 TCMs with dual inhibitory effects, might be useful for the treatment of COPD. More importantly, the results of the present research afford us an opportunity to discover new lead molecules as COPD therapeutic agents from these active TCMs.

  1. Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

    Science.gov (United States)

    Bomati, Erin K; Noel, Joseph P

    2005-05-01

    We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

  2. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis.

    Science.gov (United States)

    Nair, Ramesh B; Bastress, Kristen L; Ruegger, Max O; Denault, Jeff W; Chapple, Clint

    2004-02-01

    Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP(+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall-esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes.

  3. Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Bayliak, Maria M; Hrynkiv, Olha V; Knyhynytska, Roksolana V; Lushchak, Volodymyr I

    2018-01-01

    Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10 mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10 mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.

  4. Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae.

    Science.gov (United States)

    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.

  5. Construction and engineering of a thermostable self-sufficient cytochrome P450

    International Nuclear Information System (INIS)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-01-01

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  6. Metabolic response of Candida albicans to phenylethyl alcohol under hyphae-inducing conditions.

    Science.gov (United States)

    Han, Ting-Li; Tumanov, Sergey; Cannon, Richard D; Villas-Boas, Silas G

    2013-01-01

    Phenylethyl alcohol was one of the first quorum sensing molecules (QSMs) identified in C. albicans. This extracellular signalling molecule inhibits the hyphal formation of C. albicans at high cell density. Little is known, however, about the underlying mechanisms by which this QSM regulates the morphological switches of C. albicans. Therefore, we have applied metabolomics and isotope labelling experiments to investigate the metabolic changes that occur in C. albicans in response to phenylethyl alcohol under defined hyphae-inducing conditions. Our results showed a global upregulation of central carbon metabolism when hyphal development was suppressed by phenylethyl alcohol. By comparing the metabolic changes in response to phenylethyl alcohol to our previous metabolomic studies, we were able to short-list 7 metabolic pathways from central carbon metabolism that appear to be associated with C. albicans morphogenesis. Furthermore, isotope-labelling data showed that phenylethyl alcohol is indeed taken up and catabolised by yeast cells. Isotope-labelled carbon atoms were found in the majority of amino acids as well as in lactate and glyoxylate. However, isotope-labelled carbon atoms from phenylethyl alcohol accumulated mainly in the pyridine ring of NAD(+)/NADH and NADP(-/)NADPH molecules, showing that these nucleotides were the main products of phenylethyl alcohol catabolism. Interestingly, two metabolic pathways where these nucleotides play an important role, nitrogen metabolism and nicotinate/nicotinamide metabolism, were also short-listed through our previous metabolomics works as metabolic pathways likely to be closely associated with C. albicans morphogenesis.

  7. Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11.

    Directory of Open Access Journals (Sweden)

    Xin Tang

    Full Text Available The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA; however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11 isolated in this laboratory can produce lipid up to 36% (w/w cell dry weight (CDW. We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.

  8. Effects of 20 standard amino acids on the growth, total fatty acids production, and γ-linolenic acid yield in Mucor circinelloides.

    Science.gov (United States)

    Tang, Xin; Zhang, Huaiyuan; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2014-12-01

    Twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of γ-linolenic acid (GLA) in Mucor circinelloides. Of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high GLA yield with respective values of 17.8 g/L, 23 % (w/w, dry cell weight, DCW), and 0.81 g/L, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. To find out the potential mechanism underlying the increased lipid accumulation of M. circinelloides when grown on tyrosine, the activity of lipogenic enzymes of the fungus during lipid accumulation phase was measured. The enzyme activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase were up-regulated, while NADP-isocitrate dehydrogenase was down-regulated by tyrosine during the lipid accumulation phase of the fungus which suggested that these enzymes may be involved in the increased lipid biosynthesis by tyrosine in this fungus.

  9. Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11.

    Science.gov (United States)

    Tang, Xin; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Garre, Victoriano; Song, Yuanda; Ratledge, Colin

    2015-01-01

    The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.

  10. A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

    Science.gov (United States)

    Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

    2012-03-01

    A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

  11. Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification▿

    Science.gov (United States)

    Bowman, Michael J.; Jordan, Douglas B.; Vermillion, Karl E.; Braker, Jay D.; Moon, Jaewoong; Liu, Z. Lewis

    2010-01-01

    Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-2H]NADPD and (4S)-[4-2H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP+. Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-2H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters kcatNADPH and kcat/KmNADPH, respectively, indicating that hydride transfer is partially rate limiting to catalysis. PMID:20525870

  12. The Use of Maleic Hydrazide for Effective Hybridization of Setaria viridis.

    Directory of Open Access Journals (Sweden)

    Govinda Rizal

    Full Text Available An efficient method for crossing green foxtail (Setaria viridis is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH, and kinetin. We found that 500 μM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria.

  13. The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae.

    Science.gov (United States)

    Sacomboio, Euclides Nenga Manuel; Kim, Edson Yu Sin; Correa, Henrique Leonardo Ruchaud; Bonato, Paloma; Pedrosa, Fabio de Oliveira; de Souza, Emanuel Maltempi; Chubatsu, Leda Satie; Müller-Santos, Marcelo

    2017-10-19

    The NTR system is the major regulator of nitrogen metabolism in Bacteria. Despite its broad and well-known role in the assimilation, biosynthesis and recycling of nitrogenous molecules, little is known about its role in carbon metabolism. In this work, we present a new facet of the NTR system in the control of NADPH concentration and the biosynthesis of molecules dependent on reduced coenzyme in Herbaspirillum seropedicae SmR1. We demonstrated that a ntrC mutant strain accumulated high levels of polyhydroxybutyrate (PHB), reaching levels up to 2-fold higher than the parental strain. In the absence of NtrC, the activity of glucose-6-phosphate dehydrogenase (encoded by zwf) increased by 2.8-fold, consequently leading to a 2.1-fold increase in the NADPH/NADP + ratio. A GFP fusion showed that expression of zwf is likewise controlled by NtrC. The increase in NADPH availability stimulated the production of polyhydroxybutyrate regardless the C/N ratio in the medium. The mutant ntrC was more resistant to H 2 O 2 exposure and controlled the propagation of ROS when facing the oxidative condition, a phenotype associated with the increase in PHB content.

  14. Short-chain dehydrogenase/reductase catalyzing the final step of noscapine biosynthesis is localized to laticifers in opium poppy.

    Science.gov (United States)

    Chen, Xue; Facchini, Peter J

    2014-01-01

    The final step in the biosynthesis of the phthalideisoquinoline alkaloid noscapine involves a purported dehydrogenation of the narcotinehemiacetal keto moiety. A short-chain dehydrogenase/reductase (SDR), designated noscapine synthase (NOS), that catalyzes dehydrogenation of narcotinehemiacetal to noscapine was identified in opium poppy and functionally characterized. The NOS gene was isolated using an integrated transcript and metabolite profiling strategy and subsequently expressed in Escherichia coli. Noscapine synthase is highly divergent from other characterized members of the NADPH-dependent SDR superfamily involved in benzylisoquinoline alkaloid metabolism, and it exhibits exclusive substrate specificity for narcotinehemiacetal. Kinetic analyses showed that NOS exhibits higher catalytic efficiency with NAD+ as the cofactor compared with NADP+. Suppression of NOS transcript levels in opium poppy plants subjected to virus-induced gene silencing resulted in a corresponding reduction in the accumulation of noscapine and an increase in narcotinehemiacetal levels in the latex. Noscapine and NOS transcripts were detected in all opium poppy organs, but both were most abundant in stems. Unlike other putative biosynthetic genes clustered in the opium poppy genome, and their corresponding proteins, NOS transcripts and the cognate enzyme were abundant in latex, indicating that noscapine metabolism is completed in a distinct cell type compared with the rest of the pathway.

  15. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

    Science.gov (United States)

    Villalobo, A; Lehninger, A L

    1980-03-25

    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

  16. 1987 wet deposition temporal and spatial patterns in North America

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, J.C.; Olsen, A.R.

    1990-03-01

    The focus of this report is on North American wet deposition temporal patterns from 1979 to 1987 and spatial patterns for 1987. The report investigates the patterns of annual precipitation-weighted average concentration and annual deposition for nine ion species: hydrogen, sulfate, nitrate, ammonium, calcium, chloride, sodium, potassium, and magnesium. Data are from the Acid Deposition System (ADS) for the statistical reporting of North American deposition data which includes the National Atmospheric Deposition Program/National Trends Network (NADP/NTN), the MAP3S precipitation chemistry network, the Utility Acid Precipitation Study Program (UAPSP), the Canadian Precipitation Monitoring Network (CAPMoN), and the daily and 4-weekly Acidic Precipitation in Ontario Study (APIOS-D and APIOS-C). Mosaic maps, based on surface estimation using kriging, display concentration and deposition spatial patterns of pH, hydrogen, sulfate, nitrate, ammonium, and calcium ion species for 1987 annual, winter, and summer periods. The temporal pattern analyses use a subset of 39 sites over a 9-year (1979--1987) period and an expanded subset of 140 sites with greater spatial coverage over a 6-year (1982--1987) period. 68 refs., 15 figs., 15 tabs.

  17. S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site.

    Science.gov (United States)

    Kanda, Hironori; Toyama, Takashi; Shinohara-Kanda, Azusa; Iwamatsu, Akihiro; Shinkai, Yasuhiro; Kaji, Toshiyuki; Kikushima, Makoto; Kumagai, Yoshito

    2012-11-01

    We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

  18. Reducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: a hypoxia stress?

    Science.gov (United States)

    Saher, Shady; Fernández-García, Nieves; Piqueras, Abel; Hellín, Eladio; Olmos, Enrique

    2005-06-01

    Hyperhydricity is considered as a physiological disorder that can be induced by different stressing conditions. In the present work we have studied the metabolic and energetic states of hyperhydric carnation shoots. We have evaluated the hypothesis that hypoxia stress is the main factor affecting the metabolism of hyperhydric leaves. Our results indicate a low level of ATP in hyperhydric tissues, but only slight modifications in pyridine nucleotide contents. Concurrently, the glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) activity in hyperhydric leaves was increased but glucokinase (GK; EC 2.7.1.2) activity was unchanged. We have observed that the metabolism of pyruvate was altered in hyperhydric tissues by the induction of pyruvate synthesis via NADP-dependent malic enzyme (EC 1.1.1.40). The enzymes of the fermentative metabolism pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were highly increased in hyperhydric leaves. Sucrose metabolism was modified in hyperhydric leaves with a high increase in the activity of both synthesis and catabolic enzymes. The analysis of the sucrose, glucose and fructose contents indicated that all of these sugars were accumulated in hyperhydric leaves. However, the pinitol content was drastically decreased in hyperhydric leaves. We consider that these results suggest that hyperhydric leaves of carnation have adapted to hypoxia stress conditions by the induction of the oxidative pentose phosphate and fermentative pathways.

