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

PubMed

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

2014-11-01

A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety.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

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

PubMed

Logemann, E; Reinold, S; Somssich, I E; Hahlbrock, K

1997-08-01

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

Alcoholism and alcohol drinking habits predicted from alcohol dehydrogenase genes.

PubMed

Tolstrup, Janne Schurmann; Nordestgaard, Børge Grønne; Rasmussen, Søren; Tybjaerg-Hansen, Anne; Grønbaek, Morten

2008-06-01

Alcohol drinking habits and alcoholism are partly genetically determined. Alcohol is degraded primarily by alcohol dehydrogenase (ADH) wherein genetic variation that affects the rate of alcohol degradation is found in ADH1B and ADH1C. It is biologically plausible that these variations may be associated with alcohol drinking habits and alcoholism. By genotyping 9080 white men and women from the general population, we found that men and women with ADH1B slow vs fast alcohol degradation drank more alcohol and had a higher risk of everyday drinking, heavy drinking, excessive drinking and of alcoholism. For example, the weekly alcohol intake was 9.8 drinks (95% confidence interval (CI): 9.1-11) among men with the ADH1B.1/1 genotype compared to 7.5 drinks (95% CI: 6.4-8.7) among men with the ADH1B.1/2 genotype, and the odds ratio (OR) for heavy drinking was 3.1 (95% CI: 1.7-5.7) among men with the ADH1B.1/1 genotype compared to men with the ADH1B.1/2 genotype. Furthermore, individuals with ADH1C slow vs fast alcohol degradation had a higher risk of heavy and excessive drinking. For example, the OR for heavy drinking was 1.4 (95% CI: 1.1-1.8) among men with the ADH1C.1/2 genotype and 1.4 (95% CI: 1.0-1.9) among men with the ADH1B.2/2 genotype, compared with men with the ADH1C.1/1 genotype. Results for ADH1B and ADH1C genotypes among men and women were similar. Finally, because slow ADH1B alcohol degradation is found in more than 90% of the white population compared to less than 10% of East Asians, the population attributable risk of heavy drinking and alcoholism by ADH1B.1/1 genotype was 67 and 62% among the white population compared with 9 and 24% among the East Asian population.

Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase.

PubMed Central

Modig, Tobias; Lidén, Gunnar; Taherzadeh, Mohammad J

2002-01-01

The kinetics of furfural inhibition of the enzymes alcohol dehydrogenase (ADH; EC 1.1.1.1), aldehyde dehydrogenase (AlDH; EC 1.2.1.5) and the pyruvate dehydrogenase (PDH) complex were studied in vitro. At a concentration of less than 2 mM furfural was found to decrease the activity of both PDH and AlDH by more than 90%, whereas the ADH activity decreased by less than 20% at the same concentration. Furfural inhibition of ADH and AlDH activities could be described well by a competitive inhibition model, whereas the inhibition of PDH was best described as non-competitive. The estimated K(m) value of AlDH for furfural was found to be about 5 microM, which was lower than that for acetaldehyde (10 microM). For ADH, however, the estimated K(m) value for furfural (1.2 mM) was higher than that for acetaldehyde (0.4 mM). The inhibition of the three enzymes by 5-hydroxymethylfurfural (HMF) was also measured. The inhibition caused by HMF of ADH was very similar to that caused by furfural. However, HMF did not inhibit either AlDH or PDH as severely as furfural. The inhibition effects on the three enzymes could well explain previously reported in vivo effects caused by furfural and HMF on the overall metabolism of Saccharomyces cerevisiae, suggesting a critical role of these enzymes in the observed inhibition. PMID:11964178

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

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.

A Long-Chain Secondary Alcohol Dehydrogenase from Rhodococcus erythropolis ATCC 4277

PubMed Central

Ludwig, B.; Akundi, A.; Kendall, K.

1995-01-01

A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols. PMID:16535152

Changes in cinnamyl alcohol dehydrogenase activities from sugarcane cultivars inoculated with Sporisorium scitamineum sporidia.

PubMed

Santiago, Rocío; Alarcón, Borja; de Armas, Roberto; Vicente, Carlos; Legaz, María Estrella

2012-06-01

This study describes a method for determining cinnamyl alcohol dehydrogenase activity in sugarcane stems using reverse phase (RP) high-performance liquid chromatography to elucidate their possible lignin origin. Activity is assayed using the reverse mode, the oxidation of hydroxycinnamyl alcohols into hydroxycinnamyl aldehydes. Appearance of the reaction products, coniferaldehyde and sinapaldehyde is determined by measuring absorbance at 340 and 345 nm, respectively. Disappearance of substrates, coniferyl alcohol and sinapyl alcohol is measured at 263 and 273 nm, respectively. Isocratic elution with acetonitrile:acetic acid through an RP Mediterranea sea C18 column is performed. As case examples, we have examined two different cultivars of sugarcane; My 5514 is resistant to smut, whereas B 42231 is susceptible to the pathogen. Inoculation of sugarcane stems elicits lignification and produces significant increases of coniferyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD). Production of lignin increases about 29% in the resistant cultivar and only 13% in the susceptible cultivar after inoculation compared to uninoculated plants. Our results show that the resistance of My 5514 to smut is likely derived, at least in part, to a marked increase of lignin concentration by the activation of CAD and SAD. Copyright © Physiologia Plantarum 2012.

Alcohol Dehydrogenase and Ethanol in the Stems of Trees 1

PubMed Central

Kimmerer, Thomas W.; Stringer, Mary A.

1988-01-01

Anaerobic fermentation in plants is usually thought to be a transient phenomenon, brought about by environmental limitations to oxygen availability, or by structural constraints to oxygen transport. The vascular cambium of trees is separated from the air by the outer bark and secondary phloem, and we hypothesized that the cambium may experience sufficient hypoxia to induce anaerobic fermentation. We found high alcohol dehydrogenase activity in the cambium of several tree species. Mean activity of alcohol dehydrogenase in Populus deltoides was 165 micromoles NADH oxidized per minute per gram fresh weight in May. Pyruvate decarboxylase activity was also present in the cambium of P. deltoides, with mean activity of 26 micromoles NADH oxidized per minute per gram fresh weight in May. Lactate dehydrogenase activity was not present in any tree species we examined. Contrary to our expectation, alcohol dehydrogenase activity was inversely related to bark thickness in Acer saccharum and unrelated to bark thickness in two Populus species. Bark thickness may be less important in limiting oxygen availability to the cambium than is oxygen consumption by rapidly respiring phloem and cambium in actively growing trees. Ethanol was present in the vascular cambium of all species examined, with mean concentrations of 35 to 143 nanomoles per gram fresh weight, depending on species. Ethanol was also present in xylem sap and may have been released from the cambium into the transpiration stream. The presence in the cambium of the enzymes necessary for fermentation as well as the products of fermentation is evidence that respiration in the vascular cambium of trees may be oxygen-limited, but other biosynthetic origins of ethanol have not been ruled out. PMID:16666209

Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration

PubMed Central

Rachamin, Gloria; Macdonald, J. Alain; Wahid, Samina; Clapp, Jeremy J.; Khanna, Jatinder M.; Israel, Yedy

1980-01-01

In young (4-week-old) male and female spontaneously hypertensive (SH) rats, ethanol metabolic rate in vivo and hepatic alcohol dehydrogenase activity in vitro are high and not different in the two sexes. In males, ethanol metabolic rate falls markedly between 4 and 10 weeks of age, which coincides with the time of development of sexual maturity in the rat. Alcohol dehydrogenase activity is also markedly diminished in the male SH rat and correlates well with the changes in ethanol metabolism. There is virtually no influence of age on ethanol metabolic rate and alcohol dehydrogenase activity in the female SH rat. Castration of male SH rats prevents the marked decrease in ethanol metabolic rate and alcohol dehydrogenase activity, whereas ovariectomy has no effect on these parameters in female SH rats. Chronic administration of testosterone to castrated male SH rats and to female SH rats decreases ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in mature males. Chronic administration of oestradiol-17β to male SH rats results in marked stimulation of ethanol metabolic rate and alcohol dehydrogenase activity to values similar to those found in female SH rats. Chronic administration of ethanol to male SH rats from 4 to 11 weeks of age prevents the marked age-dependent decreases in ethanol metabolic rate and alcohol dehydrogenase activity, but has virtually no effect in castrated rats. In the intoxicated chronically ethanol-fed male SH rats, serum testosterone concentrations are significantly depressed. In vitro, testosterone has no effect on hepatic alcohol dehydrogenase activity of young male and female SH rats. In conclusion, in the male SH rat, ethanol metabolic rate appears to be limited by alcohol dehydrogenase activity and is modulated by testosterone. Testosterone has an inhibitory effect and oestradiol has a testosterone-dependent stimulatory effect on alcohol dehydrogenase activity and ethanol metabolic rate in these

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

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. PMID:15829607

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

PubMed Central

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

1993-01-01

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

Characteristics of butanol metabolism in alcohol dehydrogenase-deficient deermice.

PubMed Central

Alderman, J A; Kato, S; Lieber, C S

1989-01-01

Deermice lacking the low-Km alcohol dehydrogenase eliminated butan-1-ol, a substrate for microsomal oxidation but not for catalase, at 117 mumol/min per kg body wt. Microsomal fractions and hepatocytes metabolized butan-1-ol also (Vmax. = 6.7 nmol/min per nmol of cytochrome P-450, Km = 0.85 mM; Vmax. = 5.3 nmol/min per 10(6) cells, Km = 0.71 mM respectively). These results are consistent with alcohol oxidation by the microsomal system in these deermice. PMID:2930472

The Activity of Class I-IV Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Bladder Cancer Cells.

PubMed

Orywal, Karolina; Jelski, Wojciech; Werel, Tadeusz; Szmitkowski, Maciej

2018-01-02

The aim of this study was to determine the differences in the activity of Alcohol Dehydrogenase (ADH) isoenzymes and Aldehyde Dehydrogenase (ALDH) in normal and cancerous bladder cells. Class III, IV of ADH and total ADH activity were measured by the photometric method and class I, II ADH and ALDH activity by the fluorometric method. Significantly higher total activity of ADH was found in both, low-grade and high-grade bladder cancer, in comparison to healthy tissues. The increased activity of total ADH in bladder cancer cells may be the cause of metabolic disorders in cancer cells, which may intensify carcinogenesis.

Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol.

PubMed

Schlager, S; Neugebauer, H; Haberbauer, M; Hinterberger, G; Sariciftci, N S

2015-03-01

Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO 2 -reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate-silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO 2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO 2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %.

GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.

PubMed

Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

2006-03-01

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

Salivary alcohol dehydrogenase in non-smoking and smoking alcohol-dependent persons.

PubMed

Waszkiewicz, Napoleon; Jelski, Wojciech; Zalewska, Anna; Szulc, Agata; Szmitkowski, Maciej; Zwierz, Krzysztof; Szajda, Sławomir Dariusz

2014-09-01

Increasing attention to the importance of saliva testing is not surprising because smoking and alcohol drinking act synergistically on oral tissues, and their metabolite levels, e.g., acetaldehyde, are much higher in saliva than in blood. The activity of salivary alcohol dehydrogenase (ADH) comes from oral microbiota, mucosa, and salivary glands. The purpose of this study was to investigate the involvement of ADH in the oral health pathology of smoking (AS) and non-smoking (ANS) alcohol-dependent males. The results indicated that the AS group had a more significant and longer duration (until the 30th day of alcohol abstinence) decrease in ADH activity and output than the ANS group (until the 15th day of alcohol abstinence) compared to controls (social drinkers; C). The decreased salivary flow (SF) in alcoholics was observed longer in the ANS group (until the 30th day of alcohol abstinence), whereas in the AS group SF normalized at the 15th day, probably due to the irritating effect of tobacco smoke on the oral mucosa. Because saliva was centrifuged to remove cells and debris (including microbial cells), the detected salivary ADH activity was derived from salivary glands and/or oral mucosa. A more profound and longer decrease in ADH activity/output in smoking than non-smoking alcoholics was likely due to the damaged salivary glands and/or oral mucosa, caused by the synergistic effect of alcohol drinking and smoking. The lower values of salivary ADH in smoking than non-smoking alcoholics might also be partly due to the reversed/inhibited ADH reaction by high levels of accumulated acetaldehyde. Copyright © 2014 Elsevier Inc. All rights reserved.

Polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 and the blood and salivary ethanol and acetaldehyde concentrations of Japanese alcoholic men.

PubMed

Yokoyama, Akira; Tsutsumi, Eri; Imazeki, Hiromi; Suwa, Yoshihide; Nakamura, Chizu; Yokoyama, Tetsuji

2010-07-01

The effects of genetic polymorphism of aldehyde dehydrogenase-2 (ALDH2) on alcohol metabolism are striking in nonalcoholics, and the effects of genetic polymorphism of alcohol dehydrogenase-1B (ADH1B) are modest at most, whereas genetic polymorphisms of both strongly affect the susceptibility to alcoholism and upper aerodigestive tract (UADT) cancer of drinkers. We evaluated associations between ADH1B/ADH1C/ALDH2 genotypes and the blood and salivary ethanol and acetaldehyde levels of 168 Japanese alcoholic men who came to our hospital for the first time in the morning and had been drinking until the day before. The ethanol levels in their blood and saliva were similar, but the acetaldehyde levels in their saliva were much higher than in their blood, probably because of acetaldehyde production by oral bacteria. Blood and salivary ethanol and acetaldehyde levels were both significantly higher in the subjects with the less active ADH1B*1/*1 genotype than in the ADH1B*2 carriers, but none of the levels differed according to ALDH2 genotype. Significant linkage disequilibrium was detected between the ADH1B and ADH1C genotypes, but ADH1C genotype did not affect the blood or salivary ethanol or acetaldehyde levels. High blood acetaldehyde levels were found even in the active ALDH2*1/*1 alcoholics, which were comparable with the levels of the inactive heterozygous ALDH2*1/*2 alcoholics with less active ADH1B*1/*1. The slope of the increase in blood acetaldehyde level as the blood ethanol level increased was significantly steeper in alcoholics with inactive heterozygous ALDH2*1/*2 plus ADH1B*2 allele than with any other genotype combinations, but the slopes of the increase in salivary acetaldehyde level as the salivary ethanol level increased did not differ between the groups of subjects with any combinations of ALDH2 and ADH1B genotypes. The ADH1B/ALDH2 genotype affected the blood and salivary ethanol and acetaldehyde levels of nonabstinent alcoholics in a different manner

Heat-stable, FE-dependent alcohol dehydrogenase for aldehyde detoxification

DOEpatents

Elkins, James G.; Clarkson, Sonya

2018-04-24

The present invention relates to microorganisms and polypeptides for detoxifying aldehydes associated with industrial fermentations. In particular, a heat-stable, NADPH- and iron-dependent alcohol dehydrogenase was cloned from Thermoanaerobacter pseudethanolicus 39E and displayed activity against a number of aldehydes including inhibitory compounds that are produced during the dilute-acid pretreatment process of lignocellulosic biomass before fermentation to biofuels. Methods to use the microorganisms and polypeptides of the invention for improved conversion of bio mass to biofuel are provided as well as use of the enzyme in metabolic engineering strategies for producing longer-chain alcohols from sugars using thermophilic, fermentative microorganisms.

Increasing anaerobic acetate consumption and ethanol yields in Saccharomyces cerevisiae with NADPH-specific alcohol dehydrogenase.

PubMed

Henningsen, Brooks M; Hon, Shuen; Covalla, Sean F; Sonu, Carolina; Argyros, D Aaron; Barrett, Trisha F; Wiswall, Erin; Froehlich, Allan C; Zelle, Rintze M

2015-12-01

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter(-1) acetate during fermentation of 114 g liter(-1) glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter(-1), this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter(-1) and raised the ethanol yield to 7% above the wild-type level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

PubMed Central

Leferink, Nicole G. H.; Hendriks, Annemarie; Brouns, Stan J. J.; Hennemann, Hans-Georg; Dauβmann, Thomas; van der Oost, John

2008-01-01

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30°C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30°C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of ∼1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30°C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183. PMID:18452026

Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis

DOEpatents

Ingram, Lonnie O.; Conway, Tyrrell

1992-01-01

The alcohol dehydrogenase II gene from Zymomonas mobilis has been cloned and sequenced. This gene can be expressed at high levels in other organisms to produce acetaldehyde or to convert acetaldehyde to ethanol.

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins.

PubMed

Lapierre, Catherine; Pilate, Gilles; Pollet, Brigitte; Mila, Isabelle; Leplé, Jean-Charles; Jouanin, Lise; Kim, Hoon; Ralph, John

2004-02-01

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.

Contribution of liver alcohol dehydrogenase to metabolism of alcohols in rats.

PubMed

Plapp, Bryce V; Leidal, Kevin G; Murch, Bruce P; Green, David W

2015-06-05

The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5-20 mmol/kg. Ethanol was eliminated most rapidly, at 7.9 mmol/kgh. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5-10 mmol/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmol/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6±1 mmol/kg h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD(+) for the conversion to ketones whereas primary alcohols require two equivalents of NAD(+) for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD(+) is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Contribution of Liver Alcohol Dehydrogenase to Metabolism of Alcohols in Rats

PubMed Central

Plapp, Bryce V.; Leidal, Kevin G.; Murch, Bruce P.; Green, David W.

2015-01-01

The kinetics of oxidation of various alcohols by purified rat liver alcohol dehydrogenase (ADH) were compared with the kinetics of elimination of the alcohols in rats in order to investigate the roles of ADH and other factors that contribute to the rates of metabolism of alcohols. Primary alcohols (ethanol, 1-propanol, 1-butanol, 2-methyl-1-propanol, 3-methyl-1-butanol) and diols (1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 1,5-pentanediol) were eliminated in rats with zero-order kinetics at doses of 5–20 mmole/kg. Ethanol was eliminated most rapidly, at 7.9 mmole/kg•h. Secondary alcohols (2-propanol-d7, 2-propanol, 2-butanol, 3-pentanol, cyclopentanol, cyclohexanol) were eliminated with first order kinetics at doses of 5–10 mmole/kg, and the corresponding ketones were formed and slowly eliminated with zero or first order kinetics. The rates of elimination of various alcohols were inhibited on average 73% (55% for 2-propanol to 90% for ethanol) by 1 mmole/kg of 4-methylpyrazole, a good inhibitor of ADH, indicating a major role for ADH in the metabolism of the alcohols. The Michaelis kinetic constants from in vitro studies (pH 7.3, 37 °C) with isolated rat liver enzyme were used to calculate the expected relative rates of metabolism in rats. The rates of elimination generally increased with increased activity of ADH, but a maximum rate of 6 ± 1 mmole/kg•h was observed for the best substrates, suggesting that ADH activity is not solely rate-limiting. Because secondary alcohols only require one NAD+ for the conversion to ketones whereas primary alcohols require two equivalents of NAD+ for oxidation to the carboxylic acids, it appears that the rate of oxidation of NADH to NAD+ is not a major limiting factor for metabolism of these alcohols, but the rate-limiting factors are yet to be identified. PMID:25641189

Alcohol and aldehyde dehydrogenase gene polymorphisms influence susceptibility to esophageal cancer in Japanese alcoholics.

PubMed

Yokoyama, A; Muramatsu, T; Omori, T; Matsushita, S; Yoshimizu, H; Higuchi, S; Yokoyama, T; Maruyama, K; Ishii, H

1999-11-01

Studies have consistently demonstrated that inactive aldehyde dehydrogenase-2 (ALDH2), encoded by ALDH2*1/2*2, is closely associated with alcohol-related carcinogenesis. Recently, the contributions of alcohol dehydrogenase-2 (ADH2) polymorphism to alcoholism, esophageal cancer, and the flushing response have also been described. To determine the effects of ALDH2 and ADH2 genotypes in genetically based cancer susceptibility, lymphocyte DNA samples from 668 Japanese alcoholic men more than 40 years of age (91 with and 577 without esophageal cancer) were genotyped and the results were expressed as odds ratios (ORs). This study also tested 82 of the alcoholics with esophageal cancer to determine whether cancer susceptibility is associated with patients' responses to simple questions about current or former flushing after drinking a glass of beer. The frequencies of ADH2*1/2*1 and ALDH2*1/2*2 were significantly higher in alcoholics with, than in those without, esophageal cancer (0.473 vs. 0.289 and 0.560 vs. 0.099, respectively). After adjustment for drinking and smoking, the analysis showed significantly increased cancer risk for alcoholics with either ADH2*1/2*I (OR = 2.03) or ALDH2*1/2*2 (OR = 12.76). For those having ADH2*1/2*1 combined with ALDH2*1/2*2, the esophageal cancer risk was enhanced in a multiplicative fashion (OR = 27.66). Responses to flushing questions showed that only 47.8% of the ALDH2*1/2*2 heterozygotes with ADH2*1/ 2*1, compared with 92.3% of those with ALDH2*1/2*2 and the ADH2*2 allele, reported current or former flushing. Genotyping showed that for alcoholics who reported ever flushing, the questionnaire was 71.4% correct in identifying ALDH2*1/2*2 and 87.9% correct in identifying ALDH2*1/2*1. Japanese alcoholics can be divided into cancer susceptibility groups on the basis of their combined ADH2 and ALDH2 genotypes. The flushing questionnaire can predict high risk ALDH2*1/2*2 fairly accurately in persons with ADH2*2 allele, but a reliable

[Polymorphism of alcohol dehydrogenase gene ADH1B in eastern Slavic and Iranian-speaking populations].

PubMed

2005-11-01

Frequencies of alleles and genotypes for alcohol dehydrogenase gene ADH1B (arg47his polymorphism), associated with alcohol tolerance/sensitivity, were determined. It was demonstrated that the frequency of allele ADH1B*47his, corresponding to atypical alcohol dehydrogenase variant in Russians, Ukrainians, Iranians, and mountain-dwellers of the Pamirs constituted 3, 7, 24, and 22%, respectively. The frequencies established were consistent with the allele frequency distribution pattern among the populations of Eurasia. Russians and Ukrainians were indistinguishable from other European populations relative to the frequency of allele ADH1B*47his, and consequently, relative to specific features of ethanol metabolic pathways. The data obtained provide refinement of the geographic pattern of ADH1B*47his frequency distribution in Eurasia.

GOLD HULL AND INTERNODE2 Encodes a Primarily Multifunctional Cinnamyl-Alcohol Dehydrogenase in Rice1

PubMed Central

Zhang, Kewei; Qian, Qian; Huang, Zejun; Wang, Yiqin; Li, Ming; Hong, Lilan; Zeng, Dali; Gu, Minghong; Chu, Chengcai; Cheng, Zhukuan

2006-01-01

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis. PMID:16443696

Isolation and characterization of full-length putative alcohol dehydrogenase genes from polygonum minus

NASA Astrophysics Data System (ADS)

Hamid, Nur Athirah Abd; Ismail, Ismanizan

2013-11-01

Polygonum minus, locally named as Kesum is an aromatic herb which is high in secondary metabolite content. Alcohol dehydrogenase is an important enzyme that catalyzes the reversible oxidation of alcohol and aldehyde with the presence of NAD(P)(H) as co-factor. The main focus of this research is to identify the gene of ADH. The total RNA was extracted from leaves of P. minus which was treated with 150 μM Jasmonic acid. Full-length cDNA sequence of ADH was isolated via rapid amplification cDNA end (RACE). Subsequently, in silico analysis was conducted on the full-length cDNA sequence and PCR was done on genomic DNA to determine the exon and intron organization. Two sequences of ADH, designated as PmADH1 and PmADH2 were successfully isolated. Both sequences have ORF of 801 bp which encode 266 aa residues. Nucleotide sequence comparison of PmADH1 and PmADH2 indicated that both sequences are highly similar at the ORF region but divergent in the 3' untranslated regions (UTR). The amino acid is differ at the 107 residue; PmADH1 contains Gly (G) residue while PmADH2 contains Cys (C) residue. The intron-exon organization pattern of both sequences are also same, with 3 introns and 4 exons. Based on in silico analysis, both sequences contain "classical" short chain alcohol dehydrogenases/reductases ((c) SDRs) conserved domain. The results suggest that both sequences are the members of short chain alcohol dehydrogenase family.

