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NAD
Kinds of NAD Selected AbstractsReactive oxygen species in rostral ventrolateral medulla modulate cardiac sympathetic afferent reflex in ratsACTA PHYSIOLOGICA, Issue 4 2009M.-K. Zhong Abstract Aim:, The aim of the present study was to investigate whether reactive oxygen species (ROS) in rostral ventrolateral medulla (RVLM) modulate cardiac sympathetic afferent reflex (CSAR) and the enhanced CSAR response caused by microinjection of angiotensin II (Ang II) into the paraventricular nucleus (PVN). Methods:, Under urethane and ,-chloralose anaesthesia, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic-denervated and cervical-vagotomized rats. The CSAR was evaluated by the RSNA response to epicardial application of capsaicin (1.0 nmol). Results:, Bilateral RVLM microinjection of tempol (a superoxide anion scavenger) or polyethylene glycol-superoxide dismutase (PEG-SOD, an analogue of endogenous superoxide dismutase) attenuated the CSAR, but did not cause significant change in baseline RSNA and MAP. NAD(P)H oxidase inhibitors apocynin or phenylarsine oxide (PAO) also showed similar effects, but SOD inhibitor diethyldithio-carbamic acid (DETC) enhanced the CSAR and baseline RSNA, and increased the baseline MAP. Bilateral PVN microinjection of Ang II (0.3 nmol) enhanced the CSAR and increased RSNA and MAP, which was inhibited by the pre-treatment with RVLM administration of tempol, PEG-SOD, apocynin or PAO. The pre-treatment with DETC in the RVLM only showed a tendency in potentiating the CSAR response of Ang II in the PVN, but significantly potentiated the RSNA and MAP responses of Ang II. Conclusion:, These results suggest that the NAD(P)H oxidase-derived ROS in the RVLM modulate the CSAR. The ROS in the RVLM is necessary for the enhanced CSAR response caused by Ang II in the PVN. [source] Evaluating Enzyme Cascades for Methanol/Air Biofuel Cells Based on NAD+ -Dependent EnzymesELECTROANALYSIS, Issue 7-8 2010Abstract Previous work by the group has entailed encapsulating enzymes in polymeric micelles at bioelectrode surfaces by utilizing hydrophobically modified Nafion membranes, which are modified in order to eliminate the harsh acidity of Nafion while tailoring the size of the polymer micelles to optimize for the encapsulation of an individual enzyme. This polymer encapsulation has been shown to provide high catalytic activity and enzyme stability. In this study, we employed this encapsulation technique in developing a methanol/air biofuel cell through the combined immobilization of NAD+ -dependent alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AldDH) and formate dehydrogenase (FDH) within a tetrabutylammonium bromide (TBAB) modified Nafion to oxidize methanol to carbon dioxide with poly(methylene green) acting as the NADH electrocatalyst electropolymerized on the surface of the electrode. The methanol biofuel/air cell resulted in a maximum power density of 261±7.6,,W/cm2 and current density of 845±35.5,,A/cm2. This system was characterized for the effects of degree of oxidation, temperature, pH, and concentration of fuel and NAD. [source] Manganese speciation in human cerebrospinal fluid using CZE coupled to inductively coupled plasma MSELECTROPHORESIS, Issue 9 2007Bernhard Michalke Dr. Abstract The neurotoxic effects of manganese (Mn) at elevated concentrations are well known. This raises the question, which of the Mn species can cross neural barriers and appear in cerebrospinal fluid (CSF). CSF is the last matrix in a living human organism available for analysis before a compound reaches the brain cells and therefore it is assumed to reflect best the internal exposure of brain tissue to Mn species. A previously developed CE method was modified for separation of albumin, histidine, tyrosine, cystine, fumarate, malate, inorganic Mn, oxalacetate, ,-keto-glutarate, nicotinamide-dinucleotide (NAD), citrate, adenosine, glutathione, and glutamine. These compounds are supposed in the literature to act as potential Mn carriers. In a first attempt, these compounds were analyzed by CZE-UV to check whether they are present in CSF. The CZE-UV method was simpler than the coupled CZE-inductively coupled plasma (ICP)-dynamic reaction cell (DRC)-MS method and it was therefore chosen to obtain a first overview information. In a second step, the coupled method (CZE-ICP-DRC-MS) was used to analyze, in detail, which of the compounds found in CSF by CZE-UV were actually bound to Mn. Finally, 13 Mn species were monitored in CSF samples, most of them being identified: Mn-histidine, Mn-fumarate, Mn-malate, inorganic Mn, Mn-oxalacetate, Mn-,-keto glutarate, Mn-carrying NAD, Mn-citrate and Mn-adenosine. By far the most abundant Mn species was Mn-citrate showing a concentration of 0.7,±,0.13,µg,Mn/L. Interestingly, several other Mn species can be related to the citric acid cycle. [source] Enhancement of the NAD(P)(H) Pool in Saccharomyces cerevisiaeENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008A. Knepper Abstract Asymmetric biosyntheses allow for an efficient production of chiral building blocks. The application of whole cells as biocatalysts for asymmetric syntheses is advantageous because they already contain the essential coenzymes NAD(H) or