  19. Formation of Lignans(-)-Secoisolariciresinol and (-)-Matairesinol with Forsythia intermedia Cell-Free Extracts

    Science.gov (United States)

    Umezawa, Toshiaki; Davin, Laurence B.; Lewis, Norman G.

    1991-01-01

    In vivo labeling experiments of Forsythia intermedia plant tissue with [8-(C-14)]- and [9,9-(2)H2,OC(2)H3]coniferyl alcohols revealed that the lignans, (-)-secoisolariciresinol and (-)-matairesinol, were derived from two coniferyl alcohol molecules; no evidence for the formation of the corresponding (+)-enantiomers was found. Administration of (+/-)-[Ar-(H-3)] secoisolariciresinols to excised shoots of F.intermedia resulted in a significant conversion into (-)-matairesinol; again, the (+)-antipode was not detected. Experiments using cell-free extracts of F.intermedia confirmed and extended these findings. In the presence of NAD(P)H and H2O2, the cell-free extracts catalyzed the formation of (-)- secoisolariciresinol, with either [8-(C-14)]- or [9,9-(2)H2,OC(2)H3]coniferyl alcohols as substrates. The (+)- enantiomer was not formed. Finally, when either (-)-[Ar-(H-3)] or (+/-)-[Ar-(H-2)]secoisolariciresinols were used as substrates, in the presence of NAD(P), only (-)- and not (+)-matairesinol formation occurred. The other antipode, (+)-secoisolariciresinol, did not serve as a substrate for the formation of either (+)- or (-)-matairesinol. Thus, in F.intermedia, the formation of the lignan, (-)-secoisolariciresinol, occurs under strict stereochemical control, in a reaction or reactions requiring NAD(P)H and H2O2 as cofactors. This stereoselectivity is retained in the subsequent conversion into (-)-matairesinol, since (+)-secoisolariciresinol is not a substrate. These are the first two enzymes to be discovered in lignan formation.

  20. Antioxidant Systems from Pepper (Capsicum annuum L.): Involvement in the Response to Temperature Changes in Ripe Fruits

    Science.gov (United States)

    Mateos, Rosa M.; Jiménez, Ana; Román, Paloma; Romojaro, Félix; Bacarizo, Sierra; Leterrier, Marina; Gómez, Manuel; Sevilla, Francisca; del Río, Luis A.; Corpas, Francisco J.; Palma, José M.

    2013-01-01

    Sweet pepper is susceptible to changes in the environmental conditions, especially temperatures below 15 °C. In this work, two sets of pepper fruits (Capsicum annuum L.) which underwent distinct temperature profiles in planta were investigated. Accordingly, two harvesting times corresponding to each set were established: Harvest 1, whose fruits developed and ripened at 14.9 °C as average temperature; and Harvest 2, with average temperature of 12.4 °C. The oxidative metabolism was analyzed in all fruits. Although total ascorbate content did not vary between Harvests, a shift from the reduced to the oxidized form (dehydroascorbate), accompanied by a higher ascorbate peroxidase activity, was observed in Harvest 2 with respect to Harvest 1. Moreover, a decrease of the ascorbate-generating enzymatic system, the γ-galactono-lactone dehydrogenase, was found at Harvest 2. The activity values of the NADP-dependent dehydrogenases analyzed seem to indicate that a lower NADPH synthesis may occur in fruits which underwent lower temperature conditions. In spite of the important changes observed in the oxidative metabolism in fruits subjected to lower temperature, no oxidative stress appears to occur, as indicated by the lipid peroxidation and protein oxidation profiles. Thus, the antioxidative systems of pepper fruits seem to be involved in the response against temperature changes. PMID:23644886

  1. Dissecting protein loops with a statistical scalpel suggests a functional implication of some structural motifs.

    Science.gov (United States)

    Regad, Leslie; Martin, Juliette; Camproux, Anne-Claude

    2011-06-20

    One of the strategies for protein function annotation is to search particular structural motifs that are known to be shared by proteins with a given function. Here, we present a systematic extraction of structural motifs of seven residues from protein loops and we explore their correspondence with functional sites. Our approach is based on the structural alphabet HMM-SA (Hidden Markov Model - Structural Alphabet), which allows simplification of protein structures into uni-dimensional sequences, and advanced pattern statistics adapted to short sequences. Structural motifs of interest are selected by looking for structural motifs significantly over-represented in SCOP superfamilies in protein loops. We discovered two types of structural motifs significantly over-represented in SCOP superfamilies: (i) ubiquitous motifs, shared by several superfamilies and (ii) superfamily-specific motifs, over-represented in few superfamilies. A comparison of ubiquitous words with known small structural motifs shows that they contain well-described motifs as turn, niche or nest motifs. A comparison between superfamily-specific motifs and biological annotations of Swiss-Prot reveals that some of them actually correspond to functional sites involved in the binding sites of small ligands, such as ATP/GTP, NAD(P) and SAH/SAM. Our findings show that statistical over-representation in SCOP superfamilies is linked to functional features. The detection of over-represented motifs within structures simplified by HMM-SA is therefore a promising approach for prediction of functional sites and annotation of uncharacterized proteins.

  2. Photosynthetic pathways and the geographical distribution of grasses in South West Africa/Namibia

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, R P [Department of Agriculture and Fisheries, Pretoria (South Africa). Botanical Research Institute; Vogel, J C; Fuls, A [Council for Scientific and Industrial Research, Pretoria (South Africa). National Physical Research Lab.

    1980-07-01

    Analysis of floristic lists for South West Africa/Namibia shows that, throughout the territory, more than 95% of the grass species occurring in any given area display the C/sub 4/ photosynthetic pathway. Exceptions are areas in the north-east and southwest where between 5% and 18% of the grass species are of the C/sub 3/ type. The south-western district of Luderitz falls within the winter rainfall area and it is only here that temperate C/sub 3/ genera are found. The C/sub 3/ species in the north-east belong to tropical groups. Most of the South West African C/sub 3/ grasses grow in specialized habitats and are either hydrophytes or sciophytes. Subdivision of the C/sub 4/ grasses into the three subtypes of the C/sub 4/ pathway reveals distinctive distributional trends. Malate formers or NADP-me species clearly become more abundant with increasing rainfall, whereas the aspartate formers show the opposite tendency. However, within the aspartate forming group the results show that it is specifically the NAD-me type of species which dominate in areas of very low precipitation, notably in the Namib and pre-Namib areas where rainfall is less than 200 mm/yr. The PEP-ck species form a group intermediate between the malate formers and the NAD-me grasses, especially as far as their water requirements are concerned.

  3. A 50-year record of NOx and SO{sub 2} sources in precipitation in the Northern Rocky Mountains, USA - article no. 4

    Energy Technology Data Exchange (ETDEWEB)

    Naftz, D.L.; Schuster, P.F.; Johnson, C.A. [US Geological Survey, Salt Lake City, UT (United States)

    2011-03-07

    Ice-core samples from Upper Fremont Glacier (UFG), Wyoming, were used as proxy records for the chemical composition of atmospheric deposition. Results of analysis of the ice-core samples for stable isotopes of nitrogen ({delta}{sup 15}, NO{sub 3}{sup -}) and sulfur ({delta}{sup 34}S, SO{sub 4}{sup 2}), as well as NO{sub 3}{sup -} and SO{sub 4}{sup 2} deposition rates from the late-1940s through the early-1990s, were used to enhance and extend existing National Atmospheric Deposition Program/National Trends Network (NADP/NTN) data in western Wyoming. The most enriched {delta}{sup 34}S value in the UFG ice-core samples coincided with snow deposited during the 1980 eruption of Mt. St. Helens, Washington. The remaining {delta}{sup 34}S values were similar to the isotopic composition of coal from southern Wyoming. The {delta}{sup 15}N values in ice-core samples representing a similar period of snow deposition were negative, ranging from -5.9 to -3.2 parts per thousand and all fall within the {delta}{sup 15}N values expected from vehicle emissions. Ice-core nitrate and sulfate deposition data reflect the sharply increasing U.S. emissions data from 1950 to the mid-1970s.

  4. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    Science.gov (United States)

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  5. Effect of agronomical practices on carpology, fruit and oil composition, and oil sensory properties, in olive (Olea europaea L.).

    Science.gov (United States)

    Rosati, Adolfo; Cafiero, Caterina; Paoletti, Andrea; Alfei, Barbara; Caporali, Silvia; Casciani, Lorena; Valentini, Massimiliano

    2014-09-15

    We examined whether some agronomical practices (i.e. organic vs. conventional) affect olive fruit and oil composition, and oil sensory properties. Fruit characteristics (i.e. fresh and dry weight of pulp and pit, oil content on a fresh and dry weight basis) did not differ. Oil chemical traits did not differ except for increased content of polyphenols in the organic treatments, and some changes in the acidic composition. Sensory analysis revealed increased bitterness (both cultivars) and pungency (Frantoio) and decreased sweetness (Frantoio) in the organic treatment. Fruit metabolomic analysis with HRMAS-NMR indicated significant changes in some compounds including glycocholate, fatty acids, NADPH, NADP+, some amino acids, thymidine, trigonelline, nicotinic acid, 5,6-dihydrouracil, hesanal, cis-olefin, β-D-glucose, propanal and some unassigned species. The results suggest that agronomical practices may have effects on fruit composition that may be difficult to detect unless a broad-spectrum analysis is used. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

    Science.gov (United States)

    Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

    2011-07-01

    Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

  7. Differential Contribution of Malic Enzymes during Soybean and Castor Seeds Maturation.

    Directory of Open Access Journals (Sweden)

    Mariel Claudia Gerrard Wheeler

    Full Text Available Malic enzymes (ME catalyze the decarboxylation of malate generating pyruvate, CO2 and NADH or NADPH. In some organisms it has been established that ME is involved in lipids biosynthesis supplying carbon skeletons and reducing power. In this work we studied the MEs of soybean and castor, metabolically different oilseeds. The comparison of enzymatic activities, transcript profiles and organic acid contents suggest different metabolic strategies operating in soybean embryo and castor endosperm in order to generate precursors for lipid biosynthesis. In castor, the malate accumulation pattern agrees with a central role of this metabolite in the provision of carbon to plastids, where the biosynthesis of fatty acids occurs. In this regard, the genome of castor possesses a single gene encoding a putative plastidic NADP-ME, whose expression level is high when lipid deposition is active. On the other hand, NAD-ME showed an important contribution to the maturation of soybean embryos, perhaps driving the carbon relocation from mitochondria to plastids to support the fatty acids synthesis in the last stages of seed filling. These findings provide new insights into intermediary metabolism in oilseeds and provide new biotechnological targets to improve oil yields.

  8. Pre-steady-state kinetic analysis of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Mycobacterium tuberculosis reveals partially rate-limiting product release by parallel pathways.