An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity.

PubMed

Wuxiuer, Yimingjiang; Morgunova, Ekaterina; Cols, Neus; Popov, Alexander; Karshikoff, Andrey; Sylte, Ingebrigt; Gonzàlez-Duarte, Roser; Ladenstein, Rudolf; Winberg, Jan-Olof

2012-08-01

All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis. © 2012 The Authors Journal compilation © 2012 FEBS.

Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase.

PubMed

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

2011-11-01

Ethanol is a renewable biofuel, and it can be produced from lignocellulosic biomass. The biomass is usually converted to hydrolysates that consist of sugar and sugar derivatives, such as furfural. Yeast ferments sugar to ethanol, but furfural higher than 3 mM is inhibitory. It can take several days for yeast cells to reduce furfural to non-inhibitory furfuryl alcohol before producing ethanol. Bioreduction of furfural to furfuryl alcohol before fermentation may relieve yeast from furfural toxicity. We observed that Cupriavidus necator JMP134, a strict aerobe, rapidly reduced 17 mM furfural to less than 3 mM within 14 min with cell turbidity of 1.0 at 600 nm at 50°C. The rapid reduction consumed ethanol. The "furfural reductase" (FurX) was purified, and it oxidized ethanol to acetaldehyde and reduced furfural to furfuryl alcohol with NAD(+) as the cofactor. The protein was identified with mass spectrometry fingerprinting to be a hypothetical protein belonging to Zn-dependent alcohol dehydrogenase family. The furX-inactivation mutant of C. necator JMP134 lost the ability to rapidly reduce furfural, and Escherichia coli producing recombinant FurX gained the ability. Thus, an alcohol dehydrogenase enabled bacteria to rapidly reduce furfural with ethanol as the reducing power.

In vivo relationship between monoamine oxidase type B and alcohol dehydrogenase: effects of ethanol and phenylethylamine

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

Aliyu, S.U.; Upahi, L.

The role of acute ethanol and phenylethylamine on the brain and platelet monoamine oxidase activities, hepatic cytosolic alcohol dehydrogenase, redox state and motor behavior were studied in male rats. Ethanol on its own decreased the redox couple ratio, as well as, alcohol dehydrogenase activity in the liver while at the same time it increased brain and platelet monoamine oxidase activity due to lower Km with no change in Vmax. The elevation in both brain and platelet MAO activity was associated with ethanol-induced hypomotility in the rats. Co-administration of phenylethylamine and ethanol to the animals, caused antagonism of the ethanol-induced effectsmore » described above. The effects of phenylethylamine alone, on the above mentioned biochemical and behavioral indices, are more complex. Phenylethylamine on its own, like ethanol, caused reduction of the cytosolic redox, ratio and elevation of monoamine oxidase activity in the brain and platelets. However, in contrast to ethanol, this monoamine produced hypermotility and activation of the hepatic cytosolic alcohol dehydrogenase activity in the animals.« less

Oxidation of Two Hydroxylated Ochratoxin A Metabolites by Alcohol Dehydrogenase

PubMed Central

Syvertsen, Christian; Størmer, Fredrik C.

1983-01-01

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. Km and turnover number for 10-hydroxyochratoxin A were 110 μM and 0.1 s−1, respectively. PMID:6347065

Extreme halophilic alcohol dehydrogenase mediated highly efficient syntheses of enantiopure aromatic alcohols.

PubMed

Alsafadi, Diya; Alsalman, Safaa; Paradisi, Francesca

2017-11-07

Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (C[double bond, length as m-dash]O) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (C[double bond, length as m-dash]O) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

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

PubMed

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

2011-11-01

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

Bioelectrochemistry of non-covalent immobilized alcohol dehydrogenase on oxidized diamond nanoparticles.

PubMed

Nicolau, Eduardo; Méndez, Jessica; Fonseca, José J; Griebenow, Kai; Cabrera, Carlos R

2012-06-01

Diamond nanoparticles are considered a biocompatible material mainly due to their non-cytotoxicity and remarkable cellular uptake. Model proteins such as cytochrome c and lysozyme have been physically adsorbed onto diamond nanoparticles, proving it to be a suitable surface for high protein loading. Herein, we explore the non-covalent immobilization of the redox enzyme alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae (E.C.1.1.1.1) onto oxidized diamond nanoparticles for bioelectrochemical applications. Diamond nanoparticles were first oxidized and physically characterized by X-ray diffraction (XRD), FT-IR and TEM. Langmuir isotherms were constructed to investigate the ADH adsorption onto the diamond nanoparticles as a function of pH. It was found that a higher packing density is achieved at the isoelectric point of the enzyme. Moreover, the relative activity of the immobilized enzyme on diamond nanoparticles was addressed under optimum pH conditions able to retain up to 70% of its initial activity. Thereafter, an ethanol bioelectrochemical cell was constructed by employing the immobilized alcohol dehydrogenase onto diamond nanoparticles, this being able to provide a current increment of 72% when compared to the blank solution. The results of this investigation suggest that this technology may be useful for the construction of alcohol biosensors or biofuel cells in the near future. Copyright © 2011 Elsevier B.V. All rights reserved.

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco.

PubMed

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N; Marshall, David; Hancock, Robert D; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

2011-12-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.

The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

PubMed

Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G; Lynd, Lee R

2015-04-01

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be

Alcohol consumption and type 2 diabetes: influence of genetic variation in alcohol dehydrogenase.

PubMed

Beulens, Joline W J; Rimm, Eric B; Hendriks, Henk F J; Hu, Frank B; Manson, JoAnn E; Hunter, David J; Mukamal, Kenneth J

2007-09-01

We sought to investigate whether a polymorphism in the alcohol dehydrogenase 1c (ADH1C) gene modifies the association between alcohol consumption and type 2 diabetes. In nested case-control studies of 640 women with incident diabetes and 1,000 control subjects from the Nurses' Health Study and 383 men with incident diabetes and 382 control subjects from the Health Professionals Follow-Up Study, we determined associations between the ADH1C polymorphism, alcohol consumption, and diabetes risk. Moderate to heavy alcohol consumption (>5 g/day for women and >10 g/day for men) was associated with a decreased risk of diabetes among women (odds ratio [OR] 0.45 [95% CI 0.33-0.63]) but not men (1.08 [0.67-1.75]). ADH1C genotype modified the relation between alcohol consumption and diabetes for women (P(interaction) = 0.02). The number of ADH1C*2 alleles, related to a slower rate of ethanol oxidation, attenuated the lower risk of diabetes among women consuming >/=5 g alcohol/day (P(trend) = 0.002). These results were not significant among men. Results were similar in pooled analyses (P(interaction) = 0.02) with ORs for diabetes among moderate drinkers of 0.44 (95% CI 0.21-0.94) in ADH1C*1 homozygotes, 0.65 (0.39-1.06) for heterozygotes, and 0.78 (0.50-1.22) for ADH1C*2 homozygotes compared with those for ADH1C*1 homozygote abstainers (P(trend) = 0.02). ADH1C genotype modifies the association between alcohol consumption and diabetes. The ADH1C*2 allele, related to a slower oxidation rate, attenuates the lower diabetes risk among moderate to heavy drinkers. This suggests that the association between alcohol consumption and diabetes may be causal but mediated by downstream metabolites such as acetate rather than ethanol itself.

Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

PubMed

Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

2010-04-01

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

AAV Gene Therapy for Alcoholism: Inhibition of Mitochondrial Aldehyde Dehydrogenase Enzyme Expression in Hepatoma Cells.

PubMed

Sanchez, Anamaria C; Li, Chengwen; Andrews, Barbara; Asenjo, Juan A; Samulski, R Jude

2017-09-01

Most ethanol is broken down in the liver in two steps by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) enzymes, which metabolize down ethanol into acetaldehyde and then acetate. Some individuals from the Asian population who carry a mutation in the aldehyde dehydrogenase gene (ALDH2*2) cannot metabolize acetaldehyde as efficiently, producing strong effects, including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2 expression by genetic technologies could result in a similar phenotype. scAAV2 vectors encoding ALDH2 small hairpin RNA (shRNA) were utilized to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. Human cell lines HEK-293 and HepG2 were transduced with scAAV2/shRNA, showing a reduction in ALDH2 RNA and protein expression with the two viral concentration assayed (1 × 10 4 and 1 × 10 5 vg/cell) at two different time points. In both cell lines, ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 90% and 52%, respectively, 5 days post infection. Transduced HepG2 VL17A cells (ADH+) exposed to ethanol resulted in a 50% increase in acetaldehyde levels. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors.

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

PubMed

Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

2011-06-01

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

The Alcohol Dehydrogenase Kinetics Laboratory: Enhanced Data Analysis and Student-Designed Mini-Projects

ERIC Educational Resources Information Center

Silverstein, Todd P.

2016-01-01

A highly instructive, wide-ranging laboratory project in which students study the effects of various parameters on the enzymatic activity of alcohol dehydrogenase has been adapted for the upper-division biochemistry and physical biochemistry laboratory. Our two main goals were to provide enhanced data analysis, featuring nonlinear regression, and…

Syringyl Lignin Is Unaltered by Severe Sinapyl Alcohol Dehydrogenase Suppression in Tobacco[W

PubMed Central

Barakate, Abdellah; Stephens, Jennifer; Goldie, Alison; Hunter, William N.; Marshall, David; Hancock, Robert D.; Lapierre, Catherine; Morreel, Kris; Boerjan, Wout; Halpin, Claire

2011-01-01

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference–inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem. PMID:22158465

Fast internal dynamics in alcohol dehydrogenase

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

Monkenbusch, M.; Stadler, A., E-mail: a.stadler@fz-juelich.de; Biehl, R.

2015-08-21

Large-scale domain motions in alcohol dehydrogenase (ADH) have been observed previously by neutron spin-echo spectroscopy (NSE). We have extended the investigation on the dynamics of ADH in solution by using high-resolution neutron time-of-flight (TOF) and neutron backscattering (BS) spectroscopy in the incoherent scattering range. The observed hydrogen dynamics were interpreted in terms of three mobility classes, which allowed a simultaneous description of the measured TOF and BS spectra. In addition to the slow global protein diffusion and domain motions observed by NSE, a fast internal process could be identified. Around one third of the protons in ADH participate in themore » fast localized diffusive motion. The diffusion coefficient of the fast internal motions is around two third of the value of the surrounding D{sub 2}O solvent. It is tempting to associate the fast internal process with solvent exposed amino acid residues with dangling side chains.« less

The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

PubMed Central

Heinstra, P W; Geer, B W; Seykens, D; Langevin, M

1989-01-01

Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway. Images Fig. 1. Fig. 2. PMID:2499314

Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae

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

Conway, T.; Ingram, L.O.

1989-07-01

The gene that encodes 1,2-propanediol oxidoreductase (fucO) from Escherichia coli was sequenced. The reading frame specified a protein of 383 amino acids (including the N-terminal methionine), with an aggregate molecular weight of 40,642. The induction of fucO transcription, which occurred in the presence of fucose, was confirmed by Northern blot analysis. In E. coli, the primary fucO transcript was approximately 2.1 kilobases in length. The 5{prime} end of the transcript began more than 0.7 kilobase upstream of the fucO start codon within or beyond the fucA gene. Propanediol oxidoreductase exhibited 41.7% identity with the iron-containing alcohol dehydrogenase II from Zymomonasmore » mobilis and 39.5% identity with ADH4 from Saccharomyces cerevisiae. These three proteins did not share homology with either short-chain or long-chain zinc-containing alcohol dehydrogenase enzymes. We propose that these three unusual alcohol dehydrogenases define a new family of enzymes.« less

Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

PubMed Central

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

Genetic polymorphisms of alcohol dehydrogense-1B and aldehyde dehydrogenase-2, alcohol flushing, mean corpuscular volume, and aerodigestive tract neoplasia in Japanese drinkers.

PubMed

Yokoyama, Akira; Mizukami, Takeshi; Yokoyama, Tetsuji

2015-01-01

Genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) modulate exposure levels to ethanol/acetaldehyde. Endoscopic screening of 6,014 Japanese alcoholics yielded high detection rates of esophageal squamous cell carcinoma (SCC; 4.1%) and head and neck SCC (1.0%). The risks of upper aerodigestive tract SCC/dysplasia, especially of multiple SCC/dysplasia, were increased in a multiplicative fashion by the presence of a combination of slow-metabolizing ADH1B*1/*1 and inactive heterozygous ALDH2*1/*2 because of prolonged exposure to higher concentrations of ethanol/acetaldehyde. A questionnaire asking about current and past facial flushing after drinking a glass (≈180 mL) of beer is a reliable tool for detecting the presence of inactive ALDH2. We invented a health-risk appraisal (HRA) model including the flushing questionnaire and drinking, smoking, and dietary habits. Esophageal SCC was detected at a high rate by endoscopic mass-screening in high HRA score persons. A total of 5.0% of 4,879 alcoholics had a history of (4.0%) or newly diagnosed (1.0%) gastric cancer. Their high frequency of a history of gastric cancer is partly explained by gastrectomy being a risk factor for alcoholism because of altered ethanol metabolism, e.g., by blood ethanol level overshooting. The combination of H. pylori-associated atrophic gastritis and ALDH2*1/*2 showed the greatest risk of gastric cancer in alcoholics. High detection rates of advanced colorectal adenoma/carcinoma were found in alcoholics, 15.7% of 744 immunochemical fecal occult blood test (IFOBT)-negative alcoholics and 31.5% of the 393 IFOBT-positive alcoholics. Macrocytosis with an MCV≥106 fl increased the risk of neoplasia in the entire aerodigestive tract of alcoholics, suggesting that poor nutrition as well as ethanol/acetaldehyde exposure plays an important role in neoplasia.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

PubMed

Rivera-Meza, Mario; Quintanilla, María Elena; Tampier, Lutske; Mura, Casilda V; Sapag, Amalia; Israel, Yedy

2010-01-01

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.

Evaluation of the influence of alcohol dehydrogenase polymorphisms on alcohol elimination rates in African Americans.

PubMed

Marshall, Vanessa J; Ramchandani, Vijay A; Kalu, Nnenna; Kwagyan, John; Scott, Denise M; Ferguson, Clifford L; Taylor, Robert E

2014-01-01

The relationship between alcohol dehydrogenase (ADH) polymorphisms and alcohol use disorders in populations of African descent has not been clearly established. This study examined the effect of ADH1B polymorphisms on alcohol metabolism and subjective response, following intravenous (IV) alcohol administration, and the influence of gender, recent drinking history, and family history of alcoholism (FHA), in nondependent African American drinkers. The sample included eighty-seven 21- to 35-year-old, light social drinkers of African descent. Participants included 39 sib pairs, 2 sibships with 3 siblings each, and 3 individuals who were not part of a sibship. Participants received infusions via the use of the clamp method that refers to the goal of controlling breath alcohol concentration in 2 randomized sessions at 0.06 g% ethanol and 0 mg% (placebo), and a battery of subjective scales at predefined time points. Dependent measures included alcohol elimination rates (AERs), alcohol disappearance rates (ADRs), subjective measures peak scores, and area under the curve. General linear model and mixed models were performed to examine the relationship between ADH1B genotype, dependent measures, and influence of covariates. Participants with ADH1B1/1 genotypes showed higher number of drinks (p = 0.023) and drinks per drinking day (p = 0.009) compared with the persons with ADH1B1/3 genotype. AER (adjusted for body weight) was higher in ADH1B*1 homozygotes (p = 0.045) compared with ADH1B1/3 heterozygotes. ADR differed significantly between males and females (p = 0.002), regardless of body weight (p = 0.004) and lean body mass (p < 0.001) adjustments. Although a few subjective measures differed across genotype, all measures were higher in alcohol sessions compared with placebo sessions (p < 0.001). These observations were mediated by drinks per drinking day, gender, and FHA. ADH1B polymorphism had a marginal effect on alcohol pharmacokinetics following IV alcohol administration

Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

ERIC Educational Resources Information Center

Nash, Barbara T.

2007-01-01

SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

Cloning, expression, and characterization of a novel (S)-specific alcohol dehydrogenase from Lactobacillus kefir.

PubMed

Chen, Qilei; Hu, Youjia; Zhao, Wenjie; Zhu, Chunbao; Zhu, Baoquan

2010-01-01

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-beta-D-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.

Alcohol and aldehyde dehydrogenase gene polymorphisms and oropharyngolaryngeal, esophageal and stomach cancers in Japanese alcoholics.

PubMed

Yokoyama, A; Muramatsu, T; Omori, T; Yokoyama, T; Matsushita, S; Higuchi, S; Maruyama, K; Ishii, H

2001-03-01

Alcohol dehydrogenase-2 (ADH2) and aldehyde dehydrogenase-2 (ALDH2) gene polymorphisms play roles in ethanol metabolism, drinking behavior and esophageal carcinogenesis in Japanese; however, the combined influence of ADH2 and ALDH2 genotypes on other aerodigestive tract cancers have not been investigated. ADH2/ALDH2 genotyping was performed on lymphocyte DNA samples from Japanese alcoholic men (526 cancer-free; 159 with solitary or multiple aerodigestive tract cancers, including 33 oropharyngolaryngeal, 112 esophageal, 38 stomach and 22 multiple primary cancers in two or three organs). After adjustment for age, drinking and smoking habits, and ADH2/ALDH2 genotypes, the presence of either ADH2*1/2*1 or ALDH2*1/2*2 significantly increased the risk for oropharyngolaryngeal cancer [odds ratios (ORs), 6.68 with ADH2*1/2*1 and 18.52 with ALDH2*1/2*2] and esophageal cancer (ORs, 2.64 and 13.50, respectively). For patients with both ADH2*1/2*1 and ALDH2*1/2*2, the risks for oropharyngolaryngeal and esophageal cancers were enhanced in a multiplicative fashion (OR = 121.77 and 40.40, respectively). A positive association with ALDH2*1/2*2 alone was observed for stomach cancer patients who also had oropharyngolaryngeal and/or esophageal cancer (OR = 110.58), but it was not observed for those with stomach cancer alone. Furthermore, in the presence of ALDH2*1/2*2, the risks for multiple intra-esophageal cancers (OR = 3.43) and for esophageal cancer with oropharyngolaryngeal and/or stomach cancer (OR = 3.95) were higher than the risks for solitary intra-esophageal cancer and for esophageal cancer alone, but these tendencies were not observed for ADH2*1/2*1 genotype. Alcoholics' population attributable risks due to ADH2/ALDH2 polymorphisms were estimated to be 82.0% for oropharyngolaryngeal cancer and 63.9% for esophageal cancer.

The oxidative fermentation of ethanol in Gluconacetobacter diazotrophicus is a two-step pathway catalyzed by a single enzyme: alcohol-aldehyde Dehydrogenase (ADHa).

PubMed

Gómez-Manzo, Saúl; Escamilla, José E; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M H; Sosa-Torres, Martha Elena

2015-01-07

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2-C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

PubMed Central

Gómez-Manzo, Saúl; Escamilla, José E.; González-Valdez, Abigail; López-Velázquez, Gabriel; Vanoye-Carlo, América; Marcial-Quino, Jaime; de la Mora-de la Mora, Ignacio; Garcia-Torres, Itzhel; Enríquez-Flores, Sergio; Contreras-Zentella, Martha Lucinda; Arreguín-Espinosa, Roberto; Kroneck, Peter M. H.; Sosa-Torres, Martha Elena

2015-01-01

Gluconacetobacter diazotrophicus is a N2-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde. PMID:25574602

DFT-based prediction of reactivity of short-chain alcohol dehydrogenase

NASA Astrophysics Data System (ADS)

Stawoska, I.; Dudzik, A.; Wasylewski, M.; Jemioła-Rzemińska, M.; Skoczowski, A.; Strzałka, K.; Szaleniec, M.

2017-06-01

The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, ( S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2' ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.

Methionine-141 directly influences the binding of 4-methylpyrazole in human sigma sigma alcohol dehydrogenase.

PubMed Central

Xie, P. T.; Hurley, T. D.

1999-01-01

Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human sigma sigma isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human beta1beta1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4-methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human sigma sigma alcohol dehydrogenase (ADH) isoenzyme to 2.5 A resolution. Comparison of this structure to the human beta1beta1 isoenzyme structure indicated that the side-chain position for Met141 in sigma sigma ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in sigma sigma ADH to Leu (sigma141L) lowers the Ki for 4-methylpyrazole from 350 to 10 microM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate-binding pocket at a position close to the methyl group of 4-methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X-ray structure of the sigma141L mutant enzyme was determined to 2.4 A resolution. The three-dimensional structure of the mutant enzyme is identical to the wild-type enzyme, with the exception of the residue at position 141. Thus, the differences in 4-methylpyrazole binding between the mutant and wild-type sigma sigma ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class-specific differences in 4-methylpyrazole binding to the human ADH isoenzymes. PMID:10631979

Alcohol dehydrogenase-1B genotype (rs1229984) is a strong determinant of the relationship between body weight and alcohol intake in Japanese alcoholic men.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2013-07-01

The calories in alcoholic beverages consumed by alcoholics are a major energy source and a strong modifier of their body weight. Genetic polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) affect susceptibility to alcoholism and may affect body weight via gene-associated differences in fuel utilization in alcoholics. We evaluated associations between ADH1B/ALDH2 genotypes and the body weight and body mass index (BMI) of 1,301 Japanese alcoholic men at the time of their first visit to an addiction center. Median (25th to 75th) caloric intake in the form of alcoholic beverages was 864 (588 to 1,176) kcal/d. Age-adjusted caloric intake did not differ according to ADH1B/ALDH2 genotypes. The body weight and BMI values showed that the ADH1B*2/*2 and *1/*2 carriers (n = 939) were significantly leaner than the ADH1B*1/*1 carriers (n = 362) irrespective of age, drinking, smoking, and dietary habits. The age-adjusted body weight values of the ADH1B*2/*2, ADH1B*1/*2, and ADH1B*1/*1 carriers were 58.4 ± 0.4, 58.7 ± 0.5, and 63.6 ± 0.5 kg, respectively (ADH1B*2 vs. ADH1B*1/*1 carriers, p < 0.0001), and the corresponding BMI values were 21.0 ± 0.1, 21.0 ± 0.1, and 22.9 ± 0.2 kg/m(2) , respectively (ADH1B*2 vs. ADH1B*1/*1 carriers, p < 0.0001). No effects of inactive ALDH2 on body weight or BMI were observed. A multivariate analysis showed that BMI decreased by 0.35 per 10-year increase in age, by 1.73 in the presence of the ADH1B*2 allele, by 1.55 when the preferred beverage was whiskey, and by 0.19 per +10 cigarettes/d and that it increased by 0.10 per +22 g ethanol (EtOH)/d and by 0.41 per increase in category of frequency of milk intake (every day, occasionally, rarely, and never). The increase in BMI as alcohol consumption increased was significantly smaller in the ADH1B*2 group than in the ADH1B*1/*1 group (p = 0.002). ADH1B genotype was a strong determinant of body weight in the alcoholics. The more rapid EtOH elimination associated

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression.