NADP(H), which additionally can be regenerated in the cells. Unfortunately, reduced catalytic activity compared to the oxidoreductase activity is observed in many cases during whole-cell biotransformation. This may be caused by low intracellular coenzyme pool sizes and/or a decline in intracellular coenzyme concentrations. To enhance the intracellular coenzyme pool sizes, the effects of the precursor metabolites adenine and nicotinic acid on the intracellular accumulation of NAD(H) and NADP(H) were studied in Saccharomyces cerevisiae. Based on the results of simple batch experiments with different precursor additions, fed-batch processes for the production of yeast cells with enhanced NAD(H) or enhanced NADP(H) pool sizes were developed. Supplementation of the feed medium with 95,mM adenine and 9.5,mM nicotinic acid resulted in an increase of the intracellular NAD(H) concentration by a factor of 10 at the end of the fed-batch process compared to the reference process. The final NAD(H) concentration remains unchanged if the feed medium was solely supplemented with 95,mM adenine, but intracellular NADP(H) was increased by a factor of 4. The effects of NADP(H) pool sizes on the asymmetric reduction of ethyl-4-chloro acetoacetate (CAAE) to the corresponding (S)-4-chloro-3-hydroxybutanoate (S-CHBE) was evaluated with S.,cerevisiae,FasB,His6 as an example. An intracellular threshold concentration above 0.07,mM NADP(H) was sufficient to increase the biocatalytic S-CHBE productivity by 25,% compared to lower intracellular NADP(H) concentrations. [source] Enhancement of the NAD(P)(H) Pool in Escherichia coli for BiotransformationENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2007F. Heuser Abstract In pyridine nucleotide-dependent, reductive whole cell biotransformation with resting cells of Escherichia coli, the availability of intracellular NAD(P)(H) is a pivotal point for an efficient and highly productive substrate conversion. The question whether an increase of the intracellular NAD(P)(H) concentration could increase the productivity was discussed controversially in the past. This is the first report on an E. coli strain with an increased NAD(P)(H) pool which was tested in a reductive biotransformation system for an increased productivity. Biotransformation was performed with a strain overexpressing a gene encoding an (R)-specific alcohol dehydrogenase for the stereospecific, NADPH-dependent reduction of methyl acetoacetate (MAA) to (R)-methyl-3-hydroxybutanoate (MHB). Cofactor regeneration was implemented via glucose oxidation by coexpression of a gene encoding glucose dehydrogenase. The specific MHB productivity (mmol mg,1 cell dry weight,1h,1) enabled a comparison between the E. coli,BL21(DE3) wild-type and a genetically modified strain. The enhancement of the NAD(P)(H) pool was achieved by genetic manipulation of the NAD(H) biosynthetic pathways. After simultaneous overexpression of the pncB and nadE genes, encoding nicotinic acid phosphoribosyltransferase and NAD synthetase, measurements of the total NAD(P)(H) pool, sizes showed a 7-fold and 2-fold increased intracellular concentration of NAD(H) and NADP(H), respectively. However, the implementation of an E.,coli strain carrying a genomically integrated pncB gene with an upstream T7,promoter for biotransformation did not result in reproducible increased specific cell productivity. [source] Comparative Study of Cyanobacteria as Biocatalysts for the Asymmetric Synthesis of Chiral Building BlocksENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 2 2006J. Havel Abstract The three representative cyanobacteria, Synechococcus PCC7942, Anabaena variabilis, and Nostoc muscorum, were studied for their ability to asymmetrically reduce the prochiral ketones 2,-3,-4,-5,-6,-pentafluoroacetophenone, ethyl 4-chloroacetate, 4-chloroacetophenone, and ethylbenzoylacetate to the corresponding chiral alcohols. Photosynthesis as well as respiration was applied for intracellular regeneration of the NAD(P)H cofactor. It was shown for the first time that all cyanobacteria were able to reduce the prochiral ketones asymmetrically without light for cofactor regeneration. By comparison of the cell specific product formation capacities of cyanobacteria with typical heterotrophic whole cell biocatalysts in batch processes, it is shown that comparable or, in some cases, better performances at high enantiomeric excess (ee > 99.8,%) are obtained. As a consequence of a generally strong product inhibition, in situ product removal must be applied in order to restore process efficiency when using cyanobacteria as biocatalysts. [source] Functional role of fumarate site Glu59 involved in allosteric regulation and subunit,subunit interaction of human mitochondrial NAD(P)+ -dependent malic enzymeFEBS JOURNAL, Issue 4 2009Ju-Yi Hsieh Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+ -dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, KA,Fum and ,KA,Fum. The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The KA,Fum and ,KA,Fum of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer,tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer,tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP+ -dependent malic enzyme and cytosolic NADP+ -dependent malic