    Science.gov (United States)

    Liu, Juan; Murkin, Andrew S

    2012-07-03

    As part of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the conversion of DXP into 2-C-methyl-D-erythritol 4-phosphate (MEP) by consecutive isomerization and NADPH-dependent reduction reactions. Because this pathway is essential to many infectious organisms but is absent in humans, DXR is a target for drug discovery. In an attempt to characterize its kinetic mechanism and identify rate-limiting steps, we present the first complete transient kinetic investigation of DXR. Stopped-flow fluorescence measurements with Mycobacterium tuberculosis DXR (MtDXR) revealed that NADPH and MEP bind to the free enzyme and that the two bind together to generate a nonproductive ternary complex. Unlike the Escherichia coli orthologue, MtDXR exhibited a burst in the oxidation of NADPH during pre-steady-state reactions, indicating a partially rate-limiting step follows chemistry. By monitoring NADPH fluorescence during these experiments, the transient generation of MtDXR·NADPH·MEP was observed. Global kinetic analysis supports a model involving random substrate binding and ordered release of NADP(+) followed by MEP. The partially rate-limiting release of MEP occurs via two pathways--directly from the binary complex and indirectly via the MtDXR·NADPH·MEP complex--the partitioning being dependent on NADPH concentration. Previous mechanistic studies, including kinetic isotope effects and product inhibition, are discussed in light of this kinetic mechanism.

  9. The radiation inactivation of glutamate and isocitrate dehydrogenases

    International Nuclear Information System (INIS)

    El Failat, R.R.A.

    1980-12-01

    The reaction of free radicals produced by ionizing radiation with the enzymes glutamate dehydrogenase (GDH) and NADP + -specific isocitrate dehydrogenase (ICDH) have been studied by steady-state and pulse radiolysis techniques. In de-aerated GDH solutions, hydroxyl radicals have been found to be the most efficient of the primary radicals generated from water in causing inactivation. The effect of reaction with the enzyme of selective free radicals (SCN) 2 - , (Br) 2 - and (I) 2 - on its activity has also been studied. In neutral solutions, the order of inactivating effectiveness is (I) 2 - > (Br) 2 - > (SCN) 2 - . In the case of the thiocyanate radical anion (SCN) 2 - , the inactivation efficiency is found to depend on KSCN concentration. The radiation inactivation of GDH at both neutral and alkaline pH is accompanied by the loss of sulphydryl groups. Pulse radiolysis was also used to determine the rate constants and the transient absorption spectra following the reaction of the free radicals with GDH. 60 Co-γ-radiolysis and pulse radiolysis were also used to study the effect of ionizing radiation on the activity of ICDH. The results obtained were similar to those of GDH. (author)

  10. The Redox Code.

    Science.gov (United States)

    Jones, Dean P; Sies, Helmut

    2015-09-20

    The redox code is a set of principles that defines the positioning of the nicotinamide adenine dinucleotide (NAD, NADP) and thiol/disulfide and other redox systems as well as the thiol redox proteome in space and time in biological systems. The code is richly elaborated in an oxygen-dependent life, where activation/deactivation cycles involving O₂ and H₂O₂ contribute to spatiotemporal organization for differentiation, development, and adaptation to the environment. Disruption of this organizational structure during oxidative stress represents a fundamental mechanism in system failure and disease. Methodology in assessing components of the redox code under physiological conditions has progressed, permitting insight into spatiotemporal organization and allowing for identification of redox partners in redox proteomics and redox metabolomics. Complexity of redox networks and redox regulation is being revealed step by step, yet much still needs to be learned. Detailed knowledge of the molecular patterns generated from the principles of the redox code under defined physiological or pathological conditions in cells and organs will contribute to understanding the redox component in health and disease. Ultimately, there will be a scientific basis to a modern redox medicine.

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

    Science.gov (United States)

    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.

  12. Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism.

    Science.gov (United States)

    Rustin, P; Queiroz-Claret, C

    1985-06-01

    Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C3-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO2 fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP(+)-or a mitochondrial NAD(+)-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C3-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD(+)-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.

  13. cDNA-AFLP analysis of gene expression differences between the flower bud and sprout-shoot apical meristem of Angelica sinensis (Oliv. Diels

    Directory of Open Access Journals (Sweden)

    Yu Guang

    2011-01-01

    Full Text Available Angelica sinensis (Oliv. Diels (Umbelliferae is a well-known medicinal plant mainly distributed in Gansu Province of China. Its local and global demand is significant because of its food and medicinal applications. However, the early bolting rate of Angelica sinensis (Oliv. Diels reaches 20%-60%, which seriously affects its food and medicinal qualities. Thus, differences in gene expression between the flower bud and sprout-shoot apical meristem underwent analysis, by means of cDNA-amplified restriction fragment length polymorphism, to better understand the flowering mechanism. 64 primer sets, each of which amplified to 60 transcript-derived fragments (TDFs, were used. Among these TDFs, 26 were expressed specifically in the flower bud. After cloning and sequencing, 32 distinct sequences were obtained from these 26 TDFs, and 25 were found with homologous sequences in databases. Confirmation of differential expression of 13 sequences was obtained by semi-quantitative RT-PCR, their showing higher expression levels in flower buds. These homologous sequences encode transposable elements, pentatricopeptide repeat-containing proteins, DNA-binding transcription factors, zinc finger (B-box type family proteins, NADP-dependent sorbitol 6-phosphate dehydrogenase (S6PDH, amongst others.

  14. Artificial photosynthesis for production of hydrogen peroxide and its fuel cells.

    Science.gov (United States)

    Fukuzumi, Shunichi

    2016-05-01

    The reducing power released from photosystem I (PSI) via ferredoxin enables the reduction of NADP(+) to NADPH, which is essential in the Calvin-Benson cycle to make sugars in photosynthesis. Alternatively, PSI can reduce O2 to produce hydrogen peroxide as a fuel. This article describes the artificial version of the photocatalytic production of hydrogen peroxide from water and O2 using solar energy. Hydrogen peroxide is used as a fuel in hydrogen peroxide fuel cells to make electricity. The combination of the photocatalytic H2O2 production from water and O2 using solar energy with one-compartment H2O2 fuel cells provides on-site production and usage of H2O2 as a more useful and promising solar fuel than hydrogen. This article is part of a Special Issue entitled Biodesign for Bioenergetics--The design and engineering of electronc transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Metaproteomics analysis of the functional insights into microbial communities of combined hydrogen and methane production by anaerobic fermentation from reed straw

    Science.gov (United States)

    Yang, Yang; Wang, Yong

    2017-01-01

    A metaproteomic approach was used to analyse the proteins expressed and provide functional evidence of key metabolic pathways in the combined production of hydrogen and methane by anaerobic fermentation (CHMP-AF) for reed straw utilisation. The functions and structures of bacteria and archaea populations show significant succession in the CHMP-AF process. There are many kinds of bacterial functional proteins, mainly belonging to phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, that are involved in carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Ferredoxin-NADP reductase, present in bacteria in genus Azotobacter, is an important enzyme for NADH/NAD+ equilibrium regulation in hydrogen production. The archaeal functional proteins are mainly involved in methane metabolism in energy metabolism, such as acetyl-CoA decarboxylase, and methyl-coenzyme M reductase, and the acetic acid pathway exhibited the highest proportion of the total. The archaea of genus Methanosarcina in phylum Euryarchaeota can produce methane under the effect of multi-functional proteins through acetic acid, CO2 reduction, and methyl nutrient pathways. The study demonstrates metaproteomics as a new way of uncovering community functional and metabolic activity. The combined information was used to identify the metabolic pathways and organisms crucial for lignocellulosic biomass degradation and biogas production. This also regulates the process from its protein levels and improves the efficiency of biogas production using reed straw biomass. PMID:28817657

  16. Idh2 deficiency accelerates renal dysfunction in aged mice.

    Science.gov (United States)

    Lee, Su Jeong; Cha, Hanvit; Lee, Seoyoon; Kim, Hyunjin; Ku, Hyeong Jun; Kim, Sung Hwan; Park, Jung Hyun; Lee, Jin Hyup; Park, Kwon Moo; Park, Jeen-Woo

    2017-11-04

    The free radical or oxidative stress theory of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species (ROS) that are produced as by-products of normal metabolic processes in mitochondria. The oxidative stress may arise as a result of either increased ROS production or decreased ability to detoxify ROS. The availability of the mitochondrial NADPH pool is critical for the maintenance of the mitochondrial antioxidant system. The major enzyme responsible for generating mitochondrial NADPH is mitochondrial NADP + -dependent isocitrate dehydrogenase (IDH2). Depletion of IDH2 in mice (idh2 -/- ) shortens life span and accelerates the degeneration of multiple age-sensitive traits, such as hair grayness, skin pathology, and eye pathology. Among the various internal organs tested in this study, IDH2 depletion-induced acceleration of senescence was uniquely observed in the kidney. Renal function and structure were greatly deteriorated in 24-month-old idh2 -/- mice compared with wild-type. In addition, disruption of redox status, which promotes oxidative damage and apoptosis, was more pronounced in idh2 -/- mice. These data support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice, and thus support the oxidative stress theory of aging. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Urban wet deposition nitrate: a comparison to non-urban deposition

    International Nuclear Information System (INIS)

    Schultz, J.A.M.

    1994-01-01

    The concentration of nitrate in both wet and dry deposition has both increased historically and currently, and recently parallels emissions in NO x . Since NO x is produced in amounts comparable to SO 2 , it is an important contributor to acid deposition, and is produced in higher amounts in urban areas due to concentrated sources. Prior to to this study, national acid deposition monitoring networks in the United States have been and remain established in non-urban areas. This research study consisted of a comparison of precipitation sampling and analysis of wet deposition nitrate and pH for each deposition event in each of two urban sites over a 15 mo period. Also, a comparison of urban data and data generated at a nearby non-urban NADP site was made by examination of both monthly and seasonal data. This research suggests that national monitoring programs should consider inclusion of urban and non-urban monitoring sites in order to achieve a more representative regional assessment. 24 refs., 2 figs., 2 tabs

  18. Crystallization of a flavodoxin involved in nitrogen fixation in Rhodobacter capsulatus

    International Nuclear Information System (INIS)

    Pérez-Dorado, Inmaculada; Bortolotti, Ana; Cortez, Néstor; Hermoso, Juan A.

    2008-01-01

    The flavodoxin NifF from R. capsulatus, a candidate for nitrogenase reduction during nitrogen fixation, has been crystallized using the hanging-drop vapour-diffusion method. Preliminary X-ray data processing at 2.17 Å resolution allowed determination of the crystal system and unit-cell parameters. Flavodoxins are small electron-transfer proteins that contain one molecule of noncovalently bound flavin mononucleotide (FMN). The flavodoxin NifF from the photosynthetic bacterium Rhodobacter capsulatus is reduced by one electron from ferredoxin/flavodoxin:NADP(H) reductase and was postulated to be an electron donor to nitrogenase in vivo. NifF was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of PEG 3350 and PEG 400 at pH 5.5 and belong to the tetragonal space group P4 1 2 1 2, with unit-cell parameters a = b = 66.49, c = 121.32 Å. X-ray data sets have been collected to 2.17 Å resolution

  19. Glycogen Metabolic Genes Are Involved in Trehalose-6-Phosphate Synthase-Mediated Regulation of Pathogenicity by the Rice Blast Fungus Magnaporthe oryzae

    Science.gov (United States)

    Wilson, Richard A.; Wang, Zheng-Yi; Kershaw, Michael J.; Talbot, Nicholas J.