PubMed

Goffner, D; Van Doorsselaere, J; Yahiaoui, N; Samaj, J; Grima-Pettenati, J; Boudet, A M

1998-03-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.

Two types of alcohol dehydrogenase from Perilla can form citral and perillaldehyde.

PubMed

Sato-Masumoto, Naoko; Ito, Michiho

2014-08-01

Studies on the biosynthesis of oil compounds in Perilla will help in understanding regulatory systems of secondary metabolites and in elucidating reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, an aldo-keto reductase (AKR) and a geraniol dehydrogenase (GeDH), which are thought to participate in the biosynthesis of perilla essential oil components, such as citral and perillaldehyde, were isolated from three pure lines of perilla. These enzymes shared high amino acid sequence identity within the genus Perilla, and were expressed regardless of oil type. The overall reaction from geranyl diphosphate to citral was performed in vitro using geraniol synthase and GeDH to form a large proportion of citral and relatively little geraniol as reaction products. The biosynthetic pathway from geranyl diphosphate to citral, the main compound of citral-type perilla essential oil, was established in this study. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression pattern of two paralogs encoding cinnamyl alcohol dehydrogenases in Arabidopsis. Isolation and characterization of the corresponding mutants.

PubMed

Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-06-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cbeta. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units.

Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.

PubMed

Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

2015-01-01

Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Enzymatic conversion of tetradecanol in heterogenous phase by yeast-alcohol dehydrogenase].

PubMed

Rothe, U; Schöpp, W; Aurich, H

1976-01-01

Alcohol dehydrogenase from yeast converts long-chain primary alcohols not only in the dissolved state, but also at the surface of undissolved particles. Tetradecanol beads with a defined surface can be produced and employed as model substrate. The reaction rate was determined by the proton release accomplished in the reaction. The initial reaction rate depends on the enzyme concentration. The relation is nonlinear (vi = k-[e]0,4); the numerical value of the exponent (n = 0.4) argues in favour of a reaction occurring at the interface. The Lineweaver-Burk plots become linear if the substrate concentrations are based on the molar surface concentrations of the particles. The pH optimum for the reaction at the surface is displaced by 0.25 pH units towards the alkaline region (compared with ethanol as substrate). The activation energy of the reaction with tetradecanol beads as substrate is 30% lower than that for the ethanol oxydation.

Proteins Differentially Expressed in the Pancreas of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice Fed Ethanol For 3 Months.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-07-01

The aim of this study was to identify differentially expressed proteins in the pancreatic tissue of hepatic alcohol dehydrogenase-deficient deer mice fed ethanol to understand metabolic basis and mechanism of alcoholic chronic pancreatitis. Mice were fed liquid diet containing 3.5 g% ethanol daily for 3 months, and differentially expressed pancreatic proteins were identified by protein separation using 2-dimensional gel electrophoresis and identification by mass spectrometry. Nineteen differentially expressed proteins were identified by applying criteria established for protein identification in proteomics. An increased abundance was found for ribosome-binding protein 1, 60S ribosomal protein L31-like isoform 1, histone 4, calcium, and adenosine triphosphate (ATP) binding proteins and the proteins involved in antiapoptotic processes and endoplasmic reticulum function, stress, and/or homeostasis. Low abundance was found for endoA cytokeratin, 40S ribosomal protein SA, amylase 2b isoform precursor, serum albumin, and ATP synthase subunit β and the proteins involved in cell motility, structure, and conformation. Chronic ethanol feeding in alcohol dehydrogenase-deficient deer mice differentially expresses pancreatic functional and structural proteins, which can be used to develop biomarker(s) of alcoholic chronic pancreatitis, particularly amylase 2b precursor, and 60 kDa heat shock protein and those involved in ATP synthesis and blood osmotic pressure.

DOWNREGULATION OF CINNAMYL-ALCOHOL DEHYDROGENASE IN SWITCHGRASS BY RNA SILENCING RESULTS IN ENHANCED GLUCOSE RELEASE AFTER CELLULASE TREATMENT

USDA-ARS?s Scientific Manuscript database

Cinnamyl alcohol dehydrogenase (CAD), catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switch...

Expression Pattern of Two Paralogs Encoding Cinnamyl Alcohol Dehydrogenases in Arabidopsis. Isolation and Characterization of the Corresponding Mutants1

PubMed Central

Sibout, Richard; Eudes, Aymerick; Pollet, Brigitte; Goujon, Thomas; Mila, Isabelle; Granier, Fabienne; Séguin, Armand; Lapierre, Catherine; Jouanin, Lise

2003-01-01

Studying Arabidopsis mutants of the phenylpropanoid pathway has unraveled several biosynthetic steps of monolignol synthesis. Most of the genes leading to monolignol synthesis have been characterized recently in this herbaceous plant, except those encoding cinnamyl alcohol dehydrogenase (CAD). We have used the complete sequencing of the Arabidopsis genome to highlight a new view of the complete CAD gene family. Among nine AtCAD genes, we have identified the two distinct paralogs AtCAD-C and AtCAD-D, which share 75% identity and are likely to be involved in lignin biosynthesis in other plants. Northern, semiquantitative restriction fragment-length polymorphism-reverse transcriptase-polymerase chain reaction and western analysis revealed that AtCAD-C and AtCAD-D mRNA and protein ratios were organ dependent. Promoter activities of both genes are high in fibers and in xylem bundles. However, AtCAD-C displayed a larger range of sites of expression than AtCAD-D. Arabidopsis null mutants (Atcad-D and Atcad-C) corresponding to both genes were isolated. CAD activities were drastically reduced in both mutants, with a higher impact on sinapyl alcohol dehydrogenase activity (6% and 38% of residual sinapyl alcohol dehydrogenase activities for Atcad-D and Atcad-C, respectively). Only Atcad-D showed a slight reduction in Klason lignin content and displayed modifications of lignin structure with a significant reduced proportion of conventional S lignin units in both stems and roots, together with the incorporation of sinapaldehyde structures ether linked at Cβ. These results argue for a substantial role of AtCAD-D in lignification, and more specifically in the biosynthesis of sinapyl alcohol, the precursor of S lignin units. PMID:12805615

Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol.

PubMed

Damgaard, S E

1981-09-29

The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

Role of alcohol dehydrogenase activity and the acetaldehyde in ethanol- induced ethane and pentane production by isolated perfused rat liver.

PubMed Central

Müller, A; Sies, H

1982-01-01

The volatile hydrocarbons ethane and n-pentane are produced at increased rates by isolated perfused rat liver during the metabolism of acutely ethanol. The effect is half-maximal at 0.5 mM-ethanol, and its is not observed when inhibitors of alcohol dehydrogenase such as 4-methyl- or 4-propyl-pyrazole are also present. Propanol, another substrate for the dehydrogenase, is also active. Increased alkane production can be initiated by adding acetaldehyde in the presence of 4-methyl- or 4-propyl-pyrazole. An antioxidant, cyanidanol, suppresses the ethanol-induced alkane production. The data obtained with the isolated organ demonstrate that products known to arise from the peroxidation of polyunsaturated fatty acids are formed in the presence of ethanol and that the activity of alcohol dehydrogenase is required for the generation of the active radical species. The mere presence of ethanol, e.g. at binding sites of special form(s) of cytochrome P-450, it not sufficient to elicit an increased production of volatile hydrocarbons by rat liver. PMID:6751324

Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2015-01-01

Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67-2.94] and 1.39 [0.99-1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28-0.49] and 0.51 [0.37-0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45-0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast-metabolizing ADH1B was associated with lower serum LDL

Genetic polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 and liver cirrhosis, chronic calcific pancreatitis, diabetes mellitus, and hypertension among Japanese alcoholic men.

PubMed

Yokoyama, Akira; Mizukami, Takeshi; Matsui, Toshifumi; Yokoyama, Tetsuji; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2013-08-01

The presence of the less-active form of alcohol dehydrogenase-1B encoded by ADH1B*1/*1 (vs. *2 allele) and active form of aldehyde dehydrogenase-2 (ALDH2) encoded by ALDH2*1/*1 (vs. *2 allele) increases the risk of alcoholism in East Asians. The subjects in this cross-sectional survey were 1,902 Japanese alcoholic men (≥40 years) who underwent ADH1B/ALDH2 genotyping. Age-adjusted daily alcohol consumption did not differ according to the ADH1B/ALDH2 genotypes. The age-adjusted odds ratios (AORs; 95% confidence interval) for liver cirrhosis (LC; n = 359, 1.58 [1.19 to 2.09]), chronic calcific pancreatitis (CP; n = 80, 2.24 [1.20 to 4.20]), and diabetes mellitus (DM; n = 383, 1.51 [1.15 to 1.99]) were higher in the ADH1B*2 allele carriers than in the ADH1B*1/*1 carriers. The AORs for LC (1.43 [1.01 to 2.02]), CP (1.68 [0.80 to 3.53]), DM (1.63 [1.15 to 2.30]), and hypertension (HT; n = 495, 1.52 [1.11 to 2.07]) were higher in the ALDH2*1/*1 carriers than in the ALDH2*1/*2 carriers. The ADH1B*2-associated AOR for LC was 2.08 (1.46 to 2.94) among those aged 40 to 59 years, but 0.89 (0.56 to 1.43) among those aged 60 years or over, and the interaction between ADH1B genotype and age on the LC risk was significant (p = 0.009). When the group with non-LC and no/mild fibrosis was used as controls, the ADH1B*2-associated AORs increased according to the severity of their liver disease: 1.67 (1.32 to 2.11) for the group with non-LC and serum type IV collagen values ≥200 ng/ml, 1.81 (1.24 to 2.63) for the group of Child-Pugh class A LC, and 3.17 (1.98 to 5.07) for the group with Child-Pugh class B/C LC. Anti-hepatitis C virus (HCV) antibody was positive in 103 patients, and the groups with a high anti-HCV antibody titer and either the ADH1B*2/*2 genotype or the ALDH2*1/*1 genotype had the highest AORs (8.83 and 4.90, respectively). The population attributable fraction (PAF) due to the ADH1B*2 allele was 29% for LC, 47% for CP, and 27% for DM, and the PAF due to the ALDH2

Suppression of Cellulase and Polygalacturonase and Induction of Alcohol Dehydrogenase Isoenzymes in Avocado Fruit Mesocarp Subjected to Low Oxygen Stress

PubMed Central

Kanellis, Angelos K.; Solomos, Theophanes; Roubelakis-Angelakis, Kalliopi A.

1991-01-01

Expression of polygalacturonase and cellulase, two hydrolytic enzymes of avocado (Persea americana, cv Hass) fruit which are synthesized de novo during ripening, and alcohol dehydrogenase, a known anaerobic protein, were studied under different O2 regimes. Low O2 concentrations (2.5-5.5%) diminished the accumulation of polygalacturonase and cellulase proteins and the expression of their isoenzymes. This pattern of change in cellulase protein was also reflected in the steady-state amount of its mRNA. In contrast, 7.5 and 10% O2 did not alter the changes observed in fruits ripened in air. On the other hand, alcohol dehydrogenase was induced in 2.5, 3.5, and 5.5% O2 but not in 7.5 or 10% O2. The recovery from the hypoxic stress upon returning the fruits back to air for 24 hours, was also a function of O2 tensions under which the fruits were kept. Thus, the synthesis of polygalacturonase and cellulase was directly related to O2 levels, while the activity of the isoenzymes of alcohol dehydrogenase was inversely related to O2 levels. The results indicate that hypoxia exerts both negative and positive effects on the expression of certain genes and that these effects are initiated at the same levels of O2. ImagesFigure 2Figure 3Figure 4Figure 5Figure 6Figure 7 PMID:16668163

Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

PubMed Central

2012-01-01

Background The white-rot fungus Phanerochaete chrysosporium is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme. Results We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli and the biochemical characterization of the encoded GST and His6 tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher Km (220 μM) compared to that of NADPH (39 μM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP + at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction. Conclusions In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde. PMID:22742413

Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

NASA Astrophysics Data System (ADS)

Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

2015-12-01

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

Characterization and further stabilization of a new anti-prelog specific alcohol dehydrogenase from Thermus thermophilus HB27 for asymmetric reduction of carbonyl compounds.

PubMed

Rocha-Martín, Javier; Vega, Daniel; Bolivar, Juan M; Hidalgo, Aurelio; Berenguer, José; Guisán, José M; López-Gallego, Fernando

2012-01-01

The use of dehydrogenases in asymmetric chemistry has exponentially grown in the last decades facilitated by the genome mining. Here, a new short-chain alcohol dehydrogenase from Thermus thermophilus HB27 has been expressed, purified, characterized and stabilized by immobilization on solid supports. The enzyme catalyzes both oxidative and reductive reactions at neutral pH with a broad range of substrates. Its highest activity was found towards the reduction of 2,2',2″-trifluoroacetophenone (85 U/mg at 65 °C and pH 7). Moreover, the enzyme was stabilized more than 200-fold by multipoint covalent immobilization on agarose matrixes via glyoxyl chemistry. Such heterogeneous catalyst coupled to an immobilized cofactor recycling partner performed the quantitative asymmetric reduction of 2,2',2″-trifluoroacetophenone and rac-2-phenylpropanal to (S)-(+)-α-(trifluoromethyl)benzyl alcohol and (R)-2-phenyl-1-propanol with enantiomeric excesses of 96% and 71%, respectively. To our knowledge this is the first alcohol dehydrogenase from a thermophilic source with anti-Prelog selectivity for aryl ketones and that preferentially produces R-profens. Copyright © 2011 Elsevier Ltd. All rights reserved.

Inhibition of human alcohol and aldehyde dehydrogenases by cimetidine and assessment of its effects on ethanol metabolism.

PubMed

Lai, Ching-Long; Li, Yeung-Pin; Liu, Chiu-Ming; Hsieh, Hsiu-Shan; Yin, Shih-Jiun

2013-02-25

Previous studies have reported that cimetidine, an H2-receptor antagonist used to treat gastric and duodenal ulcers, can inhibit alcohol dehydrogenases (ADHs) and ethanol metabolism. Human alcohol dehydrogenases and aldehyde dehydrogenases (ALDHs), the principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition by cimetidine of alcohol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and aldehyde oxidation by ALDH1A1 and ALDH2 at pH 7.5 and a cytosolic NAD(+) concentration. Cimetidine acted as competitive or noncompetitive inhibitors for the ADH and ALDH isozymes/allozymes with near mM inhibition constants. The metabolic interactions between cimetidine and ethanol/acetaldehyde were assessed by computer simulation using the inhibition equations and the determined kinetic constants. At therapeutic drug levels (0.015 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μM) in target tissues, cimetidine could weakly inhibit (<5%) the activities of ADH1B2 and ADH1B3 in liver, ADH2 in liver and small intestine, ADH4 in stomach, and ALDH1A1 in the three tissues, but not significantly affect ADH1A, ADH1B1, ADH1C1/2, or ALDH2. At higher drug levels, which may accumulate in cells (0.2 mM), the activities of the weakly-inhibited enzymes may be decreased more significantly. The quantitative effects of cimetidine on metabolism of ethanol and other physiological substrates of ADHs need further investigation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism of Saccharomyces cerevisiae.

PubMed

de Smidt, Olga; du Preez, James C; Albertyn, Jacobus

2012-02-01

The physiological role and possible functional substitution of each of the five alcohol dehydrogenase (Adh) isozymes in Saccharomyces cerevisiae were investigated in five quadruple deletion mutants designated strains Q1-Q5, with the number indicating the sole intact ADH gene. Their growth in aerobic batch cultures was characterised in terms of kinetic and stoichiometric parameters. Cultivation with glucose or ethanol as carbon substrate revealed that Adh1 was the only alcohol dehydrogenase capable of efficiently catalysing the reduction of acetaldehyde to ethanol. The oxidation of produced or added ethanol could also be attributed to Adh1. Growth of strains lacking the ADH1 gene resulted in the production of glycerol as a major fermentation product, concomitant with the production of a significant amount of acetaldehyde. Strains Q2 and Q3, expressing only ADH2 or ADH3, respectively, produced ethanol from glucose, albeit less than strain Q1, and were also able to oxidise added ethanol. Strains Q4 and Q5 grew poorly on glucose and produced ethanol, but were neither able to utilise the produced ethanol nor grow on added ethanol. Transcription profiles of the ADH4 and ADH5 genes suggested that participation of these gene products in ethanol production from glucose was unlikely. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Preparation of convection interaction media isobutyl disc monolithic column and its application to purification of secondary alcohol dehydrogenase and alcohol oxidase.

PubMed

Isobe, Kimiyasu; Kawakami, Yoshimitsu

2007-03-09

A convection interaction media (trade name CIM, BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.

A new principle of figure-ground segregation: The accentuation.

PubMed

Pinna, Baingio; Reeves, Adam; Koenderink, Jan; van Doorn, Andrea; Deiana, Katia

2018-02-01

The problem of perceptual organization was studied by Gestalt psychologists in terms of figure-ground segregation. In this paper we explore a new principle of figure-ground segregation: accentuation. We demonstrate the effectiveness of accentuation relative to other Gestalt principles, and also consider it autonomous as it can agree with or oppose them. We consider three dynamic aspects of the principle, namely: attraction, accentuation and assignment. Each creature needs to attract, fascinate, seduce, draw attention (e.g., a mate or a prey animal) or distract, refuse, dissuade, discourage, repulse (e.g., a predator). Similarly, each organism needs to accentuate, highlight, stress, underline, emphasize or distract from another. Thus, accentuation assigns meaning to a visual pattern such as a coat, a plumage or a flower. False eyes (ocelli) and dots (diematic patterns) demonstrate "deceiving camouflage by accentuation" that confuses predators/preys and hides or highlights vital body parts (butterflies/flowers). They also display the deceiving appearance and exhibition of biological fitness. The same accents may serve different or even opposite goals. We conclude that accentuation improves the adaptive fitness of organisms in multifarious ways. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed.

PubMed

Ochsner, Andrea M; Müller, Jonas E N; Mora, Carlos A; Vorholt, Julia A

2014-08-25

In the Gram-positive methylotroph Bacillus methanolicus, methanol oxidation is catalyzed by an NAD-dependent methanol dehydrogenase (Mdh) that belongs to the type III alcohol dehydrogenase (Adh) family. It was previously shown that the in vitro activity of B. methanolicus Mdh is increased by the endogenous activator protein Act, a Nudix hydrolase. Here we show that this feature is not unique, but more widespread among type III Adhs in combination with Act or other Act-like Nudix hydrolases. In addition, we studied the effect of site directed mutations in the predicted active site of Mdh and two other type III Adhs with regard to activity and activation by Act. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum.

PubMed

Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

2016-06-20

Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production.

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

PubMed

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

2002-09-20

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

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

PubMed

Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

2015-01-02

Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of an allylic/benzyl alcohol dehydrogenase from Yokenella sp. strain WZY002, an organism potentially useful for the synthesis of α,β-unsaturated alcohols from allylic aldehydes and ketones.

PubMed

Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan; Wang, Zhao

2014-04-01

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg(-1) for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg(-1) using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP(+), suggesting the nature of being an aldehyde reductase.

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

PubMed Central

Ying, Xiangxian; Wang, Yifang; Xiong, Bin; Wu, Tingting; Xie, Liping; Yu, Meilan

2014-01-01

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg−1 for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg−1 using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower Km values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP+, suggesting the nature of being an aldehyde reductase. PMID:24509923

Purification and characterization of an anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol.

PubMed

Meng, Fantao; Xu, Yan

2010-04-01

An anti-Prelog alcohol dehydrogenase from Oenococcus oeni that reduces 2-octanone to (R)-2-octanol was purified by 26-fold to homogeneity. The enzyme had a homodimeric structure consisting of 49 kDa subunits, required NADPH, but not NADH, as a cofactor and was a Zn-independent short-chain dehydrogenase. Aliphatic methyl ketones (chain length > or =6 carbon atoms) and aromatic methyl ketones were the preferred substrates for the enzyme, the best being 2-octanone. Maximum enzyme activity with 2-octanone was at 45 degrees C and at pH 8.0.

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

PubMed Central

Shimao, M; Ninomiya, K; Kuno, O; Kato, N; Sakazawa, C

1986-01-01

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction. Images PMID:3513704

Investigation of structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115.

PubMed

Zhang, Huaidong; Li, Qin; Wang, Lina; Chen, Yan

2018-05-01

Alcohol dehydrogenases (ADHs) catalyze the reversible oxidation of alcohol using NAD + or NADP + as cofactor. Three ADH homologues have been identified in Komagataella phaffii GS115 (also named Pichia pastoris GS115), ADH1, ADH2 and ADH3, among which adh3 is the only gene responsible for consumption of ethanol in Komagataella phaffii GS115. However, the relationship between structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115 (KpADH3) is still not clear yet. KpADH3 was purified, identified and characterized by multiple biophysical techniques (Nano LC-MS/MS, Enzymatic activity assay, X-ray crystallography). The crystal structure of KpADH3, which was the first ADH structure from Komagataella phaffii GS115, was solved at 1.745 Å resolution. Structural analysis indicated that KpADH3 was the sole dimeric ADH structure with face-to-face orientation quaternary structure from yeast. The major structural different conformations located on residues 100-114 (the structural zinc binding loop) and residues 337-344 (the loop between α12 and β15 which covered the catalytic domain). In addition, three channels were observed in KpADH3 crystal structure, channel 2 and channel 3 may be essential for substrate specific recognition, ingress and egress, channel 1 may be the pass-through for cofactor. KpADH3 plays an important role in the metabolism of alcohols in Komagataella phaffii GS115, and its crystal structure is the only dimeric medium-chain ADH from yeast described so far. Knowledge of the relationship between structure and function of KpADH3 is crucial for understanding the role of KpADH3 in Komagataella phaffii GS115 mitochondrial metabolism. Copyright © 2018 Elsevier B.V. All rights reserved.

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

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

Plapp, Bryce V.; Savarimuthu, Baskar Raj; Ferraro, Daniel J.

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentatemore » chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ~1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.« less

Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

PubMed Central

2017-01-01

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5′-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2′-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water. PMID:28640600

Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

PubMed

Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

2013-07-01

The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO.

Aldehyde dehydrogenase 2 polymorphism for development to hepatocellular carcinoma in East Asian alcoholic liver cirrhosis.