enzyme in fumarate activation and malate cooperativity. [source] Crystal structure of archaeal highly thermostable L -aspartate dehydrogenase/NAD/citrate ternary complexFEBS JOURNAL, Issue 16 2007Kazunari Yoneda The crystal structure of the highly thermostable l -aspartate dehydrogenase (l -aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidusl -aspDH was refined at a resolution of 1.9 Å with a crystallographic R -factor of 21.7% (Rfree = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidusl -aspDH/NAD/citrate ternary complex and the Thermotoga maritimal -aspDH/NAD binary complex showed that A. fulgidusl -aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritimal -aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidusl -aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two l -aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to l -aspDH and of the molecular basis of the high thermostability of a hyperthermophilic l -aspDH. [source] Biochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pyloriFEBS JOURNAL, Issue 20 2006Cong Han Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway. It catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate, and has been developed as a promising target for the discovery of antimicrobial agent. In this report, we identified a new aroE gene encoding SDH from Helicobacter pylori strain SS1. The recombinant H. pylori shikimate dehydrogenase (HpSDH) was cloned, expressed, and purified in Escherichia coli system. The enzymatic characterization of HpSDH demonstrates its activity with kcat of 7.7 s,1 and Km of 0.148 mm toward shikimate, kcat of 7.1 s,1 and Km of 0.182 mm toward NADP, kcat of 5.2 s,1 and Km of 2.9 mm toward NAD. The optimum pH of the enzyme activity is between 8.0 and 9.0, and the optimum temperature is around 60 °C. Using high throughput screening against our laboratory chemical library, five compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H -chromen-7-yl 3-chlorobenzoate (2), butyl 2-{[3-(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H -chromen-7-yl]oxy}propanoate (3), 2-({2-[(2-{[2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl}-1,3-benzothiazol-6-yl)amino]-2-oxoethyl}sulfanyl)- N -(2-naphthyl)acetamide (4), and maesaquinone diacetate (5) were discovered as HpSDH inhibitors with IC50 values of 15.4, 3.9, 13.4, 2.9, and 3.5 µm, respectively. Further investigation indicates that compounds 1, 2, 3, and 5 demonstrate noncompetitive inhibition pattern, and compound 4 displays competitive inhibition pattern with respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibition mode, and compounds 2 and 3 show competitive inhibition mode with respect to NADP. Antibacterial assays demonstrate that compounds 1, 2, and 5 can inhibit the growth of H. pylori with MIC of 16, 16, and 32 µg·mL,1, respectively. This current work is expected to favor better understanding the features of SDH and provide useful information for the development of novel antibiotics to treat H. pylori -associated infection. [source] Prediction of coenzyme specificity in dehydrogenases/ reductasesFEBS JOURNAL, Issue 6 2006A hidden Markov model-based method, its application on complete genomes Dehydrogenases and reductases are enzymes of fundamental metabolic importance that often adopt a specific structure known as the Rossmann fold. This fold, consisting of a six-stranded ,-sheet surrounded by ,-helices, is responsible for coenzyme binding. We have developed a method to identify Rossmann folds and predict their coenzyme specificity (NAD, NADP or FAD) using only the amino acid sequence as input. The method is based upon hidden Markov models and sequence pattern analysis. The prediction sensitivity is 79% and the selectivity close to 100%. The method was applied on a set of 68 genomes, representing the three kingdoms archaea, bacteria and eukaryota. In prokaryotes, 3% of the genes were found to code for Rossmann-fold proteins, while the corresponding ratio in eukaryotes is only around 1%. In all genomes, NAD is the most preferred cofactor (41,49%), followed by NADP with 30,38%, while FAD is the least preferred cofactor (21%). However, the NAD preponderance over NADP is most pronounced in archaea, and least in eukaryotes. In all three kingdoms, only 3,8% of the Rossmann proteins are predicted to have more than one membrane-spanning segment, which is much lower than the frequency of membrane proteins in general. Analysis of the major protein types in eukaryotes reveals that the most common type (26%) of the Rossmann proteins are short-chain dehydrogenases/reductases. In addition, the identified Rossmann proteins were analyzed with respect to further protein types, enzyme classes and redundancy. The described method is available at http://www.ifm.liu.se/bioinfo, where the preferred coenzyme and its binding region are predicted given an amino acid sequence as input. [source] Characterization of depolarization and repolarization phases of mitochondrial membrane potential fluctuations induced by tetramethylrhodamine methyl ester photoactivationFEBS JOURNAL, Issue 7 2005Angela M. Falchi Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were analyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochondrial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase). Succinic