    2013-01-01

    The filamentous fungus Magnaporthe oryzae is the causal agent of rice blast disease. Here we show that glycogen metabolic genes play an important role in plant infection by M. oryzae. Targeted deletion of AGL1 and GPH1, which encode amyloglucosidase and glycogen phosphorylase, respectively, prevented mobilisation of glycogen stores during appressorium development and caused a significant reduction in the ability of M. oryzae to cause rice blast disease. By contrast, targeted mutation of GSN1, which encodes glycogen synthase, significantly reduced the synthesis of intracellular glycogen, but had no effect on fungal pathogenicity. We found that loss of AGL1 and GPH1 led to a reduction in expression of TPS1 and TPS3, which encode components of the trehalose-6-phosphate synthase complex, that acts as a genetic switch in M. oryzae. Tps1 responds to glucose-6-phosphate levels and the balance of NADP/NADPH to regulate virulence-associated gene expression, in association with Nmr transcriptional inhibitors. We show that deletion of the NMR3 transcriptional inhibitor gene partially restores virulence to a Δagl1Δgph1 mutant, suggesting that glycogen metabolic genes are necessary for operation of the NADPH-dependent genetic switch in M. oryzae. PMID:24098112

  20. [Morphological changes in the thyroid gland of rats during various phases of the estral cycle].

    Science.gov (United States)

    Pliner, L I; Ledovskaia, S M

    1975-08-01

    The functional state of the thyroid gland and the concentration of thyroid hormones in the peripheral blood were studied in 20 mature female albino rats during their estral cycle. Evaluation of the thyroid functional state was made according to data of histological, morphological (the diameter of folliculi, the height of the thyroid epithelium) and histochemical analysis (determination of NAD and NADP-dehydrogenase, succinatedehydrogenase, lactate dehydrogenase, peroxydase, acid and alkaline phosphatase) as well as biochemical determination of iodine bound with protein (IBP) in the blood plasma and investigation of the ratio of the parameters in question under conditions of the sex cycle. The cyclic changes of the morphological state of the thyroid gland attended by the phases of the estral cycle were revealed. The activation of the organ was observed in proestrus and estrus which was evidenced by high levels of activity of the enzymes under study, high concentration of IBP in the blood and increased height of thyreocytes. A decreased function of the thyroid parenchyma was observed at the period of metaestrus-diestrus.

  1. Light-regulation of enzyme activity in anacystis nidulans (Richt.).

    Science.gov (United States)

    Duggan, J X; Anderson, L E

    1975-01-01

    The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

  2. High salt diet induces metabolic alterations in multiple biological processes of Dahl salt-sensitive rats.

    Science.gov (United States)

    Wang, Yanjun; Liu, Xiangyang; Zhang, Chen; Wang, Zhengjun

    2018-06-01

    High salt induced renal disease is a condition resulting from the interactions of genetic and dietary factors causing multiple complications. To understand the metabolic alterations associated with renal disease, we comprehensively analyzed the metabonomic changes induced by high salt intake in Dahl salt-sensitive (SS) rats using GC-MS technology and biochemical analyses. Physiological features, serum chemistry, and histopathological data were obtained as complementary information. Our results showed that high salt (HS) intake for 16 weeks caused significant metabolic alterations in both the renal medulla and cortex involving a variety pathways involved in the metabolism of organic acids, amino acids, fatty acids, and purines. In addition, HS enhanced glycolysis (hexokinase, phosphofructokinase and pyruvate kinase) and amino acid metabolism and suppressed the TCA (citrate synthase and aconitase) cycle. Finally, HS intake caused up-regulation of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase), the ratio of NADPH/NADP + , NADPH oxidase activity and ROS production, suggesting that increased oxidative stress was associated with an altered PPP pathway. The metabolic pathways identified may serve as potential targets for the treatment of renal damage. Our findings provide comprehensive biochemical details about the metabolic responses to a high salt diet, which may contribute to the understanding of renal disease and salt-induced hypertension in SS rats. Copyright © 2018. Published by Elsevier Inc.

  3. Correlation between Mitochondrial Reactive Oxygen and Severity of Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Gabriel G. Dorighello

    2016-01-01

    Full Text Available Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.

  4. Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration

    Directory of Open Access Journals (Sweden)

    Sung Hwan Kim

    2016-12-01

    Full Text Available Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF, secreted by the retinal pigment epithelium (RPE, in pathological angiogenesis and the development of choroidal neovascularization (CNV in age-related macular degeneration (AMD. RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH, which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS. Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

  5. Various cellular stress components change as the rat ages: An insight into the putative overall age-related cellular stress network.

    Science.gov (United States)

    Cueno, Marni E; Imai, Kenichi

    2018-02-01

    Cellular stress is mainly comprised of oxidative, nitrosative, and endoplasmic reticulum stresses and has long been correlated to the ageing process. Surprisingly, the age-related difference among the various components in each independent stress pathway and the possible significance of these components in relation to the overall cellular stress network remain to be clearly elucidated. In this study, we obtained blood from ageing rats upon reaching 20-, 40-, and 72-wk.-old. Subsequently, we measured representative cellular stress-linked biomolecules (H 2 O 2 , glutathione reductase, heme, NADPH, NADP, nitric oxide, GADD153) and cell signals [substance P (SP), free fatty acid, calcium, NF-κB] in either or both blood serum and cytosol. Subsequently, network analysis of the overall cellular stress network was performed. Our results show that there are changes affecting stress-linked biomolecules and cell signals as the rat ages. Additionally, based on our network analysis data, we postulate that NADPH, H 2 O 2 , GADD153, and SP are the key components and the interactions between these components are central to the overall age-related cellular stress network in the rat blood. Thus, we propose that the main pathway affecting the overall age-related cellular stress network in the rat blood would entail NADPH-related oxidative stress (involving H 2 O 2 ) triggering GADD153 activation leading to SP induction which in-turn affects other cell signals. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons.

    Science.gov (United States)

    Sun, Shanshan; Hu, Fangyuan; Wu, Jihong; Zhang, Shenghai

    2017-04-01

    Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XF e 24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP + ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Fenofibrate induces ketone body production in melanoma and glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Maja M Grabacka

    2016-02-01

    Full Text Available Ketone bodies (beta-hydroxybutyrate, bHB, acetoacetate are mainly produced in the liver during prolonged fasting or starvation. bHB is a very efficient energy substrate for sustaining ATP production in peripheral tissues; importantly its consumption is preferred over glucose. However, the majority of malignant cells, particularly cancer cells of neuroectodermal origin such as glioblastoma, are not able to use ketone bodies as a source of energy. Here, we report a novel observation that fenofibrate, a synthetic peroxisome proliferator-activated receptor alpha (PPARa agonist, induces bHB production in melanoma and glioblastoma cells, as well as in neurospheres composed of nontransformed cells. Unexpectedly, this effect is not dependent on PPARa activity or its expression level. The fenofibrate-induced ketogenesis is accompanied by growth arrest and down-regulation of transketolase, but the NADP/NADPH and GSH/GSSG ratios remain unaffected. Our results reveal a new, intriguing aspect of cancer cell biology and highlight the benefits of fenofibrate as a supplement to both canonical and dietary (ketogenic therapeutic approaches against glioblastoma.

  8. Identification of conserved drought stress responsive gene-network across tissues and developmental stages in rice.

    Science.gov (United States)

    Smita, Shuchi; Katiyar, Amit; Pandey, Dev Mani; Chinnusamy, Viswanathan; Archak, Sunil; Bansal, Kailash Chander

    2013-01-01

    Identification of genes that are coexpressed across various tissues and environmental stresses is biologically interesting, since they may play coordinated role in similar biological processes. Genes with correlated expression patterns can be best identified by using coexpression network analysis of transcriptome data. In the present study, we analyzed the temporal-spatial coordination of gene expression in root, leaf and panicle of rice under drought stress and constructed network using WGCNA and Cytoscape. Total of 2199 differentially expressed genes (DEGs) were identified in at least three or more tissues, wherein 88 genes have coordinated expression profile among all the six tissues under drought stress. These 88 highly coordinated genes were further subjected to module identification in the coexpression network. Based on chief topological properties we identified 18 hub genes such as ABC transporter, ATP-binding protein, dehydrin, protein phosphatase 2C, LTPL153 - Protease inhibitor, phosphatidylethanolaminebinding protein, lactose permease-related, NADP-dependent malic enzyme, etc. Motif enrichment analysis showed the presence of ABRE cis-elements in the promoters of > 62% of the coordinately expressed genes. Our results suggest that drought stress mediated upregulated gene expression was coordinated through an ABA-dependent signaling pathway across tissues, at least for the subset of genes identified in this study, while down regulation appears to be regulated by tissue specific pathways in rice.

  9. The Contribution of On-Road Emissions of Ammonia to Atmospheric Nitrogen Deposition

    Science.gov (United States)

    Fenn, M. E.; Schilling, S.; Bytnerowicz, A.; Bell, M. D.; Sickman, J. O.; Hanks, K.; Geiser, L.