PubMed

Abe, Hiroshi; Aida, Yuta; Seki, Nobuyoshi; Sugita, Tomonori; Tomita, Yoichi; Nagano, Tomohisa; Itagaki, Munenori; Sutoh, Satoshi; Nagatsuma, Keisuke; Itoh, Kyoko; Matsuura, Tomokazu; Aizawa, Yoshio

2015-09-01

We aimed to clarify the influences of aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 1B (ADH1B) polymorphisms, and ethanol consumption profile to hepatocellular carcinoma (HCC) development in alcoholic liver cirrhosis without chronic hepatitis B and C virus infection (non-B non-C). Of 236 freshly diagnosed non-B non-C alcoholic liver cirrhosis patients, 67 were diagnosed as HCC and the remaining 169 as not having HCC. The relationship between the genetic polymorphisms and development to HCC were evaluated in well-matched patients with HCC (HCC group, n = 67) and without HCC (non-HCC group, n = 67) using propensity scores in age, sex, and prevalence of diabetes mellitus. Daily amount of ethanol consumption was significantly lower (P = 0.005), and consumptive period was significantly longer (P = 0.003) in HCC group than non-HCC group. Of 134 well-matched patients, 113 (84.3%) had ALDH2*1/*1 genotype and 21 (15.7%) had ALDH2*1/*2 genotype. In HCC development, consumptive long period (P = 0.007) and carrying ALDH2*1/*2 genotype (P = 0.026) were identified as significant factors independently participated, while there was no relation to ADH1B polymorphism. In addition, consumptive period was significantly longer in HCC group than non-HCC group in ALDH2*1/*1 genotype patients (P = 0.0005), while there was no difference in profile of ethanol consumption in ALDH2*1/*2 genotype patients. Among HCC group, daily (P = 3.78 × 10(-6) ) and cumulative amount (P = 4.89 × 10(-6) ) of ethanol consumption were significantly higher in ALDH2*1/*1 genotype patients than ALDH2*1/*2 genotype patients. In alcoholic liver cirrhosis, investigations of ALDH2 polymorphism and ethanol consumption profile are useful for prediction of HCC development. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis.

PubMed

Oda, Takahiro; Oda, Koji; Yamamoto, Hiroaki; Matsuyama, Akinobu; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi

2013-01-10

Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing

The Alcohol Dehydrogenase Isoenzyme as a Potential Marker of Pancreatitis.

PubMed

Jelski, Wojciech; Piechota, Joanna; Orywal, Karolina; Szmitkowski, Maciej

2018-05-01

Human pancreas parenchyma contains various alcohol dehydrogenase (ADH) isoenzymes and also possesses aldehyde dehydrogenase (ALDH) activity. The altered activities of ADH and ALDH in damaged pancreatic tissue in the course of pancreatitis are reflected in the human serum. The aim of this study was to investigate a potential role of ADH and ALDH as markers for acute (AP) and chronic pancreatitis (CP). Serum samples were collected for routine biochemical investigations from 75 patients suffering from acute pancreatitis and 70 patients with chronic pancreatitis. Fluorometric methods were used to measure the activity of class I and II ADH and ALDH activity. The total ADH activity and activity of class III and IV isoenzymes were measured by a photometric method. There was a significant increase in the activity of ADH III isoenzyme (15.06 mU/l and 14.62 mU/l vs. 11.82 mU/l; p<0.001) and total ADH activity (764 mU/l and 735 mU/l vs. 568 mU/l) in the sera of patients with acute pancreatitis or chronic pancreatitis compared to the control. The diagnostic sensitivity for ADH III was about 84%, specificity was 92 %, positive and negative predictive values were 93% and 87% respectively in acute pancreatitis. Area under the Receiver Operating Curve (ROC) curve for ADH III in AP and CP was 0.88 and 0.86 respectively. ADH III has a potential role as a marker of acute and chronic pancreatitis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

PubMed

Lerchner, Alexandra; Jarasch, Alexander; Meining, Winfried; Schiefner, André; Skerra, Arne

2013-11-01

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. © 2013 Wiley Periodicals, Inc.

Pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice after subchronic exposure to ethanol

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

Kaphalia, Bhupendra S., E-mail: bkaphali@utmb.ed; Bhopale, Kamlesh K.; Kondraganti, Shakuntala

2010-08-01

Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH{sup -}) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH{sup -} and hepatic ADH-normal (ADH{sup +}) deer mice fed 1%, 2% or 3.5% ethanolmore » via Lieber-DeCarli liquid diet daily for 2 months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was {approx} 1.5-fold greater in ADH{sup -} vs. ADH{sup +} deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH{sup -} deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.« less

Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

NASA Astrophysics Data System (ADS)

Kohen, Amnon; Cannio, Raffaele; Bartolucci, Simonetta; Klinman, Judith P.; Klinman, Judith P.

1999-06-01

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65°C this is analogous to previous findings with mesophilic ADH at 25°C ( ref. 5). Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

Determining the roles of the three alcohol dehydrogenases (AdhA, AdhB and AdhE) in Thermoanaerobacter ethanolicus during ethanol formation.

PubMed

Zhou, Jilai; Shao, Xiongjun; Olson, Daniel G; Murphy, Sean Jean-Loup; Tian, Liang; Lynd, Lee R

2017-05-01

Thermoanaerobacter ethanolicus is a promising candidate for biofuel production due to the broad range of substrates it can utilize and its high ethanol yield compared to other thermophilic bacteria, such as Clostridium thermocellum. Three alcohol dehydrogenases, AdhA, AdhB and AdhE, play key roles in ethanol formation. To study their physiological roles during ethanol formation, we deleted them separately and in combination. Previously, it has been thought that both AdhB and AdhE were bifunctional alcohol dehydrogenases. Here we show that AdhE has primarily acetyl-CoA reduction activity (ALDH) and almost no acetaldehyde reduction (ADH) activity, whereas AdhB has no ALDH activity and but high ADH activity. We found that AdhA and AdhB have similar patterns of activity. Interestingly, although deletion of both adhA and adhB reduced ethanol production, a single deletion of either one actually increased ethanol yields by 60-70%.

Interaction of cytoplasmic dehydrogenases: quantitation of pathways of ethanol metabolism.

PubMed

Vind, C; Grunnet, N

1983-01-01

The interaction between xylitol, alcohol and lactate dehydrogenase has been studied in hepatocytes from rats by applying specifically tritiated substrates. A simple model, describing the metabolic fate of tritium from [2-3H] xylitol and (1R) [1-3H]ethanol is presented. The model allows calculation of the specific radioactivity of free, cytosolic NADH, based on transfer of tritium to lactate, glucose and water. From the initial labelling rate of lactate and the specific radioactivity of cytosolic NADH, we have determined the reversible flow through the lactate dehydrogenase catalyzed reaction to 1-5 mumol/min . g wet wt. The results suggest that xylitol, alcohol and lactate dehydrogenase share the same pool of NAD(H) in the cytoplasma. This finding allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-pro-R hydrogen of ethanol and the hydrogen bound to carbon 2 of xylitol or carbon 2 of lactate under identical conditions.

A Hepatocyte-Mimicking Antidote for Alcohol Intoxication.

PubMed

Xu, Duo; Han, Hui; He, Yuxin; Lee, Harrison; Wu, Di; Liu, Fang; Liu, Xiangsheng; Liu, Yang; Lu, Yunfeng; Ji, Cheng

2018-04-11

Alcohol intoxication causes serious diseases, whereas current treatments are mostly supportive and unable to remove alcohol efficiently. Upon alcohol consumption, alcohol is sequentially oxidized to acetaldehyde and acetate by the endogenous alcohol dehydrogenase and aldehyde dehydrogenase, respectively. Inspired by the metabolism of alcohol, a hepatocyte-mimicking antidote for alcohol intoxication through the codelivery of the nanocapsules of alcohol oxidase (AOx), catalase (CAT), and aldehyde dehydrogenase (ALDH) to the liver, where AOx and CAT catalyze the oxidation of alcohol to acetaldehyde, while ALDH catalyzes the oxidation of acetaldehyde to acetate. Administered to alcohol-intoxicated mice, the antidote rapidly accumulates in the liver and enables a significant reduction of the blood alcohol concentration. Moreover, blood acetaldehyde concentration is maintained at an extremely low level, significantly contributing to liver protection. Such an antidote, which can eliminate alcohol and acetaldehyde simultaneously, holds great promise for the treatment of alcohol intoxication and poisoning and can provide therapeutic benefits. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The two-step electrochemical oxidation of alcohols using a novel recombinant PQQ alcohol dehydrogenase as a catalyst for a bioanode.

PubMed

Takeda, Kouta; Matsumura, Hirotoshi; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko; Nakamura, Nobuhumi; Ohno, Hiroyuki

2013-12-01

A bioanode has been developed based on the oxidation of ethanol by the recombinant pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase from Pseudomonas putidaKT2440 heterologously expressed in Pichia pastoris. The apo form of the recombinant protein (PpADH) was purified and displayed catalytic activity for binding PQQ in the presence of Ca(2+). PpADH exhibited broad substrate specificity towards various alcohols and aldehydes. The Km values for the aldehydes of PpADH were increased compared to those for the alcohols, whereas the kcat values were unaltered. For instance, the Km values at T=298.15K (25 °C) for ethanol and acetaldehyde were 0.21 (± 0.02)mM and 5.8 (± 0.60)mM, respectively. The kcat values for ethanol and acetaldehyde were 24.8 (± 1.2) s(-1) and 31.1 (± 1.2) s(-1), respectively. The aminoferrocene was used as an electron transfer mediator between PpADH and the electrode during electrochemical experiments. The catalytic currents for the oxidation of alcohol and acetaldehyde by PpADH were also observed in this system. The electric charge for the oxidation of ethanol (Q = 2.09 × 10(-3) · C) was increased two-fold compared to that for the oxidation of acetaldehyde (Q = 0.95 × 10(-3) · C), as determined by chronoamperometric measurements. Thus, we have electrochemically demonstrated the two-step oxidation of ethanol to acetate using only PpADH. © 2013.

Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem.

PubMed

Damiani, Isabelle; Morreel, Kris; Danoun, Saïda; Goeminne, Geert; Yahiaoui, Nabila; Marque, Christiane; Kopka, Joachim; Messens, Eric; Goffner, Deborah; Boerjan, Wout; Boudet, Alain-Michel; Rochange, Soizic

2005-11-01

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

Polymorphisms in the promoter region of the human class II alcohol dehydrogenase (ADH4) gene affect both transcriptional activity and ethanol metabolism in Japanese subjects.

PubMed

Kimura, Yukiko; Nishimura, Fusae T; Abe, Shuntaro; Fukunaga, Tatsushige; Tanii, Hideji; Saijoh, Kiyofumi

2009-02-01

Class II alcohol dehydrogenase (pi-ADH), encoded by alcohol dehydrogenase (ADH4), is considered to contribute to ethanol (EtOH) oxidation in the liver at high concentration. Four single nucleotide polymorphisms (SNPs) were found in the promoter region of this gene. Analysis of genotype distribution in 102 unrelated Japanese subjects revealed that four loci were in strong linkage disequilibrium and could be classified into three haplotypes. The effects of these polymorphisms on transcriptional activity were investigated in HepG2 cells. Transcriptional activity was significantly higher in cells with the -136A allele than in those with the -136C allele. To investigate whether this difference in transcriptional activity caused a difference in EtOH elimination, previous data on blood EtOH changes after 0.4 g/kg body weight alcohol ingestion were analyzed. When analyzed based on aldehyde dehydrogenase-2 gene (ALDH2) (487)Glu/Lys genotype, the significantly lower level of EtOH at peak in subjects with -136C/A and -136A/A genotype compared with subjects with -136C/C genotype indicated that -136 bp was a suggestive locus for differences in EtOH oxidation. This effect was observed only in subjects with ALDH2 (487)Glu/Glu. These results suggested that the SNP at -136bp in the ADH4 promoter had an effect on transcriptional regulation, and that the higher activity of the -136A allele compared with the -136C allele caused a lower level of blood EtOH after alcohol ingestion; that is, individuals with the -136A allele may consume more EtOH and might have a higher risk for development of alcohol dependence than those without the -136A allele.

Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus.

PubMed

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.

Role of tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase.

PubMed

Pennacchio, Angela; Esposito, Luciana; Zagari, Adriana; Rossi, Mosè; Raia, Carlo A

2009-09-01

A mutant of the thermostable NAD(+)-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp --> Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD(+) and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 A resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.

Development of an alcohol dehydrogenase biosensor for ethanol determination with toluidine blue O covalently attached to a cellulose acetate modified electrode.

PubMed

Alpat, Senol; Telefoncu, Azmi

2010-01-01

In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10(-5) M and 4 × 10(-4) M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10(-6) M. At the end of the 20(th) day, the biosensor still retained 50% of its initial activity.

Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

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

Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.

1991-04-01

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose tomore » ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).« less

Development of an Alcohol Dehydrogenase Biosensor for Ethanol Determination with Toluidine Blue O Covalently Attached to a Cellulose Acetate Modified Electrode

PubMed Central

Alpat, Şenol; Telefoncu, Azmi

2010-01-01

In this work, a novel voltammetric ethanol biosensor was constructed using alcohol dehydrogenase (ADH). Firstly, alcohol dehydrogenase was immobilized on the surface of a glassy carbon electrode modified by cellulose acetate (CA) bonded to toluidine blue O (TBO). Secondly, the surface was covered by a glutaraldehyde/bovine serum albumin (BSA) cross-linking procedure to provide a new voltammetric sensor for the ethanol determination. In order to fabricate the biosensor, a new electrode matrix containing insoluble Toluidine Blue O (TBO) was obtained from the process, and enzyme/coenzyme was combined on the biosensor surface. The influence of various experimental conditions was examined for the characterization of the optimum analytical performance. The developed biosensor exhibited sensitive and selective determination of ethanol and showed a linear response between 1 × 10−5 M and 4 × 10−4 M ethanol. A detection limit calculated as three times the signal-to-noise ratio was 5.0 × 10−6 M. At the end of the 20th day, the biosensor still retained 50% of its initial activity. PMID:22315566

Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

PubMed Central

Ismaiel, A A; Zhu, C X; Colby, G D; Chen, J S

1993-01-01

Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants. Images PMID:8349550

Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

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

Brown, Steven D; Guss, Adam M; Karpinets, Tatiana V

2011-01-01

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assaysmore » confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.« less

Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2) by in silico design.

PubMed

Cassidy, Jennifer; Bruen, Larah; Rosini, Elena; Molla, Gianluca; Pollegioni, Loredano; Paradisi, Francesca

2017-01-01

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

Genetics of alcoholism.

PubMed

Edenberg, Howard J; Foroud, Tatiana

2014-01-01

Multiple lines of evidence strongly indicate that genetic factors contribute to the risk for alcohol use disorders (AUD). There is substantial heterogeneity in AUD, which complicates studies seeking to identify specific genetic factors. To identify these genetic effects, several different alcohol-related phenotypes have been analyzed, including diagnosis and quantitative measures related to AUDs. Study designs have used candidate gene analyses, genetic linkage studies, genomewide association studies (GWAS), and analyses of rare variants. Two genes that encode enzymes of alcohol metabolism have the strongest effect on AUD: aldehyde dehydrogenase 2 and alcohol dehydrogenase 1B each has strongly protective variants that reduce risk, with odds ratios approximately 0.2-0.4. A number of other genes important in AUD have been identified and replicated, including GABRA2 and alcohol dehydrogenases 1B and 4. GWAS have identified additional candidates. Rare variants are likely also to play a role; studies of these are just beginning. A multifaceted approach to gene identification, targeting both rare and common variations and assembling much larger datasets for meta-analyses, is critical for identifying the key genes and pathways important in AUD. © 2014 Elsevier B.V. All rights reserved.

Three alcohol dehydrogenase genes and one acetyl-CoA synthetase gene are responsible for ethanol utilization in Yarrowia lipolytica.

PubMed

Gatter, Michael; Ottlik, Stephanie; Kövesi, Zsolt; Bauer, Benjamin; Matthäus, Falk; Barth, Gerold

2016-10-01

The non-conventional yeast Yarrowia lipolytica is able to utilize a wide range of different substrates like glucose, glycerol, ethanol, acetate, proteins and various hydrophobic molecules. Although most metabolic pathways for the utilization of these substrates have been clarified by now, it was not clear whether ethanol is oxidized by alcohol dehydrogenases or by an alternative oxidation system inside the cell. In order to detect the genes that are required for ethanol utilization in Y. lipolytica, eight alcohol dehydrogenase (ADH) genes and one alcohol oxidase gene (FAO1) have been identified and respective deletion strains were tested for their ability to metabolize ethanol. As a result of this, we found that the availability of ADH1, ADH2 or ADH3 is required for ethanol utilization in Y. lipolytica. A strain with deletions in all three genes is lacking the ability to utilize ethanol as sole carbon source. Although Adh2p showed by far the highest enzyme activity in an in vitro assay, the availability of any of the three genes was sufficient to enable a decent growth. In addition to ADH1, ADH2 and ADH3, an acetyl-CoA synthetase encoding gene (ACS1) was found to be essential for ethanol utilization. As Y. lipolytica is a non-fermenting yeast, it is neither able to grow under anaerobic conditions nor to produce ethanol. To investigate whether Y. lipolytica may produce ethanol, the key genes of alcoholic fermentation in S. cerevisiae, ScADH1 and ScPDC1, were overexpressed in an ADH and an ACS1 deletion strain. However, instead of producing ethanol, the respective strains regained the ability to use ethanol as single carbon source and were still not able to grow under anaerobic conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular evolution and functional divergence of alcohol dehydrogenases in animals, fungi and plants.

PubMed

Thompson, Claudia E; Freitas, Loreta B; Salzano, Francisco M

2018-01-01

Alcohol dehydrogenases belong to the large superfamily of medium-chain dehydrogenases/reductases, which occur throughout the biological world and are involved with many important metabolic routes. We considered the phylogeny of 190 ADH sequences of animals, fungi, and plants. Non-class III Caenorhabditis elegans ADHs were seen closely related to tetrameric fungal ADHs. ADH3 forms a sister group to amphibian, reptilian, avian and mammalian non-class III ADHs. In fishes, two main forms are identified: ADH1 and ADH3, whereas in amphibians there is a new ADH form (ADH8). ADH2 is found in Mammalia and Aves, and they formed a monophyletic group. Additionally, mammalian ADH4 seems to result from an ADH1 duplication, while in Fungi, ADH formed clusters based on types and genera. The plant ADH isoforms constitute a basal clade in relation to ADHs from animals. We identified amino acid residues responsible for functional divergence between ADH types in fungi, mammals, and fishes. In mammals, these differences occur mainly between ADH1/ADH4 and ADH3/ADH5, whereas functional divergence occurred in fungi between ADH1/ADH5, ADH5/ADH4, and ADH5/ADH3. In fishes, the forms also seem to be functionally divergent. The ADH family expansion exemplifies a neofunctionalization process where reiterative duplication events are related to new activities.

Can We Rule Out Meningitis from Negative Jolt Accentuation? A Retrospective Cohort Study.

PubMed

Sato, Ryota; Kuriyama, Akira; Luthe, Sarah Kyuragi

2017-04-01

Jolt accentuation has been considered to be the most sensitive physical finding to predict meningitis. However, there are only a few studies assessing the diagnostic accuracy of jolt accentuation. Therefore, we aimed to evaluate the diagnostic accuracy of jolt accentuation and investigate whether it can be extended to patients with mild altered mental status. We performed a single center, retrospective observational study on patients who presented to the emergency department in a Japanese tertiary care center from January 1, 2010 to March 31, 2016. Jolt accentuation evaluated in patients with fever, headache, and mild altered mental status with Glasgow Coma Scale no lower than E2 or M4 was defined as "jolt accentuation in the broad sense." Jolt accentuation evaluated in patients with fever, headache, and no altered mental status was defined as "jolt accentuation in the narrow sense." We evaluated the sensitivity and specificity in both groups. Among 118 patients, the sensitivity and specificity of jolt accentuation in the broad sense were 70.7% (95% confidence interval (CI): 58.0%-80.8%) and 36.7% (95% CI: 25.6%-49.3%). The positive likelihood ratio and negative likelihood ratio were 1.12 (95% CI: 0.87-1.44) and 0.80 (95% CI: 0.48-1.34), respectively. Among 108 patients, the sensitivity and specificity of jot accentuation in the narrow sense were 75.0% (95% CI: 61.8%-84.8%) and 35.1% (95% CI: 24.0%-48.0%). The positive likelihood ratio and negative likelihood ratio were 1.16 (95% CI: 0.90-1.48) and 0.71 (95% CI: 0.40-1.28), respectively. Jolt accentuation itself has a limited value in the diagnosis of meningitis regardless of altered mental status. Therefore, meningitis should not be ruled out by negative jolt accentuation. © 2017 American Headache Society.

Functional characterization of SlscADH1, a fruit-ripening-associated short-chain alcohol dehydrogenase of tomato.

PubMed

Moummou, Hanane; Tonfack, Libert Brice; Chervin, Christian; Benichou, Mohamed; Youmbi, Emmanuel; Ginies, Christian; Latché, Alain; Pech, Jean-Claude; van der Rest, Benoît

2012-10-15

A tomato short-chain dehydrogenase-reductase (SlscADH1) is preferentially expressed in fruit with a maximum expression at the breaker stage while expression in roots, stems, leaves and flowers is very weak. It represents a potential candidate for the formation of aroma volatiles by interconverting alcohols and aldehydes. The SlscADH1 recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several volatile compounds present in tomato flavour with a strong preference for the NAD/NADH co-factors. The strongest activity was observed for the reduction of hexanal (K(m)=0.175mM) and phenylacetaldehyde (K(m)=0.375mM) in the presence of NADH. The oxidation process of hexanol and 1-phenylethanol was much less efficient (K(m)s of 2.9 and 23.0mM, respectively), indicating that the enzyme preferentially acts as a reductase. However activity was observed only for hexanal, phenylacetaldehyde, (E)-2-hexenal and acetaldehyde and the corresponding alcohols. No activity could be detected for other aroma volatiles important for tomato flavour, such as methyl-butanol/methyl-butanal, 5-methyl-6-hepten-2-one/5-methyl-6-hepten-2-ol, citronellal/citronellol, neral/nerol, geraniol. In order to assess the function of the SlscADH1 gene, transgenic plants have been generated using the technique of RNA interference (RNAi). Constitutive down-regulation using the 35S promoter resulted in the generation of dwarf plants, indicating that the SlscADH1 gene, although weakly expressed in vegetative tissues, had a function in regulating plant development. Fruit-specific down-regulation using the 2A11 promoter had no morphogenetic effect and did not alter the aldehyde/alcohol balance of the volatiles compounds produced by the fruit. Nevertheless, SlscADH1-inhibited fruit unexpectedly accumulated higher concentrations of C5 and C6 volatile compounds of the lipoxygenase pathway, possibly as an indirect effect of the suppression of SlscADH1 on the catabolism of

Characterization of an Arxula adeninivorans alcohol dehydrogenase involved in the metabolism of ethanol and 1-butanol.

PubMed

Kasprzak, Jakub; Rauter, Marion; Riechen, Jan; Worch, Sebastian; Baronian, Kim; Bode, Rüdiger; Schauer, Frieder; Kunze, Gotthard

2016-05-01

In this study, alcohol dehydrogenase 1 from Arxula adeninivorans (Aadh1p) was identified and characterized. Aadh1p showed activity with short and medium chain length primary alcohols in the forward reaction and their aldehydes in the reverse reaction. Aadh1p has 64% identity with Saccharomyces cerevisiae Adh1p, is localized in the cytoplasm and uses NAD(+) as cofactor. Gene expression analysis showed a low level increase in AADH1 gene expression with ethanol, pyruvate or xylose as the carbon source. Deletion of the AADH1 gene affects growth of the cells with 1-butanol, ethanol and glucose as the carbon source, and a strain which overexpressed the AADH1 gene metabolized 1-butanol more rapidly. An ADH activity assay indicated that Aadh1p is a major enzyme for the synthesis of ethanol and the degradation of 1-butanol in A. adeninivorans. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system.

PubMed

Heinstra, P W

1993-01-01

Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).

Fiber-Optic Bio-sniffer (Biochemical Gas Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde.