acid (mitochondrial complex II substrate, given in the permeable form of dimethyl ester) abolished the rotenone-induced inhibition of R phases. Taken together, these findings indicate that the activity of both respiratory chain and ATP synthase were required for the recovery of the mitochondrial potential. The frequency of D phases prevailed over that of R phases in all experimental conditions, resulting in a progressive depolarization of mitochondria accompanied by NAD(P)H oxidation and Ca2+ influx. D phases were not blocked by cyclosporin A (inhibitor of the permeability transition pore) or o -phenyl-EGTA (a Ca2+ chelator), suggesting that the permeability transition pore was not involved in mitochondrial potential fluctuations. [source] Kinetic study of sn -glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1FEBS JOURNAL, Issue 3 2002Jin-Suk Han A gene having high sequence homology (45,49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94,96 °C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H- dependent dihydroxyacetone phosphate reduction and NAD+ -dependent,glycerol-1-phosphate (Gro1P) oxidation. NADP+ -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi,bi mechanism. [source] A novel NADPH-dependent oxidoreductase with a unique domain structure in the hyperthermophilic Archaeon, Thermococcus litoralisFEMS MICROBIOLOGY LETTERS, Issue 1 2008András Tóth Abstract Thermococcus litoralis, a hyperthermophilic Archaeon, is able to reduce elemental sulfur during fermentative growth. An unusual gene cluster (nsoABCD) was identified in this organism. In silico analysis suggested that three of the genes (nsoABC) probably originated from Eubacteria and one gene (nsoD) from Archaea. The putative NsoA and NsoB are similar to NuoE- and NuoF-type electron transfer proteins, respectively. NsoC has a unique domain structure and contains a GltD domain, characteristic of glutamate synthase small subunits, which seems to be integrated into a NuoG-type sequence. Flavin and NAD(P)H binding sites and conserved cysteines forming iron,sulfur clusters binding motifs were identified in the protein sequences deduced. The purified recombinant NsoC contains one FAD cofactor per protein molecule and catalyzes the reduction of polysulfide with NADPH as an electron donor and it also reduces oxygen. It was concluded that the Nso complex is a new type of NADPH-oxidizing enzyme using sulfur and/or oxygen as an electron acceptor. [source] KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactisFEMS YEAST RESEARCH, Issue 8 2006Michele Saliola Abstract A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene. [source] Chromosome band 16q22-linked familial AML: Exclusion of candidate genes, and possible disease risk modification by NQO1 polymorphismsGENES, CHROMOSOMES AND CANCER, Issue 3 2004Robert Escher Analyses of chromosomal translocation and inversion breakpoints in sporadic acute myeloid leukemias have identified many transcription factors as playing a role in leukemogenesis. Studies of families with a Mendelian predisposition to hematological malignancies have identified the gene coding for the transcription factor RUNX1 as a leukemia-predisposing gene involved in the first steps of leukemogenesis. Using two families, another autosomal dominant familial leukemia locus was linked to chromosome band 16q22 where the CBFB gene maps. Although CBFB forms a core-binding factor transcriptional complex with RUNX1, previous analyses have excluded the CBFB gene as the leukemia-predisposing gene in these families. In the current study, we performed an extended molecular analysis in these families of the four other transcription factor genes in the 16q22 critical region as well as of two other genes with a known association with leukemia. Several previously undescribed but nonpathogenic sequence variants were identified. We demonstrated that the transcription factors E2F4, CTCF, NFATC3, and NFAT5, and the genes coding for NAD(P)H:quinone oxido-reductase 1 (NQO1) and for E-cadherin are not responsible for the leukemia susceptibility in these families. The presence of NQO1 polymorphisms may suggest a role for this gene in disease risk modification in these families. © 2004 Wiley-Liss, Inc. [source] Synthesis of Nicotinamide Adenine Dinucleotide (NAD) Analogues with a Sugar Modified Nicotinamide MoietyHELVETICA CHIMICA ACTA, Issue 7 2007Natasha Goulioukina Abstract The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described. [source] Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection,HEPATOLOGY, Issue 1 2010Nabora Soledad Reyes de Mochel Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV),induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGF,1). TGF,1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGF,1. Conclusion: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis. Hepatology 2010 [source] Indoleamine 2,3-dioxygenase in T-cell tolerance and tumoral immune escapeIMMUNOLOGICAL REVIEWS, Issue 1 2008Jessica B. Katz Summary: Indoleamine 2, 3-dioxygenase (IDO) degrades the essential amino acid tryptophan in mammals, catalyzing the initial and rate-limiting step in the de novo biosynthesis nicotinamide adenine dinucleotide (NAD). Broad evidence implicates IDO and