    2017-12-01

    Emissions control technologies for NOx result in increased production of NH3. Emissions inventories and simulated deposition of NHx frequently underestimate reduced forms of N. Herein we provide updated spatial distribution and inventory data for on-road NH3 emissions for the continental U.S. On-road NH3 emissions were determined from on-road CO2 emissions data and published empirical NH3:CO2 vehicle emissions ratios. Emissions of NH3 in urbanized regions are typically 0.1 - 1.3 t/km2/yr. By comparison, NH3 emissions in agricultural regions generally range from 0.4 - 5.5 t/km2/yr, with a few hotspots as high as 5.5 - 11.2 t/km2/yr. We identified 500 counties that receive at least 30% of the NH3 emissions from on-road sources. Counties with higher vehicle NH3 emissions than from agriculture include 41% of the U.S. population. Within CONUS the percent of wet inorganic N deposition from the NADP/NTN as NH4+ ranged from 37 to 83% with a mean of 59.5%. Only 13% of the NADP sites across the U.S. had less than 45% of the N deposition as NH4+ based on data from 2014-2016, illustrating the near-universal occurrence of NH4+ deposition across the U.S., regardless of the primary sources of NH3 emissions. The relative importance of urban and on-road NH3 emissions versus emissions from agriculture varies regionally. In some areas both are important and should be considered when evaluating the principal sources of N deposition to affected ecosystems.Case studies of on-road NH3 emissions in relation to N deposition include four urban sites in Oregon and Washington where the NH4-N:NO3-N ratio in throughfall was 1.0 compared to an average ratio of 2.3 in bulk deposition. At urban sites in the Los Angeles Basin bulk deposition of NH4-N and NO3-N were equivalent, while NH4-N:NO3-N in throughfall under shrubs in the greater LA Basin ranged from 0.7 to 1.5. The NH4-N:NO3-N ratio at ten sites in the Lake Tahoe Basin averaged 1.4 and 1.6 in bulk deposition and throughfall. Throughfall and

  10. The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

    Directory of Open Access Journals (Sweden)

    Mariana Igoillo-Esteve

    2007-12-01

    Full Text Available Trypanosoma cruzi is highly sensitive to oxidative stress caused by reactive oxygen species. Trypanothione, the parasite's major protection against oxidative stress, is kept reduced by trypanothione reductase, using NADPH; the major source of the reduced coenzyme seems to be the pentose phosphate pathway. Its seven enzymes are present in the four major stages in the parasite's biological cycle; we have cloned and expressed them in Escherichia coli as active proteins. Glucose 6-phosphate dehydrogenase, which controls glucose flux through the pathway by its response to the NADP/NADPH ratio, is encoded by a number of genes per haploid genome, and is induced up to 46-fold by hydrogen peroxide in metacyclic trypomastigotes. The genes encoding 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, transaldolase and transketolase are present in the CL Brener clone as a single copy per haploid genome. 6-phosphogluconate dehydrogenase is very unstable, but was stabilized introducing two salt bridges by site-directed mutagenesis. Ribose-5-phosphate isomerase belongs to Type B; genes encoding Type A enzymes, present in mammals, are absent. Ribulose-5-phosphate epimerase is encoded by two genes. The enzymes of the pathway have a major cytosolic component, although several of them have a secondary glycosomal localization, and also minor localizations in other organelles.Trypanosoma cruzi é altamente sensível ao estresse oxidativo causado por espécies reativas do oxigênio. Tripanotiona, o principal protetor do parasita contra o estresse oxidativo, é mantido reduzido pela tripanotiona redutase, pela presença deNADPH; a principal fonte da coenzima reduzida parece ser a via da pentose fosfato. As sete enzimas dessa via estão presentes nos quatro principais estágios do ciclo biológico do parasita; nós clonamos e expressamos as enzimas em Escherichia coli como proteínas ativas. Glucose 6-fosfato desidrogenase, que controla o fluxo da glucose da

  11. Attributes for NHDPlus Catchments (Version 1.1) for the Conterminous United States: Normalized Atmospheric Deposition for 2002, Ammonium (NH4)

    Science.gov (United States)

    Wieczorek, Michael; LaMotte, Andrew E.

    2010-01-01

    This data set represents the average normalized atmospheric (wet) deposition, in kilograms, of Ammonium (NH4) for the year 2002 compiled for every catchment of NHDPlus for the conterminous United States. Estimates of NH4 deposition are based on National Atmospheric Deposition Program (NADP) measurements (B. Larsen, U.S. Geological Survey, written commun., 2007). De-trending methods applied to the year 2002 are described in Alexander and others, 2001. NADP site selection met the following criteria: stations must have records from 1995 to 2002 and have a minimum of 30 observations. The NHDPlus Version 1.1 is an integrated suite of application-ready geospatial datasets that incorporates many of the best features of the National Hydrography Dataset (NHD) and the National Elevation Dataset (NED). The NHDPlus includes a stream network (based on the 1:100,00-scale NHD), improved networking, naming, and value-added attributes (VAAs). NHDPlus also includes elevation-derived catchments (drainage areas) produced using a drainage enforcement technique first widely used in New England, and thus referred to as "the New England Method." This technique involves "burning in" the 1:100,000-scale NHD and when available building "walls" using the National Watershed Boundary Dataset (WBD). The resulting modified digital elevation model (HydroDEM) is used to produce hydrologic derivatives that agree with the NHD and WBD. Over the past two years, an interdisciplinary team from the U.S. Geological Survey (USGS), and the U.S. Environmental Protection Agency (USEPA), and contractors, found that this method produces the best quality NHD catchments using an automated process (USEPA, 2007). The NHDPlus dataset is organized by 18 Production Units that cover the conterminous United States. The NHDPlus version 1.1 data are grouped by the U.S. Geologic Survey's Major River Basins (MRBs, Crawford and others, 2006). MRB1, covering the New England and Mid-Atlantic River basins, contains NHDPlus

  12. A complete structural characterization of the desferrioxamine E biosynthetic pathway from the fire blight pathogen Erwinia amylovora.

    Science.gov (United States)

    Salomone-Stagni, Marco; Bartho, Joseph D; Polsinelli, Ivan; Bellini, Dom; Walsh, Martin A; Demitri, Nicola; Benini, Stefano

    2018-02-08

    The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP + binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS). Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Differential metabolism of 4-hydroxynonenal in liver, lung and brain of mice and rats

    International Nuclear Information System (INIS)

    Zheng, Ruijin; Dragomir, Ana-Cristina; Mishin, Vladimir; Richardson, Jason R.; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    The lipid peroxidation end-product 4-hydroxynonenal (4-HNE) is generated in tissues during oxidative stress. As a reactive aldehyde, it forms Michael adducts with nucleophiles, a process that disrupts cellular functioning. Liver, lung and brain are highly sensitive to xenobiotic-induced oxidative stress and readily generate 4-HNE. In the present studies, we compared 4-HNE metabolism in these tissues, a process that protects against tissue injury. 4-HNE was degraded slowly in total homogenates and S9 fractions of mouse liver, lung and brain. In liver, but not lung or brain, NAD(P)+ and NAD(P)H markedly stimulated 4-HNE metabolism. Similar results were observed in rat S9 fractions from these tissues. In liver, lung and brain S9 fractions, 4-HNE formed protein adducts. When NADH was used to stimulate 4-HNE metabolism, the formation of protein adducts was suppressed in liver, but not lung or brain. In both mouse and rat tissues, 4-HNE was also metabolized by glutathione S-transferases. The greatest activity was noted in livers of mice and in lungs of rats; relatively low glutathione S-transferase activity was detected in brain. In mouse hepatocytes, 4-HNE was rapidly taken up and metabolized. Simultaneously, 4-HNE-protein adducts were formed, suggesting that 4-HNE metabolism in intact cells does not prevent protein modifications. These data demonstrate that, in contrast to liver, lung and brain have a limited capacity to metabolize 4-HNE. The persistence of 4-HNE in these tissues may increase the likelihood of tissue injury during oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a highly reactive aldehyde. • Rodent liver, but not lung or brain, is efficient in degrading 4-hydroxynonenal. • 4-hydroxynonenal persists in tissues with low metabolism, causing tissue damage

  14. Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Barwal Indu

    2011-12-01

    Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

  15. Atmospheric deposition of nutrients to north Florida rivers: A multivariate statistical analysis. Final report. Master's thesis

    International Nuclear Information System (INIS)

    Fu, J.

    1991-01-01

    Atmospheric nutrient input to the Apalachicola Bay estuary was studied because it has been demonstrated that atmospheric deposition can be a major source of nutrients to eastern U.S. estuaries. Besides the Apalachicola River, the Sopchoppy and the Ochlockonee were also selected for a comparative analysis. Receptor model, absolute principal of component analysis (APCA), and mass balance methods were applied in the study. The results of the study show that nitrogen is probably not a limiting nutrient in the three rivers because their N:P mole ratios are nearly 3 times higher than the Redfield ratio for photosynthesis. The total atmospheric nitrogen depositions in the three river watershed are at least as great as their river fluxes. In the Apalachicola River, the atmospheric source of nitrogen is found to be several times higher than the largest possible input of urban sewage. Atmospheric deposition, therefore, might be the dominant nitrogen source entering the estuary. The results of APCA show that Apalachicola River water is mainly a mixture of components that correspond in their compositions to aged rain, ground water, and fresh rain. Atmospheric nitrate deposition is the result of the air pollution, i.e., acid rain. The studies also show that the annual average deposition of nitrate has a narrow range, mainly from 5.8 to 11.5 kg/ha/yr in most of the NADP sites in the 8 southeastern states. Since all the software and data sets employed in the study are accessible nationwide, the methods could be applied in other watersheds

  16. Selective inhibition of sheep kidney 11 beta-hydroxysteroid dehydrogenase isoform 2 activity by 5 alpha-reduced (but not 5 beta) derivatives of adrenocorticosteroids.

    Science.gov (United States)

    Latif, S A; Sheff, M F; Ribeiro, C E; Morris, D J

    1997-02-01

    We have previously reported that 5 alpha and 5 beta pathways of steroid metabolism are controlled in vivo by dietary Na+ and glycyrrhetinic acid, see Gorsline et al. 1988; Latif et al. 1990. The present investigations provide evidence supporting the suggestion that endogenous substances may regulate the glucocorticoid inactivating isoenzymes, 11 beta-HSD (hydroxysteroid dehydrogenase) 1 (liver) and 11 beta-HSD2 (kidney). The activity of 11 beta-HSD is impaired in essential hypertension, following licorice ingestion, and in patients with apparent mineralocorticoid excess where 11 beta-HSD2 is particularly affected. In all three conditions, excretion of the less common 5 alpha metabolites is elevated in urine. We now report on the differential abilities of a series of Ring A reduced (5 alpha and 5 beta) adrenocorticosteroid and progesterone metabolites to inhibit these isoenzymes. Using liver microsomes with NADP+ as co-factor (11 beta-HSD1), and sheep kidney microsomes with NAD+ as co-factor (11 beta-HSD2), we have systematically investigated the abilities of a number of adrenocorticosteroids and their derivatives to inhibit the individual isoforms of 11 beta-HSD. A striking feature is the differential sensitivity of the two isoenzymes to inhibition by 5 alpha and 5 beta derivatives. 11 beta-HSD1 is inhibited by both 5 alpha and certain 5 beta derivatives. 11 beta-HSD-2 was selectively inhibited only by 5 alpha derivatives: 5 beta derivatives were without inhibitory activity toward this isoform of 11 beta-HSD. These results indicate the importance of the structural conformation of the A and B Rings in conferring specific inhibitory properties on these compounds. In addition, we discuss the effects of additions or substitutions of other functional groups on the inhibitory potency of these steroid molecules against 11 beta-HSD1 and 11 beta-HSD2.

  17. Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

    Science.gov (United States)

    Moreno, S N; Mason, R P; Docampo, R

    1984-12-10

    At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

  18. Differential effect of adrenocorticosteroids on 11 beta-hydroxysteroid dehydrogenase bioactivity at the anterior pituitary and hypothalamus in rats.