PubMed

Iitani, Kenta; Chien, Po-Jen; Suzuki, Takuma; Toma, Koji; Arakawa, Takahiro; Iwasaki, Yasuhiko; Mitsubayashi, Kohji

2018-02-23

Volatile organic compounds (VOCs) exhaled in breath have huge potential as indicators of diseases and metabolisms. Application of breath analysis for disease screening and metabolism assessment is expected since breath samples can be noninvasively collected and measured. In this research, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the AcH bio-sniffer, a reverse reaction of alcohol dehydrogenase (ADH) was employed for reducing AcH to ethanol and simultaneously consuming a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH). The concentration of AcH can be quantified by fluorescence detection of NADH that was consumed by reverse reaction of ADH. The AcH bio-sniffer was composed of an ultraviolet light-emitting diode (UV-LED) as an excitation light source, a photomultiplier tube (PMT) as a fluorescence detector, and an optical fiber probe, and these three components were connected with a bifurcated optical fiber. A gas-sensing region of the fiber probe was developed with a flow-cell and an ADH-immobilized membrane. In the experiment, after optimization of the enzyme reaction conditions, the selectivity and dynamic range of the AcH bio-sniffer were investigated. The AcH bio-sniffer showed a short measurement time (within 2 min) and a broad dynamic range for determination of gaseous AcH, 0.02-10 ppm, which encompassed a typical AcH concentration in exhaled breath (1.2-6.0 ppm). Also, the AcH bio-sniffer exhibited a high selectivity to gaseous AcH based on the specificity of ADH. The sensor outputs were observed only from AcH-contained standard gaseous samples. Finally, the AcH bio-sniffer was applied to measure the concentration of AcH in exhaled breath from healthy subjects after ingestion of alcohol. As a result, a significant difference of AcH concentration between subjects with different aldehyde dehydrogenase type 2 (ALDH2) phenotypes was observed. The AcH bio-sniffer can be

Molecular phylogeny and evolution of alcohol dehydrogenase (Adh) genes in legumes

PubMed Central

Fukuda, Tatsuya; Yokoyama, Jun; Nakamura, Toru; Song, In-Ja; Ito, Takuro; Ochiai, Toshinori; Kanno, Akira; Kameya, Toshiaki; Maki, Masayuki

2005-01-01

Background Nuclear genes determine the vast range of phenotypes that are responsible for the adaptive abilities of organisms in nature. Nevertheless, the evolutionary processes that generate the structures and functions of nuclear genes are only now be coming understood. The aim of our study is to isolate the alcohol dehydrogenase (Adh) genes in two distantly related legumes, and use these sequences to examine the molecular evolutionary history of this nuclear gene. Results We isolated the expressed Adh genes from two species of legumes, Sophora flavescens Ait. and Wisteria floribunda DC., by a RT-PCR based approach and found a new Adh locus in addition to homologues of the Adh genes found previously in legumes. To examine the evolution of these genes, we compared the species and gene trees and found gene duplication of the Adh loci in the legumes occurred as an ancient event. Conclusion This is the first report revealing that some legume species have at least two Adh gene loci belonging to separate clades. Phylogenetic analyses suggest that these genes resulted from relatively ancient duplication events. PMID:15836788

Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases*

PubMed Central

Nagel, Zachary D.; Cun, Shujian; Klinman, Judith P.

2013-01-01

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases. PMID:23525111

A novel alcohol dehydrogenase biosensor based on solid-state electrogenerated chemiluminescence by assembling dehydrogenase to Ru(bpy)(3)2+-Au nanoparticles aggregates.

PubMed

Zhang, Lihua; Xu, Zhiai; Sun, Xuping; Dong, Shaojun

2007-01-15

Based on electrogenerated chemiluminescence (ECL), a novel method for fabrication of alcohol dehydrogenase (ADH) biosensor by self-assembling ADH to Ru(bpy)(3)(2+)-AuNPs aggregates (Ru-AuNPs) on indium tin oxide (ITO) electrode surface has been developed. Positively charged Ru(bpy)(3)(2+) could be immobilized stably on the electrode surface with negatively charged AuNPs in the form of aggregate via electrostatic interaction. On the other hand, AuNPs are favourable candidates for the immobilization of enzymes because amine groups and cysteine residues in the enzymes are known to bind strongly with AuNPs. Moreover, AuNPs can act as tiny conduction centers to facilitate the transfer of electrons. Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection for quantification of enzyme substrate, and it displayed wide linear range, high sensitivity and good stability.

Methoxyflurane enhances allyl alcohol hepatotoxicity in rats. Possible involvement of increased acrolein formation.

PubMed

Kershaw, W C; Barsotti, D A; Leonard, T B; Dent, J G; Lage, G L

1989-01-01

The effect of methoxyflurane anesthesia on allyl alcohol-induced hepatotoxicity and the metabolism of allyl alcohol was studied in male rats. Hepatotoxicity was assessed by the measurement of serum alanine aminotransferase activity and histopathological examination. Allyl alcohol-induced hepatotoxicity was enhanced when allyl alcohol (32 mg/kg) was administered 4 hr before or up to 8 days after a single 10-min exposure to methoxyflurane vapors. The possibility that methoxyflurane increases alcohol dehydrogenase-dependent oxidation of allyl alcohol to acrolein, the proposed toxic metabolite, was evaluated by measuring the rate of acrolein formation in the presence of allyl alcohol and liver cytosol. The effect of methoxyflurane on alcohol dehydrogenase activity in liver cytosol was also assessed by measuring the rate of NAD+ utilization in the presence of ethyl alcohol or allyl alcohol. Alcohol dehydrogenase activity and rate of acrolein formation were elevated in methoxyflurane-pretreated rats. The results suggest that a modest increase in alcohol dehydrogenase activity and rate of acrolein formation markedly enhances allyl alcohol-induced hepatotoxicity.

Alcohol and aldehyde dehydrogenase polymorphisms and a new strategy for prevention and screening for cancer in the upper aerodigestive tract in East Asians.

PubMed

Yokoyama, Akira; Omori, Tai; Yokoyama, Tetsuji

2010-01-01

The ethanol in alcoholic beverages and the acetaldehyde associated with alcohol consumption are Group 1 human carcinogens (WHO, International Agency for Research on Cancer). The combination of alcohol consumption, tobacco smoking, the inactive heterozygous aldehyde dehydrogenase-2 genotype (ALDH2*1/*2) and the less-active homozygous alcohol dehydrogenase-1B genotype (ADH1B*1/*1) increases the risk of squamous cell carcinoma (SCC) in the upper aerodigestive tract (UADT) in a multiplicative fashion in East Asians. In addition to being exposed to locally high levels of ethanol, the UADT is exposed to a very high concentration of acetaldehyde from a variety of sources, including that as an ingredient of alcoholic beverages per se and that found in tobacco smoke; acetaldehyde is also produced by salivary microorganisms and mucosal enzymes and is present as blood acetaldehyde. The inefficient degradation of acetaldehyde by weakly expressed ALDH2 in the UADT may be cri! tical to the local accumulation of acetaldehyde, especially in ALDH2*1/*2 carriers. ADH1B*1/*1 carriers tend to experience less intense alcohol flushing and are highly susceptible to heavy drinking and alcoholism. Heavy drinking by persons with the less-active ADH1B*1/*1 leads to longer exposure of the UADT to salivary ethanol and acetaldehyde. The ALDH2*1/*2 genotype is a very strong predictor of synchronous and metachronous multiple SCCs in the UADT. High red cell mean corpuscular volume (MCV), esophageal dysplasia, and melanosis in the UADT, all of which are frequently found in ALDH2*1/*2 drinkers, are useful for identifying high-risk individuals. We invented a simple flushing questionnaire that enables prediction of the ALDH2 phenotype. New health appraisal models that include ALDH2 genotype, the simple flushing questionnaire, or MCV are powerful tools for devising a new strategy for prevention and screening for UADT cancer in East Asians.

Mitochondria-targeted ubiquinone (MitoQ) enhances acetaldehyde clearance by reversing alcohol-induced posttranslational modification of aldehyde dehydrogenase 2: A molecular mechanism of protection against alcoholic liver disease.

PubMed

Hao, Liuyi; Sun, Qian; Zhong, Wei; Zhang, Wenliang; Sun, Xinguo; Zhou, Zhanxiang

2018-04-01

Alcohol metabolism in the liver generates highly toxic acetaldehyde. Breakdown of acetaldehyde by aldehyde dehydrogenase 2 (ALDH2) in the mitochondria consumes NAD + and generates reactive oxygen/nitrogen species, which represents a fundamental mechanism in the pathogenesis of alcoholic liver disease (ALD). A mitochondria-targeted lipophilic ubiquinone (MitoQ) has been shown to confer greater protection against oxidative damage in the mitochondria compared to untargeted antioxidants. The present study aimed to investigate if MitoQ could preserve mitochondrial ALDH2 activity and speed up acetaldehyde clearance, thereby protects against ALD. Male C57BL/6J mice were exposed to alcohol for 8 weeks with MitoQ supplementation (5mg/kg/d) for the last 4 weeks. MitoQ ameliorated alcohol-induced oxidative/nitrosative stress and glutathione deficiency. It also reversed alcohol-reduced hepatic ALDH activity and accelerated acetaldehyde clearance through modulating ALDH2 cysteine S-nitrosylation, tyrosine nitration and 4-hydroxynonenol adducts formation. MitoQ ameliorated nitric oxide (NO) donor-mediated ADLH2 S-nitrosylation and nitration in Hepa-1c1c7 cells under glutathion depletion condition. In addition, alcohol-increased circulating acetaldehyde levels were accompanied by reduced intestinal ALDH activity and impaired intestinal barrier. In accordance, MitoQ reversed alcohol-increased plasma endotoxin levels and hepatic toll-like receptor 4 (TLR4)-NF-κB signaling along with subsequent inhibition of inflammatory cell infiltration. MitoQ also reversed alcohol-induced hepatic lipid accumulation through enhancing fatty acid β-oxidation. Alcohol-induced ER stress and apoptotic cell death signaling were reversed by MitoQ. This study demonstrated that speeding up acetaldehyde clearance by preserving ALDH2 activity critically mediates the beneficial effect of MitoQ on alcohol-induced pathogenesis at the gut-liver axis. Copyright © 2017 The Authors. Published by Elsevier B

Leucaena sp. recombinant cinnamyl alcohol dehydrogenase: purification and physicochemical characterization.

PubMed

Patel, Parth; Gupta, Neha; Gaikwad, Sushama; Agrawal, Dinesh C; Khan, Bashir M

2014-02-01

Cinnamyl alcohol dehydrogenase is a broad substrate specificity enzyme catalyzing the final step in monolignol biosynthesis, leading to lignin formation in plants. Here, we report characterization of a recombinant CAD homologue (LlCAD2) isolated from Leucaena leucocephala. LlCAD2 is 80 kDa homo-dimer associated with non-covalent interactions, having substrate preference toward sinapaldehyde with Kcat/Km of 11.6×10(6) (M(-1) s(-1)), and a possible involvement of histidine at the active site. The enzyme remains stable up to 40 °C, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 0.002 and 5h, respectively. LlCAD2 showed optimal activity at pH 6.5 and 9 for reduction and oxidation reactions, respectively, and was stable between pH 7 and 9, with the deactivation rate constant (Kd(*)) and half-life (t1/2) of 7.5×10(-4) and 15 h, respectively. It is a Zn-metalloenzyme with 4 Zn(2+) per dimer, however, was inhibited in presence of externally supplemented Zn(2+) ions. The enzyme was resistant to osmolytes, reducing agents and non-ionic detergents. Copyright © 2013 Elsevier B.V. All rights reserved.

Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

PubMed

Doody, Erin E; Groebner, Jennifer L; Walker, Jetta R; Frizol, Brittnee M; Tuma, Dean J; Fernandez, David J; Tuma, Pamela L

2017-12-01

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P 450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol-fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion, and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we coincubated cells with ethanol and diallyl sulfide (DAS; a CYP2E1 inhibitor) or N -acetyl cysteine (NAC; an antioxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both DAS and NAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted growth hormone-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity. NEW & NOTEWORTHY Impaired growth hormone-mediated signaling is observed in ethanol

Redox self-sufficient whole cell biotransformation for amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2014-10-15

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD(+) was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

PubMed Central

Li, Laigeng; Cheng, Xiao Fei; Leshkevich, Jacqueline; Umezawa, Toshiaki; Harding, Scott A.; Chiang, Vincent L.

2001-01-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography–mass spectrometry-based enzyme functional analysis and substrate level–controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was ∼60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin–enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin–enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms. PMID:11449052

The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase.

PubMed

Li, L; Cheng, X F; Leshkevich, J; Umezawa, T; Harding, S A; Chiang, V L

2001-07-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93

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

Kim, Keehyuk; Plapp, Bryce V.

The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/Km) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD+ and TFE or PFB are very similar to the structures formore » the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/Km) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/Km for (S)-2-alcohols without changing V/Km for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93.

PubMed

Kim, Keehyuk; Plapp, Bryce V

2017-10-01

The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/K m ) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD + and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/K m ) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/K m for (S)-2-alcohols without changing V/K m for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.

PubMed

Van Noorden, C J

1984-01-01

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity

Nitric oxide inhibition of alcohol dehydrogenase in fresh-cut apples ( Malus domestica Borkh).

PubMed

Amissah, Joris Gerald Niilante; Hotchkiss, Joseph H; Watkins, Chris B

2013-11-20

The effects of nitric oxide (NO) and nitrite treatment on alcohol dehydrogenase activity and the shelf life of apple tissue were investigated. Fresh-cut apple slices were stored for 2 days at 6 °C in 0.25-1% NO (v/v, balance N2) or 100% N2 atmospheres. Slices were also treated with 1% NO or 2 mM sodium nitrite (NaNO2) for 20 min, stored for 6 weeks in 100% N2 at 6 °C, and analyzed for acetaldehyde, ethanol, and ethyl acetate accumulation, firmness, and color. Compared with N2 or deionized water controls, treatment with 1% NO or 2 mM NaNO2 inhibited ethanol accumulation, whereas that of acetaldehyde increased. Ethyl acetate accumulation was inhibited only by NO. Slice firmness was not affected by NO or NaNO2 treatment, but slices were darker than the untreated controls. NO and nitrite may extend the shelf life of fresh-cut produce with low concentrations of phenolic compounds.

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

PubMed

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

2017-12-01

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

Association between alcohol dehydrogenase 1C gene *1/*2 polymorphism and pancreatitis risk: a meta-analysis.

PubMed

Fang, F; Pan, J; Su, G H; Xu, L X; Li, G; Li, Z H; Zhao, H; Wang, J

2015-11-30

Numerous studies have focused on the relationship be-tween alcohol dehydrogenase 1C gene (ADH1C) *1/*2 polymorphism (Ile350Val, rs698, also known as ADH1C *1/*2) and pancreatitis risk, but the results have been inconsistent. Thus, we conducted a meta-anal-ysis to more precisely estimate this association. Relevant publications were searched in several widely used databases and 9 eligible studies were included in the meta-analysis. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association. Significant associations between ADH1C *1/*2 poly-morphism and pancreatitis risk were observed in both overall meta-analysis for 12 vs 22 (OR = 1.53, 95%CI = 1.12-2.10) and 11 + 12 vs 22 (OR = 1.44, 95%CI = 1.07-1.95), and the chronic alcoholic pancre-atitis subgroup for 12 vs 22 (OR = 1.64, 95%CI = 1.17-2.29) and 11 + 12 vs 22 (OR = 1.53, 95%CI = 1.11-2.11). Significant pancreatitis risk variation was also detected in Caucasians for 11 + 12 vs 22 (OR = 1.45, 95%CI = 1.07-1.98). In conclusion, the ADH1C *1/*2 polymorphism is likely associated with pancreatitis risk, particularly chronic alcoholic pancreatitis risk, with the *1 allele functioning as a risk factor.

In vitro expression of Candida albicans alcohol dehydrogenase genes involved in acetaldehyde metabolism.

PubMed

Bakri, M M; Rich, A M; Cannon, R D; Holmes, A R

2015-02-01

Alcohol consumption is a risk factor for oral cancer, possibly via its conversion to acetaldehyde, a known carcinogen. The oral commensal yeast Candida albicans may be one of the agents responsible for this conversion intra-orally. The alcohol dehydrogenase (Adh) family of enzymes are involved in acetaldehyde metabolism in yeast but, for C. albicans it is not known which family member is responsible for the conversion of ethanol to acetaldehyde. In this study we determined the expression of mRNAs from three C. albicans Adh genes (CaADH1, CaADH2 and CaCDH3) for cells grown in different culture media at different growth phases by Northern blot analysis and quantitative reverse transcription polymerase chain reaction. CaADH1 was constitutively expressed under all growth conditions but there was differential expression of CaADH2. CaADH3 expression was not detected. To investigate whether CaAdh1p or CaAdh2p can contribute to alcohol catabolism in C. albicans, each gene from the reference strain C. albicans SC5314 was expressed in Saccharomyces cerevisiae. Cell extracts from an CaAdh1p-expressing S. cerevisiae recombinant, but not an CaAdh2p-expressing recombinant, or an empty vector control strain, possessed ethanol-utilizing Adh activity above endogenous S. cerevisiae activity. Furthermore, expression of C. albicans Adh1p in a recombinant S. cerevisiae strain in which the endogenous ScADH2 gene (known to convert ethanol to acetaldehyde in this yeast) had been deleted, conferred an NAD-dependent ethanol-utilizing, and so acetaldehyde-producing, Adh activity. We conclude that CaAdh1p is the enzyme responsible for ethanol use under in vitro growth conditions, and may contribute to the intra-oral production of acetaldehyde. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa.

PubMed

Chao, Nan; Liu, Shu-Xin; Liu, Bing-Mei; Li, Ning; Jiang, Xiang-Ning; Gai, Ying

2014-11-01

Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

PubMed

Jun, Se-Young; Walker, Alexander M; Kim, Hoon; Ralph, John; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

2017-08-01

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p -coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum ( Sorghum bicolor ), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis ( Arabidopsis thaliana ) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p -coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing. © 2017 American Society of Plant Biologists. All Rights Reserved.

Geraniol and Geranial Dehydrogenases Induced in Anaerobic Monoterpene Degradation by Castellaniella defragrans

PubMed Central

Lüddeke, Frauke; Wülfing, Annika; Timke, Markus; Germer, Frauke; Weber, Johanna; Dikfidan, Aytac; Rahnfeld, Tobias; Linder, Dietmar; Meyerdierks, Anke

2012-01-01

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (kcat/Km = 2.02 × 106 M−1 s−1), followed by geraniol (kcat/Km = 1.57 × 106 M−1 s−1). Apparent Km values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid. PMID:22286981

Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans.

PubMed

Lüddeke, Frauke; Wülfing, Annika; Timke, Markus; Germer, Frauke; Weber, Johanna; Dikfidan, Aytac; Rahnfeld, Tobias; Linder, Dietmar; Meyerdierks, Anke; Harder, Jens

2012-04-01

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k(cat)/K(m) = 2.02 × 10(6) M(-1) s(-1)), followed by geraniol (k(cat)/K(m) = 1.57 × 10(6) M(-1) s(-1)). Apparent K(m) values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.

Expression of catalase, alcohol dehydrogenase, and malate dehydrogenase in rot grains upon fungicide use on maize hybrids grown at different spacings.

PubMed

Kluge, E R; Mendes, M C; Faria, M V; Santos, H O; Santos, L A; Sandini, I E

2017-04-20

In this study, we evaluated the fungicide effect on the incidence of rot grains and expression of catalase (CAT), alcohol dehydrogenase (ADH), and malate dehydrogenase (MDH) enzymes in commercial maize hybrids grown with conventional and reduced spacing in Guarapuava, PR, Brazil. The experiment was designed in random blocks with a 3 × 8-factorial scheme, totaling 24 treatments. The first factor constituted three levels, the first with foliar fungicide application [150.0 g/L trifloxystrobin (15.0%, w/v) + 175.0 g/L prothioconazole (17.5%, w/v)] at a dose of 0.4 L/ha at V8-stage eight expanded leaves and the second with an application of 0.5 L/ha at VT-tasseling and check (no fungicide application) stage. The second factor comprised eight maize hybrids that were divided into two groups, complex (AG 9045PRO, AG 8041PRO, DKB245PRO2, and 2B707PW) and susceptible (P 32R48H, DKB390PRO, P 30F53H, and P 30R50H), according to their reaction to the causative fungus, totaling 72 plots at each site in the crop of 2013/2014. The percentage of rot grains and the expression of CAT, ADH, and MDH were evaluated for each hybrid. The percentage of rot grains was influenced by the hybrid and fungicide used. The (trifloxystrobin + prothioconazole) reduced the incidence of rot grains, with relatively higher reduction in the hybrids considered susceptible. The higher expression of CAT enzyme was related to the higher incidence of rot grains because of grain deterioration, depending on the hybrids evaluated. A higher expression of ADH and MDH enzymes was observed in the maize hybrids belonging to the group considered tolerant.

Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

PubMed Central

Mårdh, G; Dingley, A L; Auld, D S; Vallee, B L

1986-01-01

Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine. PMID:3466164

Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

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

Plapp, Bryce V.; Ramaswamy, S.; Iowa)

2013-01-16

Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 andmore » Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.« less

Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

PubMed Central

Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

2010-01-01

Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

PubMed Central

de Vries, G E; Arfman, N; Terpstra, P; Dijkhuizen, L

1992-01-01

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed. Images PMID:1644761

Catalytic Mechanism of Short Ethoxy Chain Nonylphenol Dehydrogenase Belonging to a Polyethylene Glycol Dehydrogenase Group in the GMC Oxidoreductase Family

PubMed Central

Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

2013-01-01

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor. PMID:23306149

Catalytic mechanism of short ethoxy chain nonylphenol dehydrogenase belonging to a polyethylene glycol dehydrogenase group in the GMC oxidoreductase family.

PubMed

Liu, Xin; Ohta, Takeshi; Kawabata, Takeshi; Kawai, Fusako

2013-01-10

Ethoxy (EO) chain nonylphenol dehydrogenase (NPEO-DH) from Ensifer sp. AS08 and EO chain octylphenol dehydrogenase from Pseudomonas putida share common molecular characteristics with polyethylene glycol (PEG) dehydrogenases (PEG-DH) and comprise a PEG-DH subgroup in the family of glucose-methanol-choline (GMC) oxidoreductases that includes glucose/alcohol oxidase and glucose/choline dehydrogenase. Three-dimensional (3D) molecular modeling suggested that differences in the size, secondary structure and hydropathy in the active site caused differences in their substrate specificities toward EO chain alkylphenols and free PEGs. Based on 3D molecular modeling, site-directed mutagenesis was utilized to introduce mutations into potential catalytic residues of NPEO-DH. From steady state and rapid kinetic characterization of wild type and mutant NPEO-DHs, we can conclude that His465 and Asn507 are directly involved in the catalysis. Asn507 mediates the transfer of proton from a substrate to FAD and His465 transfers the same proton from the reduced flavin to an electron acceptor.

The multifunctional isopropyl alcohol dehydrogenase of Phytomonas sp. could be the result of a horizontal gene transfer from a bacterium to the trypanosomatid lineage.