the tryptophan catabolic pathway in generation of immune tolerance to foreign antigens in tissue microenvironments. In particular, recent findings have established that IDO is overexpressed in both tumor cells and antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the normal physiologic state, IDO is important in creating an environment that limits damage to tissues due to an overactive immune system. However, by fostering immune suppression, IDO can facilitate the survival and growth of tumor cells expressing unique antigens that would be recognized normally as foreign. In preclinical studies, small-molecule inhibitors of IDO can reverse this mechanism of immunosuppression, complementing classical cytotoxic cancer chemotherapeutic agents' ability to trigger regression of treatment-resistant tumors. These results have encouraged the clinical translation of IDO inhibitors, the first of which entered phase I clinical trials in the fall of 2007. In this article, we survey the work defining IDO as an important mediator of peripheral tolerance, review evidence of IDO dysregulation in cancer cells, and provide an overview of the development of IDO inhibitors as a new immunoregulatory treatment modality for clinical trials. [source] Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthmaIMMUNOLOGY, Issue 2 2006Marian L. Kruzel Summary Pollen grains contain reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases and in contact with mucosal surfaces generate superoxide anion (O2,,). In the presence of iron, O2,, may be converted to more reactive oxygen radicals, such as to H2O2 and/or ,OH, which may augment antigen-induced airway inflammation. The aim of the study was to examine the impact of lactoferrin (LF), an iron-binding protein, on ragweed (Ambrosia artemisiifolia) pollen extract (RWE)-induced cellular oxidative stress levels in cultured bronchial epithelial cells and accumulation of inflammatory and mucin-producing cells in airways in a mouse model of allergic airway inflammation. Results show that LF lowered RWE-induced increase in cellular reactive oxygen species (ROS) levels in bronchial epithelial cells. Most importantly, LF significantly decreased accumulation of eosinophils into airways and subepithelium of intranasally challenged, sensitized mice. LF also prevented development of mucin-producing cells. Amb a 1, the major allergenic ragweed pollen antigen lacking NAD(P)H oxidase activity, induced low-grade airway inflammation. When administered along with glucose oxidase (G-ox), a superoxide-generating enzyme, Amb a 1 induced robust airway inflammation, which was significantly lowered by LF. Surprisingly, LF decreased also inflammation caused by Amb a 1 alone. Iron-saturated hololactoferrin had only a marginal effect on RWE-induced cellular ROS levels and RWE- or Amb a 1 plus G-ox-induced inflammation. We postulate that free iron in the airways chemically reduces O2,, to more reactive species which augment antigen-induced inflammation in a mouse model of asthma. Our results suggest the utility of LF in human allergic inflammatory disorders. [source] Desorption kinetics of model polar stratospheric cloud films measured using Fourier Transform Infrared Spectroscopy and Temperature-Programmed DesorptionINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 5 2001Birgit G. Koehler This study combines Fourier transform infrared (FTIR) spectroscopy and temperature-programmed desorption to examine the evaporation kinetics of thin films of crystalline nitric acid hydrates, solid amorphous H2O/HNO3 mixtures, H2O,ice, ice coated with HCl, and solid HNO3. IR spectroscopy measured the thickness of each film as it evaporated, either at constant temperature or during a linear temperature ramp (temperature-programmed infrared, TPIR). Simultaneously, a mass spectrometer measured the rate of evaporation directly by monitoring the evolution of the molecules into the gas phase (temperature-programmed desorption, TPD). Both TPIR and TPD data provide a measurement of the desorption rate and yield the activation energy and preexponential factor for desorption. TPD measurements have the advantage of producing many data points but are subject to interference from experimental difficulties such as uneven heating from the edge of a sample and sample-support as well as pumping-speed limitations. TPIR experiments give clean but fewer data points. Evaporation occurred between 170 and 215 K for the various films. Ice evaporates with an activation energy of 12.9 ± 1 kcal/mol and a preexponential factor of 1 × 1032±1.5 molec/cm2 s, in good agreement with the literature. The beta form of nitric acid trihydrate, ,,NAT, has an Edes of 15.6 ± 2 kcal/mol with log A = 34.3 ± 2.3; the alpha form of nitric acid trihydrate, ,,NAT, is around 17.7 ± 3 kcal/mol with log A = 37.2 ± 4. For nitric acid dihydrate, NAD, Edes is 17.3 ± 2 kcal/mol with log A = 35.9 ± 2.6; for nitric acid monohydrate, NAM, Edes is 13 ± 3 kcal/mol with log A = 31.4 ± 3. The ,,NAT converts to ,,NAT during evaporation, and the amorphous solid H2O/HNO3 mixtures crystallize during evaporation. The barrier to evaporation for pure nitric acid is 14.6 ± 3 kcal/mol with log A = 34.4 ± 3. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 33: 295,309, 2001 [source] A New Regeneration System for Oxidized Nicotinamide CofactorsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 9 2009Seda Aksu Abstract A novel regeneration system for oxidized nicotinamide cofactors (NAD+ and NADP+) is presented. By combining 2,2,-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)-catalyzed oxidation of NAD(P)H with laccase-catalyzed utilization of molecular oxygen as terminal oxidant, a simple chemo-enzymatic NAD(P)+ regeneration method is achieved. Thus, the advantages of both worlds, chemical oxidation of reduced nicotinamide cofactors and laccase-catalyzed utilization of oxygen from air are combined in a simple and generally applicable new approach for biooxidation catalysis. This new application of the well-known laccase-mediator system (LMS) is successfully used to promote alcohol dehydrogenase-catalyzed oxidation reactions of primary and secondary alcohols. Already under non-optimized conditions, high turnover numbers of >300 and >16000 were obtained for the nicotinamide cofactor and ABTS, respectively. In this communication, we present the proof-of-principle and initial characterization of the proposed new regeneration system. [source] Structure and signaling in polyps of a colonial hydroidINVERTEBRATE BIOLOGY, Issue 1 2004Neil W. Blackstone Abstract. After feeding, polyps of colonial hydroids contract regularly, dispersing food throughout the colony via the gastrovascular fluid. Such contractions may trigger signaling pathways that allow colonies to grow in an adaptive manner, i.e., to initiate development of more polyps in food-rich areas and to suppress polyp development in food-poor areas. In this context, we investigated the structure and potential signaling of the junction between polyps and stolons in colonies of the hydroid Podocoryna carnea. Using transmission electron microscopy, we found that the density of mitochondrion-rich epitheliomuscular cells was low in polyp and stolon tissues except at or near the polyp-stolon junction, where many of these mitochondrion-rich cells occur in ectodermal tissue. In vivo fluorescence microscopy suggests that these mitochondria are a principal source of the metabolic signals of the colony. Both native fluorescence of NAD(P)H and fluorescence from peroxides (visualized with H2DCFDA) co-localize to this region of the polyp. Rhodamine 123 fluorescence suggests that both these metabolic signals emanate from mitochondria. To test whether such metabolic signals may be involved in colony pattern formation, inbred lines of P. carnea were used. Colonies of a runner-like inbred line grow with widely spaced polyps and long stolonal connections, much like wild-type colonies in a food-poor environment. Colonies of a sheet-like inbred line grow with closely spaced polyps and short stolonal connections, similar to wild-type colonies in a food-rich environment. Polyp-stolon junctions in runner-like and sheet-like colonies were imaged for the fluorescence of H2DCFDA. Densitometric analysis of this signal indicates that the mitochondria in epitheliomuscular cells of runner-like polyps emit greater amounts of peroxides. Because peroxides and other reactive oxygen species are frequently intermediaries in metabolic signaling pathways, we suspect that such signaling may indeed occur at polyp-stolon junctions, affecting colony pattern formation in these inbred lines and possibly in hydroid colonies in general. [source] Structure,function studies of glutamate synthases: A class of self-regulated iron-sulfur flavoenzymes essential for nitrogen assimilationIUBMB LIFE, Issue 5 2008Maria Antonietta Vanoni Abstract Glutamate synthases play with glutamine synthetase an essential role in nitrogen assimilation processes in microorganisms, plants, and lower animals by catalyzing the net synthesis of one molecule of L -glutamate from L -glutamine and 2-oxoglutarate. They exhibit a modular architecture with a common subunit or region, which is responsible for the L -glutamine-dependent glutamate synthesis from 2-oxoglutarate. Here, a PurF- (Type II- or Ntn-) type amidotransferase domain is coupled to the synthase domain, a (,/,)8 barrel containing FMN and one [3Fe-4S]0,+1 cluster, through a ,30 Å-long intramolecular tunnel for the transfer of ammonia between the sites. In bacterial and eukaryotic GltS, reducing equivalents are provided by reduced pyridine nucleotides thanks to the stable association with a second subunit or region, which acts as a FAD-dependent NAD(P)H oxidoreductase and is responsible for the formation of the two low potential [4Fe-4S]+1,+2 clusters of the enzyme. In photosynthetic cells, reduced ferredoxin is the physiological reductant. This review focus on the mechanism of cross-activation of the synthase and glutaminase reactions in response to the bound substrates and the redox state of the enzyme cofactors, as well as on recent information on the structure of the ,, protomer of the NADPH-dependent enzyme, which sheds light on the intramolecular electron transfer pathway between the flavin cofactors. © 2008 IUBMB IUBMB Life, 60(5): 287,300, 2008 [source] Catalytic mechanism and substrate selectivity of aldo-keto reductases: Insights from structure-function studies of Candida tenuis xylose reductaseIUBMB LIFE, Issue 