    Science.gov (United States)

    Idrus, R B; Mohamad, N B; Morat, P B; Saim, A; Abdul Kadir, K B

    1996-08-01

    11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) is a microsomal enzyme that catalyzes the dehydrogenation of cortisol (F) to cortisone (E) in man and corticosterone (B) to 11-dehydrocorticosterone (A) in rats. 11 beta-OHSD has been identified in a wide variety of tissues. The differential distribution of 11 beta-OHSD suggests that this enzyme has locally defined functions that vary from region to region. The aim of this study was to investigate the effects of the glucocorticoids B and dexamethasone (DM), the mineralocorticoid deoxycorticosterone (DOC), and the inhibitors of 11 beta-OHSD glycyrrhizic acid (Gl) and glycyrrhetinic acid (GE) on 11 beta-OHSD bioactivity at the hypothalamus (HT) and anterior pituitary (AP). Male Wistar rats were treated with GI or were adrenalectomized (ADX) and treated with either B, DM, or DOC for 7 days. All treatments were in vivo except GE, which was used in vitro. At the end of treatment, homogenates of HT and AP were assayed for 11 beta-OHSD bioactivity, expressed as the percentage conversion of B to A in the presence of NADP, 11 beta-OHSD bioactivity is significantly higher (P < 0.0001) in the AP compared with the HT. Adrenalectomy significantly increased the enzyme activity in the AP (P < 0.05), an effect reversed by B or DM. ADX rats treated with DOC showed decreased enzyme activity in the AP (P < 0.001) but increased the activity in the HT (P < 0.0001). Gl increased activity in both HT and AP, whereas GE decreased activity significantly. We conclude that the modulation of 11 beta-OHSD is both steroid specific and tissue specific.

  19. Visual cycle and its metabolic support in gecko photoreceptors.

    Science.gov (United States)

    Kolesnikov, A V; Ala-Laurila, P; Shukolyukov, S A; Crouch, R K; Wiggert, B; Estevez, M E; Govardovskii, V I; Cornwall, M C

    2007-02-01

    Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP+glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

  20. Modeling single nucleotide polymorphisms in the human AKR1C1 and AKR1C2 genes: implications for functional and genotyping analyses.

    Directory of Open Access Journals (Sweden)

    Jonathan W Arthur

    2010-12-01

    Full Text Available Enzymes encoded by the AKR1C1 and AKR1C2 genes are responsible for the metabolism of progesterone and 5α-dihydrotestosterone (DHT, respectively. The effect of amino acid substitutions, resulting from single nucleotide polymorphisms (SNPs in the AKR1C2 gene, on the enzyme kinetics of the AKR1C2 gene product were determined experimentally by Takashi et al. In this paper, we used homology modeling to predict and analyze the structure of AKR1C1 and AKR1C2 genetic variants. The experimental reduction in enzyme activity in the AKR1C2 variants F46Y and L172Q, as determined by Takahashi et al., is predicted to be due to increased instability in cofactor binding, caused by disruptions to the hydrogen bonds between NADP and AKR1C2, resulting from the insertion of polar residues into largely non-polar environments near the site of cofactor binding. Other AKR1C2 variants were shown to involve either conservative substitutions or changes taking place on the surface of the molecule and distant from the active site, confirming the experimental finding of Takahashi et al. that these variants do not result in any statistically significant reduction in enzyme activity. The AKR1C1 R258C variant is predicted to have no effect on enzyme activity for similar reasons. Thus, we provide further insight into the molecular mechanism of the enzyme kinetics of these proteins. Our data also highlight previously reported difficulties with online databases.

  1. Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications.

    Science.gov (United States)

    Holtgrefe, Simone; Gohlke, Jochen; Starmann, Julia; Druce, Samantha; Klocke, Susanne; Altmann, Bianca; Wojtera, Joanna; Lindermayr, Christian; Scheibe, Renate

    2008-06-01

    Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

  2. Effects of x-irradiation on lens reducing systems

    International Nuclear Information System (INIS)

    Giblin, F.J.; Chakrapani, B.; Reddy, V.N.

    1978-01-01

    Studies have been made of the effects of x ray on various lens reducing systems including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS), and the activities of certain enzymes including glutathion reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PD). It was found that during several weeks following x irradiation but prior to cataract formation there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the x-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the x-rayed lens was found to decrease significantly relative to controls one week prior to cataract formation and the ratio of NADPH to NADP + in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the x-rayed lens. A corresponding decrease occurred in the activity of the HMS in x-rayed lenses as measured by culture in the presence of 1- 14 C-labelled glucose. G-6-PD was partially inactivated in the x-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to x-irradiation. The data indicate that x-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that, when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high molecular weight protein aggregates may be initiated

  3. Capsicum chinensis L. growth and nutraceutical properties are enhanced by biostimulants in a long-term period: chemical and metabolomic approaches.

    Directory of Open Access Journals (Sweden)

    ANDREA eERTANI

    2014-08-01

    Full Text Available Two biostimulants, one derived from alfalfa plants (AH and the other obtained from red grape (RG, were chemically characterised using enzyme linked immuno-sorbent assays, Fourier transform infrared (FTIR and Raman spectroscopies. Two doses (50 and 100 mL L-1 for RG, and 25 and 50 mL L-1 for AH of biostimulants were applied to Capsicum chinensis L. plants cultivated in pots inside a tunnel. The experimental design consisted of the factorial combination of treatment (no biostimulant, plus AH, plus RG at three doses (zero, low and high and two time-course applications (at the second and fourth week after transplantation and the effects were recorded at flowering and maturity. Both biostimulants contained different amounts of indoleacetic acid and isopentenyladenosine; the AH spectra exhibited amino acid functional groups in the peptidic structure, while the RG spectra showed the presence of polyphenols, such as resveratrol. These results revealed that at flowering, RG and AH increased the weights of fresh leaves and fruits and the number of green fruits, whereas at maturity, the biostimulants most affected the fresh weight and number of red fruits. At flowering, the leaves of the treated plants contained high amounts of epicatechin, ascorbic acid, quercetin and dihydrocapsaicin. At maturity, the leaves of the treated plants exhibited elevated amounts of fructose, glucose, chlorogenic and ferulic acids. Moreover, green fruits exhibited a high content of chlorogenic acid, p-hydroxybenzoic acid, p-coumaric acid and antioxidant activity, while both AH- and RG-treated red fruits were highly endowed in capsaicin. The 1H high-resolution magic-angle spinning (HRMAS-nuclear magnetic resonance (NMR spectra of red fruits revealed that both products induced a high amount of NADP+, whereas RG also increased glucose, fumarate, ascorbate, thymidine and high molecular weight species. Our results suggested that AH and RG promoted plant growth and the production of

  4. Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

    Science.gov (United States)

    Jwa, Nam-Soo; Hwang, Byung Kook

    2017-01-01

    Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

  5. Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants

    Directory of Open Access Journals (Sweden)

    Nam-Soo Jwa

    2017-09-01

    Full Text Available Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP-triggered immunity (PTI and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

  6. Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.

    Science.gov (United States)

    Pennacchio, Angela; Pucci, Biagio; Secundo, Francesco; La Cara, Francesco; Rossi, Mosè; Raia, Carlo A

    2008-07-01

    The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adh(Tt)) was heterologously overexpressed in Escherichia coli, and the protein (ADH(Tt)) was purified to homogeneity and characterized. ADH(Tt) is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to approximately 73 degrees C and a 30-min half-inactivation temperature of approximately 90 degrees C, as well as good tolerance to common organic solvents. ADH(Tt) has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and alpha-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, alpha-tetralone, and alpha-methyl and alpha-ethyl benzoylformates to (S)-(-)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-alpha-(trifluoromethyl)benzyl alcohol (93% ee), (S)-alpha-tetralol (>99% ee), methyl (R)-(-)-mandelate (92% ee), and ethyl (R)-(-)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily.

  7. Non radioactive precursor import into chloroplasts

    International Nuclear Information System (INIS)

    Lombardo, V.A.; Ottado, J.

    2003-01-01

    Full text: Eukaryotic cells have a subcellular organization based on organelles. Protein transport to these organelles is quantitatively important because the majority of cellular proteins are codified in nuclear genes and then delivered to their final destination. Most of the chloroplast proteins are translated on cytoplasmic ribosomes as larger precursors with an amino terminal transit peptide that is necessary and sufficient to direct the precursor to the chloroplast. Once inside the organelle the transit peptide is cleaved and the mature protein adopts its folded form. In this work we developed a system for the expression and purification of the pea ferredoxin-NADP + reductase precursor (preFNR) for its import into chloroplasts in non radioactive conditions. We constructed a preFNR fused in its carboxy terminus to a 6 histidines peptide (preFNR-6xHis) that allows its identification using a commercial specific antibody. The construction was expressed, purified, processed and precipitated, rendering a soluble and active preFNR-6xHis that was used in binding and import into chloroplasts experiments. The reisolated chloroplasts were analyzed by SDS-PAGE, electro-blotting and revealed by immuno-detection using either colorimetric or chemiluminescent reactive. We performed also import experiments labeling preFNR and preFNR-6xHis with radioactive methionine as controls. We conclude that preFNR-6xHis is bound and imported into chloroplasts as the wild type preFNR and that both colorimetric or chemiluminescent detection methods are useful to avoid the manipulation of radioactive material. (author)

  8. Biochemical and locomotor responses of Carcinus maenas exposed to the serotonin reuptake inhibitor fluoxetine.

    Science.gov (United States)

    Mesquita, Sofia Raquel; Guilhermino, Lúcia; Guimarães, Laura

    2011-10-01

    The aim of this study was to assess the effects of the widely used anti-depressant fluoxetine on behaviour (locomotion), moulting, neuromuscular transmission, energy production and anti-oxidant defences' efficiency of the epibenthic crab Carcinus maenas. Crabs were individually exposed to fluoxetine concentrations for 7d. Effects on locomotion were assessed at the end of the exposure using an open field test adapted to C. maenas in the present study. Tissue samples were later collected to evaluate fluoxetine effects on physiological functions using the activity of key enzymes and other parameters as biomarkers, namely: N-acetyl-β-glucosaminidase (NAGase) in the epidermis (moulting) and the hepatopancreas; cholinesterases (ChE) in muscle (neuromuscular cholinergic transmission); NADP(+)-dependent isocitrate dehydrogenase (IDH) and lactate dehydrogenease (LDH) in muscle (energy production); glutathione S-transferases (GST) in hepatopancreas (biotransformation and oxidative stress system); glutathione reductase (GR), and glutathione peroxidade (GPx), total glutathione levels (TG) and lipid peroxidation levels in the hepatopancreas (anti-oxidant defences and oxidative damage). Because no information on C. maenas NAGase activity was previously available, its variation during the moult cycle was also investigated. The results showed that locomotion was significantly increased at fluoxetine concentrations equal or above 120 μg L⁻¹, with animals spending more time moving, walking longer distances than controls. Levels of NAGase activity were found to vary in relation to C. maenas moult cycle, but no alterations were observed after exposure to fluoxetine. Significant increases in the activity of ChE, GST and GR enzymes, and the levels of TG were found, with a lowest observed effect concentration (LOEC) of 120 μg L⁻¹. Effects on locomotion were significantly and positively correlated to those induced on ChE activity. The results raise concern when hypothesising

  9. Evidence for carbon flux shortage and strong carbon/nitrogen interactions in pea nodules at early stages of water stress.