PubMed

Molinas, Sara M; Altabe, Silvia G; Opperdoes, Fred R; Rider, Mark H; Michels, Paul A M; Uttaro, Antonio D

2003-09-19

Isopropyl alcohol dehydrogenase (iPDH) is a dimeric mitochondrial alcohol dehydrogenase (ADH), so far detected within the Trypanosomatidae only in the genus Phytomonas. The cloning, sequencing, and heterologous expression of the two gene alleles of the enzyme revealed that it is a zinc-dependent medium-chain ADH. Both polypeptides have 361 amino acids. A mitochondrial targeting sequence was identified. The mature proteins each have 348 amino acids and a calculated molecular mass of 37 kDa. They differ only in one amino acid, which can explain the three isoenzymes and their respective isoelectric points previously found. A phylogenetic analysis locates iPDH within a cluster with fermentative ADHs from bacteria, sharing 74% similarity and 60% identity with Ralstonia eutropha ADH. The characterization of the two bacterially expressed Phytomonas enzymes and the comparison of their kinetic properties with those of the wild-type iPDH and of the R. eutropha ADH strongly support the idea of a horizontal gene transfer event from a bacterium to a trypanosomatid to explain the origin of the iPDH in Phytomonas. Phytomonas iPDH and R. eutropha ADH are able to use a wide range of substrates with similar Km values such as primary and secondary alcohols, diols, and aldehydes, as well as ketones such as acetone, diacetyl, and acetoin. We speculate that, as for R. eutropha ADH, Phytomonas iPDH acts as a safety valve for the release of excess reducing power.

Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

PubMed

Beauchamp, Justin; Vieille, Claire

2015-01-01

N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

Mannitol and Mannitol Dehydrogenases in Conidia of Aspergillus oryzae

PubMed Central

Horikoshi, Koki; Iida, Shigeji; Ikeda, Yonosuke

1965-01-01

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

Immobilization of alcohol dehydrogenase in phospholipid Langmuir-Blodgett films to detect ethanol.

PubMed

Caseli, Luciano; Perinotto, Angelo C; Viitala, Tapani; Zucolotto, Valtencir; Oliveira, Osvaldo N

2009-03-03

Enzyme immobilization in nanostructured films may be useful for a number of biomimetic systems, particularly if suitable matrixes are identified. Here we show that alcohol dehydrogenase (ADH) has high affinity toward a negatively charged phospholipid, dimyristoylphosphatidic acid (DMPA), which forms a Langmuir monolayer at an air-water interface. Incorporation of ADH into the DMPA monolayer was monitored with surface pressure measurements and polarization-modulation infrared reflection absorption spectroscopy, with the alpha-helices from ADH being mainly oriented parallel to the water surface. ADH remained at the interface even at high surface pressures, thus allowing deposition of Langmuir-Blodgett (LB) films from the DMPA-ADH film. Indeed, interaction with DMPA enhances the transfer of ADH, where the mass transferred onto a solid support increased from 134 ng for ADH on a Gibbs monolayer to 178 ng for an LB film with DMPA. With fluorescence spectroscopy it was possible to confirm that the ADH structure was preserved even after one month of the LB deposition. ADH-containing films deposited onto gold-interdigitated electrodes were employed in a sensor array capable of detecting ethanol at concentrations down to 10 ppb (in volume), using impedance spectroscopy as the method of detection.

Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: assessment of the effects on first-pass metabolism of ethanol.

PubMed

Lee, Shou-Lun; Lee, Yung-Pin; Wu, Min-Li; Chi, Yu-Chou; Liu, Chiu-Ming; Lai, Ching-Long; Yin, Shih-Jiun

2015-05-01

Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5 mM NAD(+). Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5 mM) that the decrease of activities at 2-10 mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75-86% and 31-52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10-50 μM acetaldehyde to be 62-73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin. Copyright © 2015 Elsevier Inc. All rights reserved.

High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots.

PubMed

dos Santos, W D; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, O

2006-01-01

This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.

Genes contributing to the development of alcoholism: an overview.

PubMed

Edenberg, Howard J

2012-01-01

Genetic factors (i.e., variations in specific genes) account for a substantial portion of the risk for alcoholism. However, identifying those genes and the specific variations involved is challenging. Researchers have used both case-control and family studies to identify genes related to alcoholism risk. In addition, different strategies such as candidate gene analyses and genome-wide association studies have been used. The strongest effects have been found for specific variants of genes that encode two enzymes involved in alcohol metabolism-alcohol dehydrogenase and aldehyde dehydrogenase. Accumulating evidence indicates that variations in numerous other genes have smaller but measurable effects.

Comparative evolutionary genomics of the HADH2 gene encoding Aβ-binding alcohol dehydrogenase/17β-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10)

PubMed Central

Marques, Alexandra T; Antunes, Agostinho; Fernandes, Pedro A; Ramos, Maria J

2006-01-01

Background The Aβ-binding alcohol dehydrogenase/17β-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10) is an enzyme involved in pivotal metabolic processes and in the mitochondrial dysfunction seen in the Alzheimer's disease. Here we use comparative genomic analyses to study the evolution of the HADH2 gene encoding ABAD/HSD10 across several eukaryotic species. Results Both vertebrate and nematode HADH2 genes showed a six-exon/five-intron organization while those of the insects had a reduced and varied number of exons (two to three). Eutherian mammal HADH2 genes revealed some highly conserved noncoding regions, which may indicate the presence of functional elements, namely in the upstream region about 1 kb of the transcription start site and in the first part of intron 1. These regions were also conserved between Tetraodon and Fugu fishes. We identified a conserved alternative splicing event between human and dog, which have a nine amino acid deletion, causing the removal of the strand βF. This strand is one of the seven strands that compose the core β-sheet of the Rossman fold dinucleotide-binding motif characteristic of the short chain dehydrogenase/reductase (SDR) family members. However, the fact that the substrate binding cleft residues are retained and the existence of a shared variant between human and dog suggest that it might be functional. Molecular adaptation analyses across eutherian mammal orthologues revealed the existence of sites under positive selection, some of which being localized in the substrate-binding cleft and in the insertion 1 region on loop D (an important region for the Aβ-binding to the enzyme). Interestingly, a higher than expected number of nonsynonymous substitutions were observed between human/chimpanzee and orangutan, with six out of the seven amino acid replacements being under molecular adaptation (including three in loop D and one in the substrate binding loop). Conclusion Our study revealed that HADH2 genes maintained a

Impact of chronic low to moderate alcohol consumption on blood lipid and heart energy profile in acetaldehyde dehydrogenase 2-deficient mice.

PubMed

Fan, Fan; Cao, Quan; Wang, Cong; Ma, Xin; Shen, Cheng; Liu, Xiang-wei; Bu, Li-ping; Zou, Yun-zeng; Hu, Kai; Sun, Ai-jun; Ge, Jun-bo

2014-08-01

To investigate the roles of acetaldehyde dehydrogenase 2 (ALDH2), the key enzyme of ethanol metabolism, in chronic low to moderate alcohol consumption-induced heart protective effects in mice. Twenty-one male wild-type (WT) or ALDH2-knockout (KO) mice were used in this study. In each genotype, 14 animals received alcohol (2.5%, 5% and 10% in week 1-3, respectively, and 18% in week 4-7), and 7 received water for 7 weeks. After the treatments, survival rate and general characteristics of the animals were evaluated. Serum ethanol and acetaldehyde levels and blood lipids were measured. Metabolomics was used to characterize the heart and serum metabolism profiles. Chronic alcohol intake decreased the survival rate of KO mice by 50%, and significantly decreased their body weight, but did not affect those of WT mice. Chronic alcohol intake significantly increased the serum ethanol levels in both WT and KO mice, but KO mice had significantly higher serum acetaldehyde levels than WT mice. Chronic alcohol intake significantly increased the serum HDL cholesterol levels in WT mice, and did not change the serum HDL cholesterol levels in KO mice. After chronic alcohol intake, WT and KO mice showed differential heart and serum metabolism profiles, including the 3 main energy substrate types (lipids, glucose and amino acids) and three carboxylic acid cycles. Low to moderate alcohol consumption increases HDL cholesterol levels and improves heart energy metabolism profile in WT mice but not in ALDH2-KO mice. Thus, preserved ALDH2 function is essential for the protective effect of low to moderate alcohol on the cardiovascular system.

Human class I alcohol dehydrogenases catalyze the oxidation of glycols in the metabolism of norepinephrine.

PubMed Central

Mårdh, G; Luehr, C A; Vallee, B L

1985-01-01

Investigations of the function of human liver alcohol dehydrogenase (ADH) in norepinephrine metabolism have revealed that class I ADH catalyzes the oxidation of the intermediary alcohols 4-hydroxy-3-methoxyphenyl glycol (HMPG) and 3,4-dihydroxyphenyl glycol (DHPG) in vitro. The kcat/Km values for the individual homogeneous class I isozymes are generally in the range from 2.0 to 10 mM-1 X min-1, slightly lower than those obtained for ethanol oxidation, 16-66 mM-1 X min-1, but considerably higher than those obtained for ethylene glycol oxidation, 0.23-1.5 mM-1 X min-1. Importantly, HMPG and DHPG are not substrates for the class II or class III ADHs. 4-Methylpyrazole and 1,10-phenanthroline inhibit the class I ADH-catalyzed oxidation of HMPG, DHPG, and ethanol with inhibition constants of 75-90 nM and 19-22 microM, respectively, indicating that these substrates interact at the same catalytic site of ADH. Moreover, ethanol inhibits the oxidation of HMPG. The competition of ethanol with HMPG for ADH provides a basis for the in vivo changes observed in norepinephrine metabolism after acute ethanol intake. Any assessment of norepinephrine function through the study of metabolites in peripheral body fluid must include monitoring the oxidation of HMPG by ADH. PMID:3161078

Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis.

PubMed

Pan, Haiyun; Zhou, Rui; Louie, Gordon V; Mühlemann, Joëlle K; Bomati, Erin K; Bowman, Marianne E; Dudareva, Natalia; Dixon, Richard A; Noel, Joseph P; Wang, Xiaoqiang

2014-09-01

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. © 2014 American Society of Plant Biologists. All rights reserved.

Geraniol dehydrogenase, the key enzyme in biosynthesis of the alarm pheromone, from the astigmatid mite Carpoglyphus lactis (Acari: Carpoglyphidae).

PubMed

Noge, Koji; Kato, Makiko; Mori, Naoki; Kataoka, Michihiko; Tanaka, Chihiro; Yamasue, Yuji; Nishida, Ritsuo; Kuwahara, Yasumasa

2008-06-01

Geraniol dehydrogenase (GeDH), which plays an important role in the biosynthesis of neral, an alarm pheromone, was purified from the astigmatid mite Carpoglyphus lactis. The enzyme was obtained in an apparently homogeneous and active form after 1879-fold purification through seven steps of chromatography. Car. lactis GeDH was determined to be a monomer in its active form with a relative molecular mass of 42 800, which is a unique subunit structure in comparison with already established alcohol dehydrogenases. Car. lactis GeDH oxidized geraniol into geranial in the presence of NAD+. NADP+ was ineffective as a cofactor, suggesting that Car. lactis GeDH is an NAD+-dependent alcohol dehydrogenase. The optimal pH and temperature for geraniol oxidation were determined to be pH 9.0 and 25 degrees C, respectively. The Km values for geraniol and NAD+ were 51.0 microm and 59.5 microm, respectively. Car. lactis GeDH was shown to selectively oxidize geraniol, whereas its geometrical isomer, nerol, was inert as a substrate. The high specificity for geraniol suggests that Car. lactis GeDH specializes in the alarm pheromone biosynthesis of Car. lactis. Car. lactis GeDH is composed of 378 amino acids. Structurally, Car. lactis GeDH showed homology with zinc-dependent alcohol dehydrogenases found in mammals and a mosquito (36.6-37.6% identical), and the enzyme was considered to be a member of the medium-chain dehydrogenase/reductase family, in view of the highly conserved sequences of zinc-binding and NAD+-binding sites. Phylogenetic analyses indicate that Car. lactis GeDH could be categorized as a new class, different from other established alcohol dehydrogenases.

Environmental stresses of field growth allow cinnamyl alcohol dehydrogenase-deficient Nicotiana attenuata plants to compensate for their structural deficiencies.

PubMed

Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T

2012-08-01

The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants.

Ethanol at low concentrations protects glomerular podocytes through alcohol dehydrogenase and 20-HETE.

PubMed

McCarthy, Ellen T; Zhou, Jianping; Eckert, Ryan; Genochio, David; Sharma, Rishi; Oni, Olurinde; De, Alok; Srivastava, Tarak; Sharma, Ram; Savin, Virginia J; Sharma, Mukut

2015-01-01

Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high concentration or ADH inhibitor increased glomerular albumin permeability in vitro. 20-HETE and its metabolite produced by ADH activity, 20-carboxy-arachidonic acid, protected the glomerular permeability barrier against an ADH inhibitor, puromycin or FSGS permeability factor. We conclude that ADH activity is required for glomerular function, 20-HETE is a physiological substrate of ADH in podocytes and that podocytes are useful biosensors to understand glomeruloprotective effects of ethanol. Published by Elsevier Inc.

Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

NASA Astrophysics Data System (ADS)

Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

Ethanol at Low Concentrations Protects Glomerular Podocytes through Alcohol Dehydrogenase and 20-HETE

PubMed Central

McCarthy, Ellen T.; Zhou, Jianping; Eckert, Ryan; Genochio, David; Sharma, Rishi; Oni, Olurinde; De, Alok; Srivastava, Tarak; Sharma, Ram; Savin, Virginia J.; Sharma, Mukut

2014-01-01

Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high concentration or ADH inhibitor increased glomerular albumin permeability in vitro. 20-HETE and its metabolite produced by ADH activity, 20-carboxy-arachidonic acid, protected the glomerular permeability barrier against an ADH inhibitor, puromycin or FSGS permeability factor. We conclude that ADH activity is required for glomerular function, 20-HETE is a physiological substrate of ADH in podocytes and that podocytes are useful biosensors to understand glomeruloprotective effects of ethanol. PMID:25447342

Suitability of the hydrocarbon-hydroxylating molybdenum-enzyme ethylbenzene dehydrogenase for industrial chiral alcohol production.

PubMed

Tataruch, M; Heider, J; Bryjak, J; Nowak, P; Knack, D; Czerniak, A; Liesiene, J; Szaleniec, M

2014-12-20

The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular Control of the Induction of Alcohol Dehydrogenase by Ethanol in Drosophila Melanogaster Larvae

PubMed Central

Kapoun, A. M.; Geer, B. W.; Heinstra, PWH.; Corbin, V.; McKechnie, S. W.

1990-01-01

The activity of alcohol dehydrogenase (ADH:EC 1.1.1.1), the initial enzyme in the major pathway for ethanol degradation, is induced in Drosophila melanogaster larvae by low concentrations of dietary ethanol. Two lines of evidence indicate that the metabolic products of the ADH pathway for ethanol degradation are not directly involved in the induction of Adh. First, the accumulation of the proximal transcript in Adh(n2) larvae was increased when the intracellular level of ethanol was elevated. In addition, the ADH activity, the proximal Adh mRNA, and the intracellular concentration of ethanol were elevated coordinately in wild-type larvae fed hexadeuterated-ethanol, which is metabolized more slowly than normal ethanol. An examination of P element transformant lines with specific deletions in the 5' regulatory DNA of the Adh gene showed that the DNA sequence between +604 and +634 of the start site of transcription from the distal promoter was essential for this induction. The DNA sequence between -660 and about -5000 of the distal transcript start site was important for the down-regulation of the induction response. PMID:2157627

CvADH1, a member of short-chain alcohol dehydrogenase family, is inducible by gibberellin and sucrose in developing watermelon seeds.

PubMed

Kim, Joonyul; Kang, Hong-Gyu; Jun, Sung-Hoon; Lee, Jinwon; Yim, Jieun; An, Gynheung

2003-01-01

To understand the molecular mechanisms that control seed formation, we selected a seed-preferential gene (CvADH1) from the ESTs of developing watermelon seeds. RNA blot analysis and in situ localization showed that CvADH1 was preferentially expressed in the nucellar tissue. The CvADH1 protein shared about 50% homology with short-chain alcohol dehydrogenase including ABA2 in Arabidopsis thaliana, stem secoisolariciresinol dehydrogenase in Forsythia intermedia, and 3beta-hydroxysterol dehydrogenase in Digitalis lanata. We investigated gene-expression levels in seeds from both normally pollinated fruits and those made parthenocarpic via N-(2-chloro-4-pyridyl)-N'-phenylurea treatment, the latter of which lack zygotic tissues. Whereas the transcripts of CvADH1 rapidly started to accumulate from about the pre-heart stage in normal seeds, they were not detectable in the parthenocarpic seeds. Treating the parthenogenic fruit with GA(3) strongly induced gene expression, up to the level accumulated in pollinated seeds. These results suggest that the CvADH1 gene is induced in maternal tissues by signals made in the zygotic tissues, and that gibberellin might be one of those signals. We also observed that CvADH1 expression was induced by sucrose in the parthenocarpic seeds. Therefore, we propose that the CvADH1 gene is inducible by gibberellin, and that sucrose plays an important role in the maternal tissues of watermelon during early seed development.

Antidotes for poisoning by alcohols that form toxic metabolites.

PubMed

McMartin, Kenneth; Jacobsen, Dag; Hovda, Knut Erik

2016-03-01

The alcohols, methanol, ethylene glycol and diethylene glycol, have many features in common, the most important of which is the fact that the compounds themselves are relatively non-toxic but are metabolized, initially by alcohol dehydrogenase, to various toxic intermediates. These compounds are readily available worldwide in commercial products as well as in homemade alcoholic beverages, both of which lead to most of the poisoning cases, from either unintentional or intentional ingestion. Although relatively infrequent in overall occurrence, poisonings by metabolically-toxic alcohols do unfortunately occur in outbreaks and can result in severe morbidity and mortality. These poisonings have traditionally been treated with ethanol since it competes for the active site of alcohol dehydrogenase and decreases the formation of toxic metabolites. Although ethanol can be effective in these poisonings, there are substantial practical problems with its use and so fomepizole, a potent competitive inhibitor of alcohol dehydrogenase, was developed for a hopefully better treatment for metabolically-toxic alcohol poisonings. Fomepizole has few side effects and is easy to use in practice and it may obviate the need for haemodialysis in some, but not all, patients. Hence, fomepizole has largely replaced ethanol as the toxic alcohol antidote in many countries. Nevertheless, ethanol remains an important alternative because access to fomepizole can be limited, the cost may appear excessive, or the physician may prefer ethanol due to experience. © 2015 The British Pharmacological Society.

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

PubMed Central

Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

2014-01-01

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

Expression and characterization of a class III alcohol dehydrogenase gene from Gluconobacter frateurii in the presence of methanol during glyceric acid production from glycerol.

PubMed

Sato, Shun; Morita, Naoki; Kitamoto, Dai; Habe, Hiroshi

2013-01-01

Some acetic acid bacteria have been shown to produce large amounts of glyceric acid (GA) from glycerol, which is a by-product of biodiesel fuel (BDF) production. Previously, a Gluconobacter strain was found that produced decreased amounts of GA from glycerol in the presence of methanol, a major ingredient of raw glycerol derived from the BDF industry. Thus, a comparative transcriptome analysis of Gluconobacter frateurii NBRC103465 was performed to investigate changes in gene expression during GA production from glycerol in the presence of methanol. Cells grown with methanol showed upregulated expression of a class III alcohol dehydrogenase homolog (adhC(Gf)) and decreased GA production. adhC(Gf) was cloned and expressed heterologously in Escherichia coli, and the presence of an additional protein with an approximate molecular mass of 39 kDa in the cytosol of the recombinant E. coli cells was identified by SDS-PAGE. Activity measurements of the cytosol revealed that the translational product of adhC(Gf) exhibited formaldehyde dehydrogenase activity in the presence of nicotinamide adenine dinucleotide and glutathione. Gluconobacter frateurii cells grown in 1% methanol-containing glycerol were found to have fivefold higher formaldehyde dehydrogenase activity than cells grown without methanol, suggesting that adhC(Gf) in G. frateurii cells functions in the dissimilation of methanol-derived formaldehyde.

Alcoholism: genes and mechanisms.

PubMed

Oroszi, Gabor; Goldman, David

2004-12-01

Alcoholism is a chronic relapsing/remitting disease that is frequently unrecognized and untreated, in part because of the partial efficacy of treatment. Only approximately one-third of patients remain abstinent and one-third have fully relapsed 1 year after withdrawal from alcohol, with treated patients doing substantially better than untreated [1]. The partial effectiveness of strategies for prevention and treatment, and variation in clinical course and side effects, represent a challenge and an opportunity to better understand the neurobiology of addiction. The strong heritability of alcoholism suggests the existence of inherited functional variants of genes that alter the metabolism of alcohol and variants of other genes that alter the neurobiologies of reward, executive cognitive function, anxiety/dysphoria, and neuronal plasticity. Each of these neurobiologies has been identified as a critical domain in the addictions. Functional alleles that alter alcoholism-related intermediate phenotypes include common alcohol dehydrogenase 1B and aldehyde dehydrogenase 2 variants that cause the aversive flushing reaction; catechol-O-methyltransferase (COMT) Val158Met leading to differences in three aspects of neurobiology: executive cognitive function, stress/anxiety response, and opioid function; opioid receptor micro1 (OPRM1) Asn40Asp, which may serve as a gatekeeper molecule in the action of naltrexone, a drug used in alcoholism treatment; and HTTLPR, which alters serotonin transporter function and appears to affect stress response and anxiety/dysphoria, which are factors relevant to initial vulnerability, the process of addiction, and relapse.

Furaldehyde substrate specificity and kinetics of Saccharomyces cerevisiae alcohol dehydrogenase 1 variants.

PubMed

Laadan, Boaz; Wallace-Salinas, Valeria; Carlsson, Åsa Janfalk; Almeida, João Rm; Rådström, Peter; Gorwa-Grauslund, Marie F

2014-08-09

A previously discovered mutant of Saccharomyces cerevisiae alcohol dehydrogenase 1 (Adh1p) was shown to enable a unique NADH-dependent reduction of 5-hydroxymethylfurfural (HMF), a well-known inhibitor of yeast fermentation. In the present study, site-directed mutagenesis of both native and mutated ADH1 genes was performed in order to identify the key amino acids involved in this substrate shift, resulting in Adh1p-variants with different substrate specificities. In vitro activities of the Adh1p-variants using two furaldehydes, HMF and furfural, revealed that HMF reduction ability could be acquired after a single amino acid substitution (Y295C). The highest activity, however, was reached with the double mutation S110P Y295C. Kinetic characterization with both aldehydes and the in vivo primary substrate acetaldehyde also enabled to correlate the alterations in substrate affinity with the different amino acid substitutions. We demonstrated the key role of Y295C mutation in HMF reduction by Adh1p. We generated and kinetically characterized a group of protein variants using two furaldehyde compounds of industrial relevance. Also, we showed that there is a threshold after which higher in vitro HMF reduction activities do not correlate any more with faster in vivo rates of HMF conversion, indicating other cell limitations in the conversion of HMF.

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols.

PubMed

Yamamoto, Hiroaki; Kudoh, Masatake

2013-09-01

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99 % optical purity.

Monoterpene alcohol metabolism: identification, purification, and characterization of two geraniol dehydrogenase isoenzymes from Polygonum minus leaves.

PubMed

Hassan, Maizom; Maarof, Nur Diyana; Ali, Zainon Mohd; Noor, Normah Mohd; Othman, Roohaida; Mori, Nobuhiro

2012-01-01

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

Interaction between high-fat diet and alcohol dehydrogenase on ethanol-elicited cardiac depression in murine myocytes.