9 2006Regina Kratzer Abstract Aldo-keto reductases (AKRs) constitute a large protein superfamily of mainly NAD(P)-dependent oxidoreductases involved in carbonyl metabolism. Catalysis is promoted by a conserved tetrad of active site residues (Tyr, Lys, Asp and His). Recent results of structure-function relationship studies for xylose reductase (AKR2B5) require an update of the proposed catalytic mechanism. Electrostatic stabilization by the ,-NH3+ group of Lys is a key source of catalytic power of xylose reductase. A molecular-level analysis of the substrate binding pocket of xylose reductase provides a case of how a very broadly specific AKR achieves the requisite selectivity for its physiological substrate and could serve as the basis for the design of novel reductases with improved specificities for biocatalytic applications. iubmb Life, 58: 499-507, 2006 [source] Asymmetric Carbonyl Reductions with Microbial KetoreductasesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 14-15 2008Menno Abstract The biocatalytic reduction of ,-keto esters and some aromatic ketones in the presence of a variety of ketoreductases from different microbial origins was investigated. The prochiral selectivity was generally high and both product enantiomers could be obtained by a proper choice of enzyme. Aromatic ketones reacted slower than the esters but the prochiral selectivity was often high. The organic cosolvent tolerance of these enzymes was rather variable but useful activity could be maintained in a number of cases. Reduction of the oxidized cofactors NAD and NADP, employing 2-propanol as a sacrificial reductant, was catalyzed by the ketoreductases from Rhodococcus erythropolis and Lactobacillus kefir, respectively. [source] Catalytic Hydroxylation in Biphasic Systems using CYP102A1 MutantsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 7-8 2005Steffen Abstract Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. Their technical application is, however, limited to a small number of whole-cell processes. The use of the isolated P450 enzymes is believed to be impractical due to their low stability, stoichiometric need of the expensive cofactor NAD(P)H and low solubility of most substrates in aqueous media. We investigated the behaviour of an isolated bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with the NADP+ -dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental set-up cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent mutant of CYP102A1 was designed. For recycling of NADH an NAD+ -dependent FDH was used. The stability of the monooxygenase mutants under the reaction conditions in the biphasic system was quite high as revealed by total turnover numbers of up to 12,850 in the NADPH-dependent cyclohexane hydroxylation and up to 30,000 in the NADH-dependent myristic acid oxidation. [source] Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2001F. Mozzi Aims: The objective of this work was to study the influence of the sugar source on exopolysaccharide (EPS) production and the activities of the enzymes involved in the synthesis of sugar nucleotides in Lactobacillus casei CRL 87. The relationship between these enzymes and EPS formation was determined. Methods and Results: The concentration of EPS was estimated by the phenol/sulphuric acid method while the chemical composition of purified EPS was investigated using gas-liquid chromatography. Biosynthetic enzyme activities were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. Polysaccharide production by Lb. casei CRL 87 was 1·7 times greater on galactose than on glucose. The isolated polymer was composed of rhamnose, glucose and galactose. The activities of uridine-diphosphate (UDP)-glucose-pyrophosphorylase, thymidine-diphosphate (dTDP)-glucose-pyrophosphorylase and the dTDP-rhamnose-synthetic enzyme system were higher in galactose-grown than in glucose-grown cells. When an EPS, mutant strain was used, galactokinase activity was not detected on galactose, this sugar not being available for the formation of sugar nucleotides for further EPS production. dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities were lower than the values found for the wild type strain. Conclusions: The carbon source present in the culture medium affects EPS production by Lb. casei CRL 87. The greater polymer synthesis by galactose-grown cells is correlated with the higher UDP-glucose-pyrophosphorylase, dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities. Initial sugar metabolism is also an important step for the synthesis of EPS precursors by this strain. Significance and Impact of the Study: Knowledge of the effect of the sugar source on EPS production and the activities of biosynthetic enzymes provides information about the mechanisms of regulation of the synthesis of EPS which can contribute to improving polymer production. [source] Gender divergent expression of Nqo1 in Sprague Dawley and August Copenhagen x Irish ratsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2008Lisa M. Augustine In the mammalian liver, there is an abundance of enzymes that function to enable the safe and efficient elimination of potentially harmful xenobiotics that are encountered through environmental exposure. A variety of factors, including gender