    Science.gov (United States)

    Gálvez, Loli; González, Esther M; Arrese-Igor, Cesar

    2005-09-01

    Symbiotic N2 fixation in legume nodules declines under a wide range of environmental stresses. A high correlation between N2 fixation decline and sucrose synthase (SS; EC 2.4.1.13) activity down-regulation has been reported, although it has still to be elucidated whether a causal relationship between SS activity down-regulation and N2 fixation decline can be established. In order to study the likely C/N interactions within nodules and the effects on N2 fixation, pea plants (Pisum sativum L. cv. Sugar snap) were subjected to progressive water stress by withholding irrigation. Under these conditions, nodule SS activity declined concomitantly with apparent nitrogenase activity. The levels of UDP-glucose, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate decreased in water-stressed nodules compared with unstressed nodules. Drought also had a marked effect on nodule concentrations of malate, succinate, and alpha-ketoglutarate. Moreover, a general decline in nodule adenylate content was detected. NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42) was the only enzyme whose activity increased as a result of water deficit, compensating for a possible C/N imbalance and/or supplying NADPH in circumstances that the pentose phosphate pathway was impaired, as suggested by the decline in glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) activity. The overall results show the occurrence of strong C/N interactions in nodules subjected to water stress and support a likely limitation of carbon flux that might be involved in the decline of N2 fixation under drought.

  10. Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling

    Energy Technology Data Exchange (ETDEWEB)

    R. Quatrini; C. Appia-Ayme; Y. Denis; J. Ratouchniak; F. Veloso; J. Valdes; C. Lefimil; S. Silver; F. Roberto; O. Orellana; F. Denizot; E. Jedlicki; D. Holmes; V. Bonnefoy

    2006-09-01

    Acidithiobacillus ferrooxidans is a well known acidophilic, chemolithoautotrophic, Gram negative, bacterium involved in bioleaching and acid mine drainage. In aerobic conditions, it gains energy mainly from the oxidation of ferrous iron and/or reduced sulfur compounds present in ores. After initial oxidation of the substrate, electrons from ferrous iron or sulfur enter respiratory chains and are transported through several redox proteins to oxygen. However, the oxidation of ferrous iron and reduced sulfur compounds has also to provide electrons for the reduction of NAD(P) that is subsequently required for many metabolic processes including CO2 fixation. To help to unravel the enzymatic pathways and the electron transfer chains involved in these processes, a genome-wide microarray transcript profiling analysis was carried out. Oligonucleotides corresponding to approximately 3000 genes of the A. ferrooxidans type strain ATCC23270 were spotted onto glass-slides and hybridized with cDNA retrotranscribed from RNA extracted from ferrous iron and sulfur grown cells. The genes which are preferentially transcribed in ferrous iron conditions and those preferentially transcribed in sulfur conditions were analyzed. The expression of a substantial number of these genes has been validated by real-time PCR, Northern blot hybridization and/or immunodetection analysis. Our results support and extend certain models of iron and sulfur oxidation and highlight previous observations regarding the possible presence of alternate electron pathways. Our findings also suggest ways in which iron and sulfur oxidation may be co-ordinately regulated. An accompanying paper (Appia-Ayme et al.) describes results pertaining to other metabolic functions.

  11. Effects of x-irradiation on lens reducing systems. [Rabbits

    Energy Technology Data Exchange (ETDEWEB)

    Giblin, F.J.; Chakrapani, B.; Reddy, V.N.

    1978-01-01

    Studies have been made of the effects of x ray on various lens reducing systems including the levels of NADPH and glutathione (GSH), the activity of the hexose monophosphate shunt (HMS), and the activities of certain enzymes including glutathion reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase (G-6-PD). It was found that during several weeks following x irradiation but prior to cataract formation there was very little change in the number of reduced -SH groups per unit weight of lens protein but that, with the appearance of cataract, there was a sudden loss of protein -SH groups. In contrast, the concentration of GSH in the x-rayed lens decreased throughout the experimental period. Similarly, the concentration of NADPH in the x-rayed lens was found to decrease significantly relative to controls one week prior to cataract formation and the ratio of NADPH to NADP/sup +/ in the lens shifted at this time period from a value greater than 1.0 in the control lens to less than 1.0 in the x-rayed lens. A corresponding decrease occurred in the activity of the HMS in x-rayed lenses as measured by culture in the presence of 1-/sup 14/C-labelled glucose. G-6-PD was partially inactivated in the x-rayed lens. Of the eight enzymes studied, G-6-PD appeared to be the most sensitive to x-irradiation. The data indicate that x-irradiation results in a steady decrease in the effectiveness of lens reducing systems and that, when these systems reach a critically low point, sudden oxidation of protein -SH groups and formation of high molecular weight protein aggregates may be initiated.

  12. Alpha-eleostearic acid (9Z11E13E-18:3) is quickly converted to conjugated linoleic acid (9Z11E-18:2) in rats.

    Science.gov (United States)

    Tsuzuki, Tsuyoshi; Tokuyama, Yoshiko; Igarashi, Miki; Nakagawa, Kiyotaka; Ohsaki, Yusuke; Komai, Michio; Miyazawa, Teruo

    2004-10-01

    We previously showed that alpha-eleostearic acid (alpha-ESA; 9Z11E13E-18:3) is converted to conjugated linoleic acid (CLA; 9,11-18:2) in the liver and plasma of rats that were given diets including 1% alpha-ESA for 4 wk. In this study, we investigated this phenomenon in detail. First, the chemical structure of CLA produced by alpha-ESA administration was determined. After alpha-ESA was orally administered to rats, CLA in rat liver was isolated by HPLC. The positional and geometric isomerism was determined using GC-EI/MS and (13)C-NMR, respectively, and the CLA generated in rats after alpha-ESA feeding was confirmed to be 9Z11E-CLA. Next, the concentrations of alpha-ESA and CLA were determined 0, 3, 6, and 24 h after oral administration of alpha-ESA to rats. Moreover, we also investigated whether enteric bacteria are involved in the conversion of alpha-ESA to CLA using germ-free rats. alpha-ESA was orally administered to germ-free and normal rats and alpha-ESA and CLA were detected in the organs of both groups. In addition, to confirm that this reaction was enzyme-mediated, alpha-ESA was reacted with tissue homogenates (liver, kidney, and small intestine mucous) and coenzymes (NADH, NAD(+), NADPH, and NADP(+)), and the enzyme activities were estimated from the amount of CLA produced. CLA was detected when alpha-ESA was reacted with liver, kidney, and small intestine mucous homogenates and a coenzyme (NADPH). These results indicated that alpha-ESA is converted to 9Z11E-CLA in rats by a Delta13-saturation reaction carried out by an NADPH-dependent enzyme.

  13. Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth.

    Science.gov (United States)

    Dellero, Younès; Lamothe-Sibold, Marlène; Jossier, Mathieu; Hodges, Michael

    2015-09-01

    Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air-grown ggt1 mutants showed retarded growth and development, that was not observed at high CO2 (3000 μL L(-1) ). When compared to wild-type (WT) plants, air-grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP(+) /NADPH ratios. When compared to WT plants, their net CO2 assimilation rates (An ) were 50% lower and this mirrored decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO2 -grown ggt1 plants transferred to air revealed a rapid decrease of An and photosynthetic electron transfer rate while maintaining a high energetic state. Short-term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short-term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long-term (12 days) acclimation to air from high CO2 when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low An limits nitrogen-assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  14. Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase.

    Science.gov (United States)

    Hata, H; Shimizu, S; Hattori, S; Yamada, H

    1989-02-24

    Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.

  15. D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens

    International Nuclear Information System (INIS)

    Lessmann, D.; Schimz, K.L.; Kurz, G.

    1975-01-01

    The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300μmol NADH formed per min per mg protein, was shown to be homogeneous. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220,000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265,000. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal acivity at pH 8.9. The Entner-Doudoroff enzyme showed specificity for NAD + as well as for NADP + and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of β-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase. (orig.) [de

  16. The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation.

    Science.gov (United States)

    Molenaar, Remco J; Radivoyevitch, Tomas; Maciejewski, Jaroslaw P; van Noorden, Cornelis J F; Bleeker, Fonnet E

    2014-12-01

    Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key events in the development of glioma, acute myeloid leukemia (AML), chondrosarcoma, intrahepatic cholangiocarcinoma (ICC), and angioimmunoblastic T-cell lymphoma. They also cause D-2-hydroxyglutaric aciduria and Ollier and Maffucci syndromes. IDH1/2 mutations are associated with prolonged survival in glioma and in ICC, but not in AML. The reason for this is unknown. In their wild-type forms, IDH1 and IDH2 convert isocitrate and NADP(+) to α-ketoglutarate (αKG) and NADPH. Missense mutations in the active sites of these enzymes induce a neo-enzymatic reaction wherein NADPH reduces αKG to D-2-hydroxyglutarate (D-2HG). The resulting D-2HG accumulation leads to hypoxia-inducible factor 1α degradation, and changes in epigenetics and extracellular matrix homeostasis. Such mutations also imply less NADPH production capacity. Each of these effects could play a role in cancer formation. Here, we provide an overview of the literature and discuss which downstream molecular effects are likely to be the drivers of the oncogenic and survival-prolonging properties of IDH1/2 mutations. We discuss interactions between mutant IDH1/2 inhibitors and conventional therapies. Understanding of the biochemical consequences of IDH1/2 mutations in oncogenesis and survival prolongation will yield valuable information for rational therapy design: it will tell us which oncogenic processes should be blocked and which "survivalogenic" effects should be retained. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2.

    Directory of Open Access Journals (Sweden)

    Jian-Guo Ren

    2010-09-01

    Full Text Available Malic enzyme 2 (ME2 is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel

  18. Thermodynamic and NMR analyses of NADPH binding to lipocalin-type prostaglandin D synthase

    International Nuclear Information System (INIS)

    Qin, Shubin; Shimamoto, Shigeru; Maruno, Takahiro; Kobayashi, Yuji; Kawahara, Kazuki; Yoshida, Takuya; Ohkubo, Tadayasu

    2015-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) is one of the most abundant proteins in human cerebrospinal fluid (CSF) with dual functions as a prostaglandin D_2 (PGD_2) synthase and a transporter of lipophilic ligands. Recent studies revealed that L-PGDS plays important roles in protecting against various neuronal diseases induced by reactive oxygen species (ROS). However, the molecular mechanisms of such protective actions of L-PGDS remain unknown. In this study, we conducted thermodynamic and nuclear magnetic resonance (NMR) analyses, and demonstrated that L-PGDS binds to nicotinamide coenzymes, including NADPH, NADP"+, and NADH. Although a hydrophilic ligand is not common for L-PGDS, these ligands, especially NADPH showed specific interaction with L-PGDS at the upper pocket of its ligand-binding cavity with an unusually bifurcated shape. The binding affinity of L-PGDS for NADPH was comparable to that previously reported for NADPH oxidases and NADPH in vitro. These results suggested that L-PGDS potentially attenuates the activities of NADPH oxidases through interaction with NADPH. Given that NADPH is the substrate for NADPH oxidases that play key roles in neuronal cell death by generating excessive ROS, these results imply a novel linkage between L-PGDS and ROS. - Highlights: • Interactions of L-PGDS with nicotinamide coenzymes were studied by ITC and NMR. • The binding affinity of L-PGDS was strongest to NADPH among nicotinamide coenzymes. • NADPH binds to the upper part of L-PGDS ligand-binding cavity. • L-PGDS binds to both lipophilic and hydrophilic ligands. • This study implies a novel linkage between L-PGDS and reactive oxygen species.