PubMed

Ren, Jun

2007-12-01

Consumption of high-fat diet and alcohol is associated with obesity, leading to enhanced morbidity and mortality. This study was designed to examine the interaction between high-fat diet and the alcohol metabolizing enzyme alcohol dehydrogenase (ADH) on ethanol-induced cardiac depression. Mechanical and intracellular Ca2+ properties were measured in cardiomyocytes from ADH transgenic and Friend Virus-B type (FVB) mice fed a low- or high-fat diet for 16 weeks. Expression of protein kinase B (Akt) and Foxo3a, two proteins essential for cardiac survival, was evaluated by Western blot. Cardiac damage was determined by carbonyl formation. High fat but not ADH induced obesity without hyperglycemia or hypertension, prolonged time-to-90% relengthening (TR90), and depressed peak shortening (PS) and maximal velocity of shortening/relengthening (+/- dL/dt) without affecting intracellular Ca2+ properties. Ethanol suppressed PS and intracellular Ca2+ rise in low-fat-fed FVB mouse cardiomyocytes. ADH but not high-fat diet shifted the threshold of ethanol-induced inhibition of PS and +/- dL/dt to lower levels. The amplitude of ethanol-induced cardiac depression was greater in the high-fat but not the ADH group without additive effects. Ethanol down- and up-regulated Akt and Foxo3a expression, respectively, and depressed intracellular Ca2+ rise, the effects of which were exaggerated by ADH, high-fat, or both. High-fat diet, but not ADH, enhanced Foxo3a expression and carbonyl content in non-ethanol-treated mice. Ethanol challenge significantly enhanced protein carbonyl formation, with the response being augmented by ADH, high-fat, or both. Our data suggest that high-fat diet and ADH transgene may exaggerate ethanol-induced cardiac depression and protein damage in response to ethanol.

Research on alcohol metabolism among Asians and its implications for understanding causes of alcoholism.

PubMed Central

Suddendorf, R F

1989-01-01

Research into the causes of alcoholism is a relatively recent scientific endeavor. One area of study which could lead to better understanding of the disease is the possibility of a genetic predisposition to alcoholism. Recent work has demonstrated that people have varying complements of enzymes to metabolize alcohol. Current knowledge is examined about the influence of various ethanol metabolizing enzymes on alcohol consumption by Asians and members of other ethnic groups. The two principal enzymes involved in ethanol oxidative metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH is responsible for the metabolism of ethanol to acetaldehyde. ALDH catalyzes the conversion of acetaldehyde to acetate. The different isozymes account for the diversity of alcohol metabolism among individuals. An isozyme of ADH (beta 2 beta 2) is found more frequently in Asians than in whites, and an ALDH isozyme (ALDH2), although present in Asians, often is in an inactive form. The presence of an inactive form of ALDH2 is thought to be responsible for an increase in acetaldehyde levels in the body. Acetaldehyde is considered responsible for the facial flushing reaction often observed among Asians who have consumed alcohol. A dysphoric reaction to alcohol, producing uncomfortable sensations, is believed to be a response to deter further consumption. Although the presence of an inactive ALDH2 isozyme may serve as a deterrent to alcohol consumption, its presence does not fully explain the levels of alcohol consumption by those with the inactive isozyme. Other conditions, such as social pressure, and yet undetermined biological factors, may play a significant role in alcohol consumption. PMID:2511595

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of

Alcohol dehydrogenase of acetic acid bacteria: structure, mode of action, and applications in biotechnology.

PubMed

Yakushi, Toshiharu; Matsushita, Kazunobu

2010-05-01

Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) of acetic acid bacteria is a membrane-bound enzyme involved in the acetic acid fermentation by oxidizing ethanol to acetaldehyde coupling with reduction of membranous ubiquinone (Q), which is, in turn, re-oxidized by ubiquinol oxidase, reducing oxygen to water. PQQ-ADHs seem to have co-evolved with the organisms fitting to their own habitats. The enzyme consists of three subunits and has a pyrroloquinoline quinone, 4 heme c moieties, and a tightly bound Q as the electron transfer mediators. Biochemical, genetic, and electrochemical studies have revealed the unique properties of PQQ-ADH since it was purified in 1978. The enzyme is unique to have ubiquinol oxidation activity in addition to Q reduction. This mini-review focuses on the molecular properties of PQQ-ADH, such as the roles of the subunits and the cofactors, particularly in intramolecular electron transport of the enzyme from ethanol to Q. Also, we summarize biotechnological applications of PQQ-ADH as to enantiospecific oxidations for production of the valuable chemicals and bioelectrocatalysis for sensors and fuel cells using indirect and direct electron transfer technologies and discuss unsolved issues and future prospects related to this elaborate enzyme.

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

PubMed Central

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

2012-01-01

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

Effects of Cavities at the Nicotinamide Binding Site of Liver Alcohol Dehydrogenase on Structure, Dynamics and Catalysis

PubMed Central

2015-01-01

A role for protein dynamics in enzymatic catalysis of hydrogen transfer has received substantial scientific support, but the connections between protein structure and catalysis remain to be established. Valine residues 203 and 207 are at the binding site for the nicotinamide ring of the coenzyme in liver alcohol dehydrogenase and have been suggested to facilitate catalysis with “protein-promoting vibrations” (PPV). We find that the V207A substitution has small effects on steady-state kinetic constants and the rate of hydrogen transfer; the introduced cavity is empty and is tolerated with minimal effects on structure (determined at 1.2 Å for the complex with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol). Thus, no evidence is found to support a role for Val-207 in the dynamics of catalysis. The protein structures and ligand geometries (including donor–acceptor distances) in the V203A enzyme complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol (determined at 1.1 Å) are very similar to those for the wild-type enzyme, except that the introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest, in contrast to previous studies, that the diminished tunneling and decreased rate of hydride transfer (16-fold, relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer, but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis. PMID:24437493

Genetic polymorphisms of ADH1B, ADH1C and ALDH2 in Turkish alcoholics: lack of association with alcoholism and alcoholic cirrhosis.

PubMed

Vatansever, Sezgin; Tekin, Fatih; Salman, Esin; Altintoprak, Ender; Coskunol, Hakan; Akarca, Ulus Salih

2015-05-17

No data exists regarding the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) gene polymorphisms in Turkish alcoholic cirrhotics. We studied the polymorphisms of ADH1B, ADH1C and ALDH2 genes in alcoholic cirrhotics and compared the results with non-cirrhotic alcoholics and healthy volunteers. Overall, 237 subjects were included for the study: 156 alcoholic patients (78 cirrhotics, 78 non-cirrhotic alcoholics) and 81 healthy volunteers. Three different single-nucleotide-polymorphism genotyping methods were used. ADH1C genotyping was performed using a polymerase chain reaction-restriction fragment length polymorphism method. The identified ADH1C genotypes were named according to the presence or absence of the enzyme restriction sites. ADH1B (Arg47Hys) genotyping was performed using the allele specific primer extension method, and ALDH2 (Glu487Lys) genotyping was performed by a multiplex polymerase chain reaction using two allele-specific primer pairs. For ADH1B, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 97.4%, 94.9% and 99.4%, respectively. For ADH1C, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 47%, 36.3% and 45%, respectively. There was no statistical difference between the groups for ADH1B and ADH1C (p>0.05). All alcoholic and non-alcoholic subjects (100%) had the allele *1 for ALDH2. The obtained results for ADH1B, ADH1C, and ALDH gene polymorphisms in the present study are similar to the results of Caucasian studies. ADH1B and ADH1C genetic variations are not related to the development of alcoholism or susceptibility to alcoholic cirrhosis. ALDH2 gene has no genetic variation in the Turkish population.

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

PubMed

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

2013-07-01

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

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.

PubMed

Sillers, Ryan; Al-Hinai, Mohab Ali; Papoutsakis, Eleftherios T

2009-01-01

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula.

PubMed

Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A; Hahn, Michael G; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A

2013-08-13

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

PubMed Central

Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A.; Hahn, Michael G.; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A.

2013-01-01

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure. PMID:23901113

Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Stam, L. F.; Laurie, C. C.

1996-01-01

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce ``superalleles'' with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect. PMID:8978044

Role of accentuation in the selection/rejection task framing effect.

PubMed

Chen, Jing; Proctor, Robert W

2017-04-01

Procedure invariance is a basic assumption of rational theories of choice, however, it has been shown to be violated: Different response modes, or task frames, sometimes reveal opposite preferences. The current study focused on selection and rejection task frames, involving a unique type of problem with enriched and impoverished options, which has previously led to conflicting findings and theoretical explanations: the compatibility hypothesis (Shafir, 1993) and the accentuation hypothesis (Wedell, 1997). We examined the role of task frame by distinguishing these 2 hypotheses and evaluating the information-processing basis of the choices. Experiments conducted online (Experiments 1 and 3) and in-lab (Experiment 4 with eye-tracking technique) revealed a difference between the 2 task frames in the choice data (i.e., the task-framing effect) as a function of the relative attractiveness of the options. Also, this task-framing effect was not influenced by imposed time constraints (Experiments 5 and 6) and was similarly evident with a more direct measure for the option attractiveness (obtained in Experiment 7). Experiment 2, conducted in a lab setting with verbal-protocol requirements, yielded no task-framing effect, suggesting that a requirement to verbalize reasons for choice minimizes accentuation. With this exception, the choice data are in agreement with the accentuation hypothesis, and the combined findings in choice, decision time, task confusion, and eye-tracking data provide evidence of a basis in cognitive effort rather than motivation, as Wedell proposed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Greater Strength Gains after Training with Accentuated Eccentric than Traditional Isoinertial Loads in Already Strength-Trained Men

PubMed Central

Walker, Simon; Blazevich, Anthony J.; Haff, G. Gregory; Tufano, James J.; Newton, Robert U.; Häkkinen, Keijo

2016-01-01

As training experience increases it becomes more challenging to induce further neuromuscular adaptation. Consequently, strength trainers seek alternative training methods in order to further increase strength and muscle mass. One method is to utilize accentuated eccentric loading, which applies a greater external load during the eccentric phase of the lift as compared to the concentric phase. Based upon this practice, the purpose of this study was to determine the effects of 10 weeks of accentuated eccentric loading vs. traditional isoinertial resistance training in strength-trained men. Young (22 ± 3 years, 177 ± 6 cm, 76 ± 10 kg, n = 28) strength-trained men (2.6 ± 2.2 years experience) were allocated to concentric-eccentric resistance training in the form of accentuated eccentric load (eccentric load = concentric load + 40%) or traditional resistance training, while the control group continued their normal unsupervised training program. Both intervention groups performed three sets of 6-RM (session 1) and three sets of 10-RM (session 2) bilateral leg press and unilateral knee extension exercises per week. Maximum force production was measured by unilateral isometric (110° knee angle) and isokinetic (concentric and eccentric 30°.s−1) knee extension tests, and work capacity was measured by a knee extension repetition-to-failure test. Muscle mass was assessed using panoramic ultrasonography and dual-energy x-ray absorptiometry. Surface electromyogram amplitude normalized to maximum M-wave and the twitch interpolation technique were used to examine maximal muscle activation. After training, maximum isometric torque increased significantly more in the accentuated eccentric load group than control (18 ± 10 vs. 1 ± 5%, p < 0.01), which was accompanied by an increase in voluntary activation (3.5 ± 5%, p < 0.05). Isokinetic eccentric torque increased significantly after accentuated eccentric load training only (10 ± 9%, p < 0.05), whereas concentric torque

Natural alcohol exposure: is ethanol the main substrate for alcohol dehydrogenases in animals?

PubMed

Hernández-Tobías, Aída; Julián-Sánchez, Adriana; Piña, Enrique; Riveros-Rosas, Héctor

2011-05-30

Alcohol dehydrogenase (ADH) activity is widely distributed in all phyla. In animals, three non-homologous NAD(P)(+)-dependent ADH protein families are reported. These arose independently throughout evolution and possess different structures and mechanisms of reaction: type I (medium-chain) ADHs are zinc-containing enzymes and comprise the most studied group in vertebrates; type II (short-chain) ADHs lack metal cofactor and have been extensively studied in Drosophila; and type III ADHs are iron-dependent/-activated enzymes that were initially identified only in microorganisms. The presence of these different ADHs in animals has been assumed to be a consequence of chronic exposure to ethanol. By far the most common natural source of ethanol is fermentation of fruit sugars by yeast, and available data support that this fruit trait evolved in concert with the characteristics of their frugivorous seed dispersers. Therefore, if the presence of ADHs in animals evolved as an adaptive response to dietary ethanol exposure, then it can be expected that the enzymogenesis of these enzymes began after the appearance of angiosperms with fleshy fruits, because substrate availability must precede enzyme selection. In this work, available evidence supporting this possibility is discussed. Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies (order Diptera). Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. In contrast, phyletic distribution of types I and III ADHs in animals showed that these appeared before angiosperms and land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol

Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

PubMed

Hempel, Niels; Görisch, Helmut; Mern, Demissew S

2013-09-01

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

Gene ercA, Encoding a Putative Iron-Containing Alcohol Dehydrogenase, Is Involved in Regulation of Ethanol Utilization in Pseudomonas aeruginosa

PubMed Central

Hempel, Niels; Görisch, Helmut

2013-01-01

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported. PMID:23813731

Inhibition of catalase-dependent ethanol metabolism in alcohol dehydrogenase-deficient deermice by fructose.

PubMed Central

Handler, J A; Bradford, B U; Glassman, E B; Forman, D T; Thurman, R G

1987-01-01

Hepatic microsomal fractions from ADH (alcohol dehydrogenase)-negative deermice incubated with an NADPH-generating system metabolized butanol and ethanol at rates around 10 nmol/min per mg. In contrast, cytosolic catalase from ADH-negative deermouse liver oxidized ethanol, but not butanol, when incubated with an H2O2-generating system. Thus butanol is oxidized by cytochrome P-450 in microsomal fractions, but not by cytosolic catalase, in tissues from ADH-negative deermice. In perfused livers from ADH-negative deermice, rates of ethanol uptake at low concentrations of ethanol (1.5 mM) were about 60 mumol/h per g, yet butanol (1.5 mM) uptake was undetectable (less than 4 mumol/h per g). At higher concentrations of alcohol (25-30 mM), rates of ethanol uptake were about 80 mumol/h per g, whereas rates of butanol uptake were only about 9 mumol/h per g. Because rates of butanol metabolism via cytochrome P-450 in deermice were more than an order of magnitude lower than rates of ethanol uptake in livers from ADH-negative deermice, it is concluded that ethanol uptake by perfused livers from ADH-negative deermice is catalysed predominantly via catalase-H2O2. In support of this conclusion, rates of H2O2 generation, which are rate-limiting for the peroxidation of ethanol by catalase, were about 65 mumol/h per g in livers from ADH-negative deermice, values similar to rates of ethanol uptake of about 60 mumol/h per g measured under identical conditions. Rates of ethanol uptake by perfused livers from ADH-positive, but not from ADH-negative, deermice were increased by about 50% by infusion of fructose. Thus it is concluded that the stimulation of hepatic ethanol uptake by fructose is dependent on the presence of ADH. Unexpectedly, fructose decreased rates of ethanol metabolism and H2O2 generation by about 60% in perfused livers from ADH-negative deermice, probably by decreasing activation of fatty acids and thus diminishing rates of peroxisomal beta-oxidation. PMID:3435455

Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3 alpha-hydroxysteroid dehydrogenase activity.

PubMed Central

Deyashiki, Y; Taniguchi, H; Amano, T; Nakayama, T; Hara, A; Sawada, H

1992-01-01

Two monomeric dihydrodiol dehydrogenases with pI values of 5.4 and 7.6 were co-purified with androsterone dehydrogenase activity to homogeneity from human liver. The two enzymes differed from each other on peptide mapping and in their heat-stabilities; with respect to the latter the dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities of the respective enzymes were similarly inactivated. The pI 5.4 enzyme was equally active towards trans- and cis-benzene dihydrodiols, and towards (S)- and (R)-forms of indan-1-ol and 1,2,3,4-tetrahydronaphth-1-ol and oxidized the 3 alpha-hydroxy group of C19-, C21- and C24-steroids, whereas the pI 7.6 enzyme showed high specificity for trans-benzene dihydrodiol, (S)-forms of the alicyclic alcohols and C19- and C21-steroids. Although the two enzymes reduced various xenobiotic carbonyl compounds and the 3-oxo group of C19- and C21-steroids, and were A-specific in the hydrogen transfer from NADPH, only the pI 5.4 enzyme showed reductase activity towards 7 alpha-hydroxy-5 beta-cholestan-3-one and dehydrolithocholic acid. The affinity of the two enzymes for the steroidal substrates was higher than that for the xenobiotic substrates. The two enzymes also showed different susceptibilities to the inhibition by anti-inflammatory drugs and bile acids. Whereas the pI-5.4 enzyme was highly sensitive to anti-inflammatory steroids, showing mixed-type inhibitions with respect to indan-1-ol and androsterone, the pI 7.6 enzyme was inhibited more potently by non-steroidal anti-inflammatory drugs and bile acids than by the steroidal drugs, and the inhibitions were all competitive. These structural and functional differences suggest that the two enzymes are 3 alpha-hydroxysteroid dehydrogenase isoenzymes. Images Fig. 2. PMID:1554355

Effects of a Transposable Element Insertion on Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Dunn, R. C.; Laurie, C. C.

1995-01-01

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called RI42) causes the low expression level, the insertion was excised from the cloned RI42 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(w(a)) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature. PMID:7498745

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism

PubMed Central

Crichton, Paul G.; Affourtit, Charles; Moore, Anthony L.

2006-01-01

In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria. PMID:16999687

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

PubMed

Frébortova; Matsushita; Arata; Adachi

1998-01-27

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Aldo-keto reductase and alcohol dehydrogenase contribute to benznidazole natural resistance in Trypanosoma cruzi.

PubMed

González, Laura; García-Huertas, Paola; Triana-Chávez, Omar; García, Gabriela Andrea; Murta, Silvane Maria Fonseca; Mejía-Jaramillo, Ana M

2017-12-01

The improvement of Chagas disease treatment is focused not only on the development of new drugs but also in understanding mechanisms of action and resistance to drugs conventionally used. Thus, some strategies aim to detect specific changes in proteins between sensitive and resistant parasites and to evaluate the role played in these processes by functional genomics. In this work, we used a natural Trypanosoma cruzi population resistant to benznidazole, which has clones with different susceptibilities to this drug without alterations in the NTR I gene. Using 2DE-gel electrophoresis, the aldo-keto reductase and the alcohol dehydrogenase proteins were found up regulated in the natural resistant clone and therefore their possible role in the resistance to benznidazole and glyoxal was investigated. Both genes were overexpressed in a drug sensitive T. cruzi clone and the biological changes in response to these compounds were evaluated. The results showed that the overexpression of these proteins enhances resistance to benznidazole and glyoxal in T. cruzi. Moreover, a decrease in mitochondrial and cell membrane damage was observed, accompanied by a drop in the intracellular concentration of reactive oxygen species after treatment. Our results suggest that these proteins are involved in the mechanism of action of benznidazole. © 2017 John Wiley & Sons Ltd.

Tea catechins and flavonoids from the leaves of Camellia sinensis inhibit yeast alcohol dehydrogenase.

PubMed

Manir, Md Maniruzzaman; Kim, Jeong Kee; Lee, Byeong-Gon; Moon, Surk-Sik

2012-04-01

Four new quercetin acylglycosides, designated camelliquercetisides A-D, quercetin 3-O-[α-L-arabinopyranosyl(1→3)][2-O″-(E)-p-coumaroyl][β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→6)]-β-D-glucoside (17), quercetin 3-O-[2-O″-(E)-p-coumaroyl][β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→6)]-β-D-glucoside (18), quercetin 3-O-[α-L-arabinopyranosyl(1→3)][2-O″-(E)-p-coumaroyl][α-L-rhamnopyranosyl(1→6)]-β-d-glucoside (19), and quercetin 3-O-[2-O″-(E)-p-coumaroyl][α-L-rhamnopyranosyl(1→6)]-β-D-glucoside (20), together with caffeine and known catechins, and flavonoids (1-16) were isolated from the leaves of Camellia sinensis. Their structures were determined by spectroscopic (1D and 2D NMR, IR, and HR-TOF-MS) and chemical methods. The catechins and flavonoidal glycosides exhibited yeast alcohol dehydrogenase (ADH) inhibitory activities in the range of IC(50) 8.0-70.3μM, and radical scavenging activities in the range of IC(50) 1.5-43.8 μM, measured by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Copyright © 2012 Elsevier Ltd. All rights reserved.

Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

NASA Technical Reports Server (NTRS)

Chung, H. J.; Ferl, R. J.

1999-01-01

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

The accentuation principle of figure-ground segregation and the downbeat illusion.

PubMed

Pinna, Baingio; Sirigu, Luca

2016-10-01

Pinna and Sirigu (2011) demonstrated a new principle of grouping, called the accentuation principle, stating that, all else being equal, elements tend to group in the same oriented direction of the discontinuous element placed within a whole set of continuous/homogeneous components. The discontinuous element behaves like an accent, i.e. a visual emphasis within the wholeness of components as shown in the next section. In this work, the accentuation principle has been extended to new visual domains. In particular, it is shown how this principle affects shape perception. Moreover several visual object attributes are also highlighted, among which orientation, spatial position, inner dynamics and apparent motion that determine the so-called organic segmentation and furthermore tend to induce figure-ground segregation. On the basis of the results of experimental phenomenology, the accentuation can be considered as a complex principle ruling grouping, figure-ground segregation, shape and meaning formation. Through a new musical illusion of downbeat, it is also demonstrated that this principle influences perceptual organization not only in space but also in time and, thus, in both visual and musical domains. This illusion can be heard in eight measures of Pagodes, a solo piano music by Claude Debussy (1862-1918), where a strong physical-perceptual discrepancy in terms of upbeats and downbeats inversion is strongly perceived in both staves. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Moderate Alcohol Consumption on Gene Expression Related to Colonic Inflammation and Antioxidant Enzymes in Rats

PubMed Central

Klarich, DawnKylee S.; Penprase, Jerrold; Cintora, Patricia; Medrano, Octavio; Erwin, Danielle; Brasser, Susan M.; Hong, Mee Young

2017-01-01

Excessive alcohol consumption is a risk factor associated with colorectal cancer; however, some studies have reported that moderate alcohol consumption may not contribute additional risk for developing colorectal cancer while others suggest that moderate alcohol consumption provides a protective effect that reduces colorectal cancer risk. The purpose of this study was to determine the effects of moderate voluntary alcohol (20% ethanol) intake on alternate days for 3 months in outbred Wistar rats on risk factors associated with colorectal cancer development. Colonic gene expression of cyclooxygenase-2, RelA, 8-oxoguanine DNA glycosylase 1, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase M1, and aldehyde dehydrogenase 2 were determined. Blood alcohol content, liver function enzyme activities, and 8-oxo-deoxyguanosine DNA adducts were also assessed. Alcohol-treated rats were found to have significantly lower 8-oxo-deoxyguanosine levels in blood, a marker of DNA damage. Alanine aminotransferase and lactate dehydrogenase were both significantly lower in the alcohol group. Moderate alcohol significantly decreased cyclooxygenase-2 gene expression, an inflammatory marker associated with colorectal cancer risk. The alcohol group had significantly increased glutathione-S-transferase M1 expression, an antioxidant enzyme that helps detoxify carcinogens, such as acetaldehyde, and significantly increased aldehyde dehydrogenase 2 expression, which allows for greater acetaldehyde clearance. Increased expression of glutathione-S-transferase M1 and aldehyde dehydrogenase 2 likely contributed to reduce mucosal damage that is caused by acetaldehyde accumulation. These results indicate that moderate alcohol may reduce the risk for colorectal cancer development, which was evidenced by reduced inflammation activity and lower DNA damage after alcohol exposure. PMID:28599714

Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7

PubMed Central

Singh, Vijay Shankar; Dubey, Ashutosh Prakash; Gupta, Ankush; Singh, Sudhir; Singh, Bhupendra Narain

2017-01-01

ABSTRACT Azospirillum brasilense Sp7 uses glycerol as a carbon source for growth and nitrogen fixation. When grown in medium containing glycerol as a source of carbon, it upregulates the expression of a protein which was identified as quinoprotein alcohol dehydrogenase (ExaA). Inactivation of exaA adversely affects the growth of A. brasilense on glycerol. A determination of the transcription start site of exaA revealed an RpoN-dependent −12/−24 promoter consensus. The expression of an exaA::lacZ fusion was induced maximally by glycerol and was dependent on σ54. Bioinformatic analysis of the sequence flanking the −12/−24 promoter revealed a 17-bp sequence motif with a dyad symmetry of 6 nucleotides upstream of the promoter, the disruption of which caused a drastic reduction in promoter activity. The electrophoretic mobility of a DNA fragment containing the 17-bp sequence motif was retarded by purified EraR, a LuxR-type transcription regulator that is transcribed divergently from exaA. EraR also showed a positive interaction with RpoN in two-hybrid and pulldown assays. IMPORTANCE Quinoprotein alcohol dehydrogenase (ExaA) plays an important role in the catabolism of alcohols in bacteria. Although exaA expression is thought to be regulated by a two-component system consisting of EraS and EraR, the mechanism of regulation was not known. This study shows the details of the regulation of expression of the exaA gene in A. brasilense. We have shown here that exaA of A. brasilense is maximally induced by glycerol and harbors a σ54-dependent promoter. The response regulator EraR binds to an inverted repeat located upstream of the exaA promoter. This study shows that a LuxR-type response regulator (EraR) binds upstream of the exaA gene and physically interacts with σ54. The unique feature of this regulation is that EraR is a LuxR-type transcription regulator that lacks the GAFTGA motif, a characteristic feature of the enhancer binding proteins that are known to

Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7.