and genetic polymorphisms, contribute to the variation between an individual system's detoxification capacity and thus its ability to protect itself against oxidative stress, cellular damage, cell death, etc. NAD(P)H:quinone oxidoreducatase 1 (Nqo1) is an antioxidant enzyme that plays a major role in reducing reactive electrophiles, thereby protecting cells from free-radical damage and oxidative stress. The goal of this study was to determine the gender-specific expression and inducibility of Nqo1 in the Sprague Dawley (SD) and August Copenhagen x Irish (ACI) rat strains, two strains that are commonly used in drug metabolism and drug-induced enzyme induction, toxicity, and carcinogenesis studies. Nqo1 mRNA, protein, and activity levels were determined through 96 h in SD and ACI males and females following treatment with known Nqo1 inducers oltipraz and butylated hydroxyanisole. In the SD strain, gender dimorphic expression of Nqo1 was observed with female mRNA, protein, and activity levels being significantly higher than in males. In contrast, there were minimal differences in Nqo1 mRNA, protein, and activity levels between ACI males and females. The gender dimorphic expression of Nqo1 in the SD rats was maintained through the course of induction, with female-induced levels greater than male-induced levels indicating that SD females may have a greater capacity to protect against oxidative stress and thus a decreased susceptibility to carcinogens. © 2008 Wiley Periodicals, Inc. J Biochem Mol Toxicol 22:93,100, 2008; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20224 [source] Hepatic chemoprotective enzyme responses to 2-substituted selenazolidine-4(R)-carboxylic acidsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2006Wael M. El-Sayed Abstract In epidemiology and human supplementation studies, as well as many animal models, selenium has shown antitumorigenic activity. The mechanism of action, however, has not been satisfactorily resolved. Selenium supplementation affects many enzymes in addition to those where selenocysteine is an essential component. Such enzymes include cytoprotective detoxifying enzymes, and the regulation of these enzymes by a set of 2-substituted selenazolidine-4(R)-carboxylic acids (SCAs) has been investigated. Following seven consecutive daily doses of these prodrugs of L -selenocysteine, changes in hepatic enzyme activities and/or mRNA levels of glutathione transferase (GST), microsomal epoxide hydrolase (mEH), NAD(P)H-quinone oxidoreductase (NQO), UDP-glucuronosyltransferase (UGT), glutathione peroxidase (GPx), and thioredoxin reductase (TR) have been observed. Among the enzymes examined, UGTs and GPx were found to be the least affected. Among the compounds, 2-oxoSCA produced the most changes and 2-phenylSCA produced the least, none. For no two compounds was the pattern of changes identical, and for a single compound, few changes were reproduced in common by the two routes of administration investigated. In general, more changes were elicited following intraperitoneal (i.p.) administration than with the intragastric (i.g.) route. This dominance was typified by 2-butylSCA and 2-cyclohexylSCA where enzyme activity elevations (TR and mEH with both, NQO with 2-butylSCA) were seen only with the i.p. route. With 2-oxoSCA, however, GST, TR, and NQO activities were found to be elevated independent of route. Only with GST (both routes) and TR (i.p. route), elevations in mRNAs accompanied the 2-oxoSCA elicited elevations of activities at the time of sacrifice. For some enzymes, most notably mEH with compounds administered i.p., elevations in mRNAs were not manifest as increased enzyme activity. Thus, although constituting a closely related series of compounds, each 2-substituted SCA produced its own unique pattern of changes, and for most members, changes were predominant following i.p. administration. © 2006 Wiley Periodicals, Inc. J Biochem Mol Toxicol 20:292,301, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20148 [source] Point of View: Could glucose be a proaging factor?JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2008Eva Kassi Abstract There is an ever-increasing scientific interest for the interplay between cell's environment and the aging process. Although it is known that calorie restriction affects longevity, the exact molecular mechanisms through which nutrients influence various cell signalling/modulators of lifespan remain a largely unresolved issue. Among nutrients, glucose constitutes an evolutionarily stable, precious metabolic fuel, which is catabolized through glycolytic pathway providing energy in the form of ATP and consuming NAD. Accumulating evidence shows that among the important regulators of aging process are autophagy, sirtuin activity and oxidative stress. In light of recent work indicating that glucose availability decreases lifespan whilst impaired glucose metabolism extends life expectancy, the present article deals with the potential role of glucose in the aging process by regulating , directly through its metabolism or indirectly through insulin secretion , autophagy, sirtuins as well as other modulators of aging like oxidative stress and advanced glycation end-products (AGEs). [source] | |||||||||||||||||||||||||||||||