  19. Biochemical characterization of an L-tryptophan dehydrogenase from the photoautotrophic cyanobacterium Nostoc punctiforme.

    Science.gov (United States)

    Ogura, Ryutaro; Wakamatsu, Taisuke; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa

    2014-06-10

    An NAD(+)-dependent l-tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH) was cloned and overexpressed in Escherichia coli. The recombinant NpTrpDH with a C-terminal His6-tag was purified to homogeneity using a Ni-NTA agarose column, and was found to be a homodimer with a molecular mass of 76.1kDa. The enzyme required NAD(+) and NADH as cofactors for oxidative deamination and reductive amination, respectively, but not NADP(+) or NADPH. l-Trp was the preferred substrate for deamination, though l-Phe was deaminated at a much lower rate. The enzyme exclusively aminated 3-indolepyruvate; phenylpyruvate was inert. The pH optima for the deamination of l-Trp and amination of 3-indolpyruvate were 11.0 and 7.5, respectively. For deamination of l-Trp, maximum enzymatic activity was observed at 45°C. NpTrpDH retained more than 80% of its activity after incubation for 30min at pHs ranging from 5.0 to 11.5 or incubation for 10min at temperatures up to 40°C. Unlike l-Trp dehydrogenases from higher plants, NpTrpDH activity was not activated by metal ions. Typical Michaelis-Menten kinetics were observed for NAD(+) and l-Trp for oxidative deamination, but with reductive amination there was marked substrate inhibition by 3-indolepyruvate. NMR analysis of the hydrogen transfer from the C4 position of the nicotinamide moiety of NADH showed that NpTrpDH has a pro-S (B-type) stereospecificity similar to the Glu/Leu/Phe/Val dehydrogenase family. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Biofabricated film with enzymatic and redox-capacitor functionalities to harvest and store electrons

    International Nuclear Information System (INIS)

    Liba, Benjamin D; Kim, Eunkyoung; Martin, Alexandra N; Liu Yi; Bentley, William E; Payne, Gregory F

    2013-01-01

    Exciting opportunities in bioelectronics will be facilitated by materials that can bridge the chemical logic of biology and the digital logic of electronics. Here we report the fabrication of a dual functional hydrogel film that can harvest electrons from its chemical environment and store these electrons by switching the film's redox-state. The hydrogel scaffold was formed by the anodic deposition of the aminopolysaccharide chitosan. Electron-harvesting function was conferred by co-depositing the enzyme glucose dehydrogenase (GDH) with chitosan. GDH catalyzes the transfer of electrons from glucose to the soluble redox-shuttle NADP + . Electron-storage function was conferred by the redox-active food phenolic chlorogenic acid (CA) that was enzymatically grafted to the chitosan scaffold using tyrosinase. The grafted CA undergoes redox-cycling reactions with NADPH resulting in the net transfer of electrons to the film where they are stored in the reduced state of CA. The individual and dual functionalities of these films were demonstrated experimentally. There are three general conclusions from this proof-of-concept study. First, enzymatically-grafted catecholic moieties confer redox-capacitor function to the chitosan scaffold. Second, biological materials (i.e. chitosan and CA) and mechanisms (i.e. tyrosinase-mediated grafting) allow the reagentless fabrication of functional films that should be environmentally-friendly, safe and potentially even edible. Finally, the film's ability to mediate the transfer of electrons from a biological metabolite to an electrode suggests an approach to bridge the chemical logic of biology with the digital logic of electronics. (paper)

  1. The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism.

    Science.gov (United States)

    Stenmark, Pål; Moche, Martin; Gurmu, Daniel; Nordlund, Pär

    2007-10-12

    We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

  2. Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria.

    Science.gov (United States)

    Hirschfeld, Josefine; White, Phillipa C; Milward, Michael R; Cooper, Paul R; Chapple, Iain L C

    2017-12-01

    Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii , stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated. Copyright © 2017 American Society for Microbiology.

  3. Effect Of Substrates On The Fractionation Of Hydrogen Isotopes During Lipid-Biosynthesis By Haloarcula marismortui

    Science.gov (United States)

    Dirghangi, S. S.; Pagani, M.

    2010-12-01

    Lipids form an important class of proxies for paleoclimatological research, and hydrogen isotope ratios of lipids are being increasingly used for understanding changes in the hydrological system. Proper understanding of hydrogen isotope fractionation during lipid biosynthesis is therefore important and attention has been directed toward understanding the magnitude of hydrogen isotope fractionation that occurs during lipid biosynthesis in various organisms. Hydrogen isotope ratios of lipids depend on the hydrogen isotopic composition of the ambient water, hydrogen isotopic composition of NADPH used during biosynthesis, growth conditions, pathways of lipid biosynthesis, and substrates in the case of heterotrophic organisms. Recently it has been observed that NADPH contributes a significant part of the hydrogen in fatty acids synthesized by bacteria during heterotrophic growth (Zhang et al, 2009). As NADPH is formed by reduction of NADP+ during metabolism of substrates, different metabolic pathways form NADPH with different D/H ratios, which in turn results in variation in D/H ratios of lipids (Zhang et al, 2009). Therefore, substrates play a significant role in hydrogen isotopic compositions of lipids. For this study, we are investigating the effects of substrates on hydrogen isotope fractionation during biosynthesis of isoprenoidal lipids by heterotrophically growing halophilic archaea. Haloarcula marismortui is a halophilic archaea which synthesizes Archaeol (a diether lipid) and other isoprenoidal lipids. We have grown Haloarcula marismortui in pure cultures on three different substrates and are in the process of evaluating isotopic variability of Archaeol and other lipids associated with substrate and the D/H composition of ambient water. Our results will be helpful for a better understanding of hydrogen isotope fractionations during lipid synthesis by archaea. Also, halophilic archaea are the only source of archaeol in hypersaline environments. Therefore, our

  4. Atmospheric wet deposition of mercury in North America

    Energy Technology Data Exchange (ETDEWEB)

    Sweet, C.W.; Prestbo, E.; Brunette, B.

    1999-07-01

    Currently, 39 states in the US and 5 Canadian provinces have issued advisories about the dangers of eating mercury-contaminated fish taken from waters within their boundaries. The problem is most severe in the Great Lakes region, the Northeast US states, the Canadian maritime provinces, and in south Florida where many lakes and streams contain fish with concentrations of 1 ppm or higher. For many rural and remote locations, atmospheric deposition is the primary source of mercury. In 1995, the National Atmospheric Deposition Program (NADP) initiated a program to monitor total mercury and methylmercury (MMHg) in wet deposition (rain and snow) in North America. In this program, the Mercury Deposition Network (MDN), individual monitoring sites are funded and operated by a variety of local, state, and federal agencies. However, sampling and analysis are coordinated through a central laboratory so that all of the samples are collected and analyzed using the same protocols. Weekly wet-only precipitation samples are collected using an all-glass sampling train and special handling techniques. Analysis is by cold vapor atomic fluorescence spectrometry using USEPA Method 1631 for total mercury. Nearly 40 MDN sites are in operation in 1999. Most of the sites are in the eastern US and Canada. During 1996 and 1997, the volume-weighted mean concentration of total mercury in precipitation collected at 22 sites ranged from 6.0 to 18.9 ng/L. Annual deposition varied between 2.1 and 25.3 {micro} g/m{sup 2}. The average weekly wet deposition of total mercury is more than three times higher in the summer (June-August) than in the winter (December-February). This increase is due to both higher amounts of precipitation and higher concentrations of mercury in precipitation during the summer. The highest values for mercury concentration in precipitation and wet deposition of mercury were measured in the southeastern US.

  5. Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.

    Directory of Open Access Journals (Sweden)

    Gilles Gouspillou

    Full Text Available BACKGROUND: Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria. METHODOLOGY/PRINCIPAL FINDINGS: The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically and phosphorylation (determined using the glucose-hexokinase glucose-6-phosphate dehydrogenase-NADP(+ enzymatic system rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K(mVox and phosphorylation (K(mVp rates. We also demonstrate that determination of K(mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method. CONCLUSIONS/SIGNIFICANCE: Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.

  6. A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles

    Directory of Open Access Journals (Sweden)

    Shekhawat G

    2009-07-01

    Full Text Available Abstract Background The synthesis of gold nanoparticles (GNPs has received considerable attention with their potential applications in various life sciences related applications. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system, which has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. In the present study, we have demonstrated the synthesis of gold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines in India. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization via charge capping was investigated. Results A bacterial isolate capable of gold nanoparticle synthesis was isolated and identified as a novel strain of Stenotrophomonas malophilia (AuRed02 based on its morphology and an analysis of its 16S rDNA gene sequence. After 8 hrs of incubation, monodisperse preparation of gold nanoparticles was obtained. Gold nanoparticles were characterized and found to be of ~40 nm size. Electrophoresis, Zeta potential and FTIR measurements confirmed that the particles are capped with negatively charged phosphate groups from NADP rendering them stable in aqueous medium. Conclusion The process of synthesis of well-dispersed nanoparticles using a novel microorganism isolated from the gold enriched soil sample has been reported in this study, leading to the development of an easy bioprocess for synthesis of GNPs. This is the first study in which an extensive characterization of the indigenous bacterium isolated from the actual gold enriched soil was conducted. Promising mechanism for the biosynthesis of GNPs by the strain and their stabilization via charge capping is suggested, which involves an NADPH-dependent reductase enzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process.

  7. Kinetic alteration of a human dihydrodiol/3alpha-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine.

    Science.gov (United States)

    Ohta, T; Ishikura, S; Shintani, S; Usami, N; Hara, A

    2000-01-01

    Human dihydrodiol dehydrogenase with 3alpha-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1-1C4) that belong to the aldo-keto reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1-1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the K(m) for NADP(+) and differently influenced the K(m) and k(cat) for substrates depending on their structures. The kinetic constants for bile acids with a 12alpha-hydroxy group were decreased 1.5-7-fold and those for the other substrates were increased 1.3-9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17beta-oestradiol, phenolphthalein and flufenamic acid and 3,5,3', 5'-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the K(m) for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators. PMID:11104674