PubMed

Singh, Vijay Shankar; Dubey, Ashutosh Prakash; Gupta, Ankush; Singh, Sudhir; Singh, Bhupendra Narain; Tripathi, Anil Kumar

2017-07-01

Azospirillum brasilense Sp7 uses glycerol as a carbon source for growth and nitrogen fixation. When grown in medium containing glycerol as a source of carbon, it upregulates the expression of a protein which was identified as quinoprotein alcohol dehydrogenase (ExaA). Inactivation of exaA adversely affects the growth of A. brasilense on glycerol. A determination of the transcription start site of exaA revealed an RpoN-dependent -12/-24 promoter consensus. The expression of an exaA :: lacZ fusion was induced maximally by glycerol and was dependent on σ 54 Bioinformatic analysis of the sequence flanking the -12/-24 promoter revealed a 17-bp sequence motif with a dyad symmetry of 6 nucleotides upstream of the promoter, the disruption of which caused a drastic reduction in promoter activity. The electrophoretic mobility of a DNA fragment containing the 17-bp sequence motif was retarded by purified EraR, a LuxR-type transcription regulator that is transcribed divergently from exaA EraR also showed a positive interaction with RpoN in two-hybrid and pulldown assays. IMPORTANCE Quinoprotein alcohol dehydrogenase (ExaA) plays an important role in the catabolism of alcohols in bacteria. Although exaA expression is thought to be regulated by a two-component system consisting of EraS and EraR, the mechanism of regulation was not known. This study shows the details of the regulation of expression of the exaA gene in A. brasilense We have shown here that exaA of A. brasilense is maximally induced by glycerol and harbors a σ 54 -dependent promoter. The response regulator EraR binds to an inverted repeat located upstream of the exaA promoter. This study shows that a LuxR-type response regulator (EraR) binds upstream of the exaA gene and physically interacts with σ 54 The unique feature of this regulation is that EraR is a LuxR-type transcription regulator that lacks the GAFTGA motif, a characteristic feature of the enhancer binding proteins that are known to interact with σ 54

Thermostable NADP+-Dependent Medium-Chain Alcohol Dehydrogenase from Acinetobacter sp. Strain M-1: Purification and Characterization and Gene Expression in Escherichia coli

PubMed Central

Tani, Akio; Sakai, Yasuyoshi; Ishige, Takeru; Kato, Nobuo

2000-01-01

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes. PMID:11097895

Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

PubMed Central

Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.

1997-01-01

This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

Effects of Beverages on Alcohol Metabolism: Potential Health Benefits and Harmful Impacts

PubMed Central

Wang, Fang; Zhang, Yu-Jie; Zhou, Yue; Li, Ya; Zhou, Tong; Zheng, Jie; Zhang, Jiao-Jiao; Li, Sha; Xu, Dong-Ping; Li, Hua-Bin

2016-01-01

Nonalcoholic beverages are usually consumed accompanying alcoholic drinks, and their effects on alcohol metabolism are unclear in vivo. In this study, the effects of 20 nonalcoholic beverages on alcohol metabolism and liver injury caused by alcohol were evaluated in mice. Kunming mice were orally fed with alcohol (52%, v/v) and beverages. The concentrations of ethanol and acetaldehyde in blood as well as the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in liver were assessed to indicate alcohol metabolism. The levels of aspartate aminotransferase (AST) and alanine transaminase (ALT) in serum as well as the levels of malonaldehyde (MDA) and superoxide dismutase (SOD) in liver were measured to reflect the alcohol-induced liver injury. The results showed that the treatment of soda water, green tea and honey chrysanthemum tea could accelerate ethanol metabolism and prevent liver injuries caused by alcohol when companied with excessive alcohol drinking. They might be potential dietary supplements for the alleviation of harmful effects from excessive alcohol consumption. On the contrary, some beverages such as fresh orange juice and red bull are not advised to drink when companied with alcohol consumption due to their adverse effects on ethanol induced liver injury. PMID:27005619

[Alcohol].

PubMed

Zima, T

1996-07-14

Alcohol is one of the most widely used addictive substances. It can be assumed that everybody encounters alcohol--ethanol in various forms and concentrations in the course of their lives. A global and social problem of our civilization is alcohol consumption which has a rising trend. Since 1989 the consumption of alcoholic beverages is rising and the mean annual consumption of concentrated ethanol per head is cea 10 litres. In ethanol abuse the organism is damaged not only by ethanol alone but in particular by substances formed during its metabolism. Its detailed knowledge is essential for the knowledge and investigations of the metabolic and toxic effect of ethanol on the organism. Ingested alcohol is in 90-98% eliminated from the organism by three known metabolic pathways: 1-alcohol dehydrogenase, 2-the microsomal ethanol oxidizing system and 3-catalase. Alcohol is a frequent important risk factor of serious "diseases of civilization" such as IHD, hypertension, osteoporosis, neoplastic diseases. Cirrhosis of the liver and chronic pancreatitis are the well known diseases associated with alcohol ingestion and also their most frequent cause. It is impossible to list all organs and diseases which develop as a result of alcohol consumption. It is important to realize that regular and "relatively" small amounts in the long run damage the organism and may be even fatal.

Finland Has it All? Examining the Media Accentuation of "Finnish Education" in Australia, Germany and South Korea

ERIC Educational Resources Information Center

Takayama, Keita; Waldow, Florian; Sung, Youl-Kwan

2013-01-01

Drawing on the conceptual work of externalisation in comparative education and multi-accentual signs in cultural studies, this article examines how the print news media accentuate "Finnish education" in the process of inserting this external reference into the domestic political discourses around education reform in Australia, Germany…

STRUCTURE TOXICITY IN RELATIONSHIPS FOR A,B-UNSATURATED ALCOHOLS IN FISH

EPA Science Inventory

Previous toxicity testing with fathead minnows (Pimephales promelas) indicated that some unsaturated acetylenic and allylic alcohols can be metabolically activated, via alcohol dehydrogenase, to highly toxic a,B-unsaturated aldehydes and ketones or allene derivatives. lthough sev...

Xylitol dehydrogenase from Candida tropicalis: molecular cloning of the gene and structural analysis of the protein.

PubMed

Lima, Luanne Helena Augusto; Pinheiro, Cristiano Guimarães do Amaral; de Moraes, Lídia Maria Pepe; de Freitas, Sonia Maria; Torres, Fernando Araripe Gonçalves

2006-12-01

Yeasts can metabolize xylose by the action of two key enzymes: xylose reductase and xylitol dehydrogenase. In this work, we present data concerning the cloning of the XYL2 gene encoding xylitol dehydrogenase from the yeast Candida tropicalis. The gene is present as a single copy in the genome and is controlled at the transcriptional level by the presence of the inducer xylose. XYL2 was functionally tested by heterologous expression in Saccharomyces cerevisiae to develop a yeast strain capable of producing ethanol from xylose. Structural analysis of C. tropicalis xylitol dehydrogenase, Xyl2, suggests that it is a member of the medium-chain dehydrogenase (MDR) family. This is supported by the presence of the amino acid signature [GHE]xx[G]xxxxx[G]xx[V] in its primary sequence and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD(+) and zinc ion binding domains.

Is HIV a model of accelerated or accentuated aging?

PubMed

Pathai, Sophia; Bajillan, Hendren; Landay, Alan L; High, Kevin P

2014-07-01

Antiretroviral therapy has reduced the incidence of adverse events and early mortality in HIV-infected persons. Despite these benefits, important comorbidities that increase with age (eg, diabetes, cardiovascular disease, cancer, liver disease, and neurocognitive impairment) are more prevalent in HIV-infected persons than in HIV-uninfected persons at every age, and geriatric syndromes such as falls and frailty occur earlier in HIV-infected persons. This raises a critical research question: Does HIV accelerate aging through pathways and mechanisms common to the aging process or is HIV simply an additional risk factor for a wide number of chronic conditions, thus accentuating aging? Extensive literature review. The purpose of this review is to briefly outline the evidence that age-related clinical syndromes are exacerbated by HIV, examine the ways in which HIV is similar, and dissimilar from natural aging, and assess the validity of HIV as a model of premature aging. Specific biomarkers of aging are limited in HIV-infected hosts and impacted by antiretroviral therapy, and a high rate of modifiable life style confounders (eg, smoking, substance abuse, alcohol) and coinfections (eg, hepatitis) in HIV-infected participants. There is a need for validated biomarkers of aging in the context of HIV. Despite these differences, welldesigned studies of HIV-infected participants are likely to provide new opportunities to better understand the mechanisms that lead to aging and age-related diseases. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pichia stipitis Genes for Alcohol Dehydrogenase with Fermentative and Respiratory Functions

PubMed Central

Cho, Jae-yong; Jeffries, Thomas W.

1998-01-01

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation. PMID:9546172

Conversion of alcohols to enantiopure amines through dual enzyme hydrogen-borrowing cascades

PubMed Central

Mutti, Francesco G.; Knaus, Tanja; Scrutton, Nigel S.; Breuer, Michael; Turner, Nicholas J.

2016-01-01

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds on industrial scale. Here we present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on the combination of an alcohol dehydrogenase (ADHs from Aromatoleum sp., Lactobacillus sp. and Bacillus sp.) enzyme operating in tandem with an amine dehydrogenase (AmDHs engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols (up to 96% conversion and 99% enantiomeric excess). Furthermore, primary alcohols are aminated with high conversion (up to 99%). This redox self-sufficient network possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product. PMID:26404833

Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades.

PubMed

Mutti, Francesco G; Knaus, Tanja; Scrutton, Nigel S; Breuer, Michael; Turner, Nicholas J

2015-09-25

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product. Copyright © 2015, American Association for the Advancement of Science.

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from the leaf tissues of a basin mangrove Lumnitzera racemosa Willd.

PubMed

Murugan, K; Arunkumar, N S; Mohankumar, C

2004-01-01

Cinnamyl alcohol-NADPH-dehydrogenase (CAD), the marker enzyme of lignin biosynthesis was purified from the leaf tissues of a basin mangrove Lumnitzera racemosa by ammonium sulphate precipitation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the CAD enzyme was determined as 89 kDa, by size elution chromatography. SDS-PAGE of CAD revealed two closely associated bands of 45 kDa and 42 kDa as heterogenous subunits. The optimum pH of CAD was found to be 4.0. Km for the substrates cinnamaldehyde, coniferaldehyde and sinapaldehyde was determined. Cinnamaldehyde showed higher Km value than sinapaldehyde and coniferaldehyde. The correlation of activity of CAD with the amount of lignin was found less significant in L. racemosa, compared to plant species of other habitats viz., mesophytes, xerophytes and hydrophytes, suggesting that CAD possibly exhibits physiological suppression due to the saline habitat of the plant.

Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies1[C][W][OA

PubMed Central

Kaur, Harleen; Shaker, Kamel; Heinzel, Nicolas; Ralph, John; Gális, Ivan; Baldwin, Ian T.

2012-01-01

The organized lignocellulosic assemblies of cell walls provide the structural integrity required for the large statures of terrestrial plants. Silencing two CINNAMYL ALCOHOL DEHYDROGENASE (CAD) genes in Nicotiana attenuata produced plants (ir-CAD) with thin, red-pigmented stems, low CAD and sinapyl alcohol dehydrogenase activity, low lignin contents, and rubbery, structurally unstable stems when grown in the glasshouse (GH). However, when planted into their native desert habitat, ir-CAD plants produced robust stems that survived wind storms as well as the wild-type plants. Despite efficient silencing of NaCAD transcripts and enzymatic activity, field-grown ir-CAD plants had delayed and restricted spread of red stem pigmentation, a color change reflecting blocked lignification by CAD silencing, and attained wild-type-comparable total lignin contents. The rubbery GH phenotype was largely restored when field-grown ir-CAD plants were protected from wind, herbivore attack, and ultraviolet B exposure and grown in restricted rooting volumes; conversely, it was lost when ir-CAD plants were experimentally exposed to wind, ultraviolet B, and grown in large pots in growth chambers. Transcript and liquid chromatography-electrospray ionization-time-of-flight analysis revealed that these environmental stresses enhanced the accumulation of various phenylpropanoids in stems of field-grown plants; gas chromatography-mass spectrometry and nuclear magnetic resonance analysis revealed that the lignin of field-grown ir-CAD plants had GH-grown comparable levels of sinapaldehyde and syringaldehyde cross-linked into their lignins. Additionally, field-grown ir-CAD plants had short, thick stems with normal xylem element traits, which collectively enabled field-grown ir-CAD plants to compensate for the structural deficiencies associated with CAD silencing. Environmental stresses play an essential role in regulating lignin biosynthesis in lignin-deficient plants. PMID:22645069

Surface functionalization of chitosan-coated magnetic nanoparticles for covalent immobilization of yeast alcohol dehydrogenase from Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Li, Gui-yin; Zhou, Zhi-de; Li, Yuan-jian; Huang, Ke-long; Zhong, Ming

2010-12-01

A novel and efficient immobilization of yeast alcohol dehydrogenase (YADH, EC1.1.1.1) from Saccharomyces cerevisiae has been developed by using the surface functionalization of chitosan-coated magnetic nanoparticles (Fe 3O 4/KCTS) as support. The magnetic Fe 3O 4/KCTS nanoparticles were prepared by binding chitosan alpha-ketoglutaric acid (KCTS) onto the surface of magnetic Fe 3O 4 nanoparticles. Later, covalent immobilization of YADH was attempted onto the Fe 3O 4/KCTS nanoparticles. The effect of various preparation conditions on the immobilized YADH process such as immobilization time, enzyme concentration and pH was investigated. The influence of pH and temperature on the activity of the free and immobilized YADH using phenylglyoxylic acid as substrate has also been studied. The optimum reaction temperature and pH value for the enzymatic conversion catalyzed by the immobilized YADH were 30 °C and 7.4, respectively. Compared to the free enzyme, the immobilized YADH retained 65% of its original activity and exhibited significant thermal stability and good durability.

Alcohol dehydrogenase AdhA plays a role in ethanol tolerance in model cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Vidal, Rebeca

2017-04-01

The protein AdhA from the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) has been previously reported to show alcohol dehydrogenase activity towards ethanol and both NAD and NADP. This protein is currently being used in genetically modified strains of Synechocystis capable of synthesizing ethanol showing the highest ethanol productivities. In the present work, mutant strains of Synechocystis lacking AdhA have been constructed and tested for tolerance to ethanol. The lack of AdhA in the wild-type strain reduces survival to externally added ethanol at lethal concentration of 4% (v/v). On the other hand, the lack of AdhA in an ethanologenic strain diminishes tolerance of cells to internally produced ethanol. It is also shown that light-activated heterotrophic growth (LAHG) of the wild-type strain is impaired in the mutant strain lacking AdhA (∆adhA strain). Photoautotrophic, mixotrophic, and photoheterotrophic growth are not affected in the mutant strain. Based on phenotypic characterization of ∆adhA mutants, the possible physiological function of AdhA in Synechocystis is discussed.

Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

PubMed Central

2012-01-01

Background The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied. Results The presence of S. bombicola immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of S. cerevisiae was also influenced by S. bombicola immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both PDC1 and ADH1 genes was highly induced at the initial phase of fermentation. The expression level of PDC1 at the end of fermentation was much higher in pure culture while ADH1 level was similar in both pure and mixed fermentations. Conclusion In mixed fermentation, S. bombicola immobilized cells greatly affected the fermentation behavior of S. cerevisiae and the analytical composition of wine. The influence of S. bombicola on S. cerevisiae was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during S. cerevisiae fermentation causing variation in the gene

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

PubMed Central

2009-01-01

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits. PMID:21637665

Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar.

PubMed

Baucher, M.; Chabbert, B.; Pilate, G.; Van Doorsselaere, J.; Tollier, M. T.; Petit-Conil, M.; Cornu, D.; Monties, B.; Van Montagu, M.; Inze, D.; Jouanin, L.; Boerjan, W.

1996-12-01

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

Study on immobilization of yeast alcohol dehydrogenase on nanocrystalline Ni-Co ferrites as magnetic support.

PubMed

Shakir, Mohammad; Nasir, Zeba; Khan, Mohd Shoeb; Lutfullah; Alam, Md Fazle; Younus, Hina; Al-Resayes, Saud Ibrahim

2015-01-01

The covalent binding of yeast alcohol dehydrogenase (YADH) enzyme complex in a series of magnetic crystalline Ni-Co nanoferrites, synthesized via sol-gel auto combustion technique was investigated. The structural analysis, morphology and magnetic properties of Ni-Co nanoferrites were determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), vibrating-sample magnetometer (VSM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). The comparative analysis of the HRTEM micrographs of bare magnetic nanoferrite particles and particles immobilized with enzyme revealed an uniform distribution of the particles in both the cases without undergoing change in the size which was found to be in the range 20-30 nm. The binding of YADH to Ni-Co nanoferrites and the possible binding mechanism have been suggested by comparing the FTIR results. The binding properties of the immobilized YADH enzyme were also studied by kinetic parameters, optimum operational pH, temperature, thermal stability and reusability. The immobilized YADH exhibits enhanced thermal stability as compared to the free enzyme over a wide range of temperature and pH, and showed good durability after recovery by magnetic separation for repeated use. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel roles for AhR and ARNT in the regulation of alcohol dehydrogenases in human hepatic cells.

PubMed

Attignon, Eléonore A; Leblanc, Alix F; Le-Grand, Béatrice; Duval, Caroline; Aggerbeck, Martine; Rouach, Hélène; Blanc, Etienne B

2017-01-01

The mechanisms by which pollutants participate in the development of diverse pathologies are not completely understood. The pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates the AhR (aryl hydrocarbon receptor) signaling pathway. We previously showed that TCDD (25 nM, 30 h) decreased the expression of several alcohol metabolism enzymes (cytochrome P450 2E1, alcohol dehydrogenases ADH1, 4 and 6) in differentiated human hepatic cells (HepaRG). Here, we show that, as rapidly as 8 h after treatment (25 nM TCDD) ADH expression decreased 40 % (p < 0.05). ADH1 and 4 protein levels decreased 40 and 27 %, respectively (p < 0.05), after 72 h (25 nM TCDD). The protein half-lives were not modified by TCDD which suggests transcriptional regulation of expression. The AhR antagonist CH-223191 or AhR siRNA reduced the inhibitory effect of 25 nM TCDD on ADH1A, 4 and 6 expression 50-100 % (p < 0.05). The genomic pathway (via the AhR/ARNT complex) and not the non-genomic pathway involving c-SRC mediated these effects. Other AhR ligands (3-methylcholanthrene and PCB 126) decreased ADH1B, 4 and 6 mRNAs by more than 78 and 55 %, respectively (p < 0.01). TCDD also regulated the expression of ADH4 in the HepG2 human hepatic cell line, in primary human hepatocytes and in C57BL/6J mouse liver. In conclusion, activation of the AhR/ARNT signaling pathway by AhR ligands represents a novel mechanism for regulating the expression of ADHs. These effects may be implicated in the toxicity of AhR ligands as well as in the alteration of ethanol or retinol metabolism and may be associated further with higher risk of liver diseases or/and alcohol abuse disorders.

Proteomic Profiling of Liver and Plasma in Chronic Ethanol Feeding Model of Hepatic Alcohol Dehydrogenase-Deficient Deer Mice.

PubMed

Bhopale, Kamlesh K; Amer, Samir M; Kaphalia, Lata; Soman, Kizhake V; Wiktorowicz, John E; Shakeel Ansari, Ghulam A; Kaphalia, Bhupendra S

2017-10-01

Chronic alcohol abuse, a major risk factor for such diseases as hepatitis and cirrhosis, impairs hepatic alcohol dehydrogenase (ADH; key ethanol [EtOH]-metabolizing enzyme). Therefore, differentially altered hepatic and plasma proteomes were identified in chronic EtOH feeding model of hepatic ADH-deficient (ADH - ) deer mice to understand the metabolic basis of alcoholic liver disease (ALD). ADH - deer mice were fed 3.5 g% EtOH via Lieber-DeCarli liquid diet daily for 3 months and histology of the liver assessed. Liver and plasma proteins were separated by 2-dimensional gel electrophoresis. The proteins differentially expressed were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Histology of the liver showed panlobular steatosis and infiltration of T lymphocytes. Using the criteria of ≥1.5 for fold change (p-value ≤0.05) with expectation value (E ≤10 -3 ) and protein score (≥64), 18 proteins in the livers and 5 in the plasma of EtOH-fed mice were differentially expressed and identified. Prolyl 4-hydroxylase, cytochrome b-5, endo A cytokeratin, ATP synthase, heat-shock 70 kD proteins, enoyl CoA hydratase, stress-70 protein, peroxiredoxin 1, and ornithine carbamoyl transferase were up-regulated in the livers. However, carbonic anhydrase 3, mitochondrial ATP synthase, aldolase 2, actin γ, laminin receptor, and carbamoyl phosphate synthase were down-regulated. Contrary to the increased expression of creatine kinase M-type, a decreased expression of serine protease inhibitor A3A precursor, sulfated glycoprotein-2 (clusterin), and apolipoprotein E isoforms were found in the plasma of EtOH group. Chronic EtOH feeding in ADH - deer mice causes steatosis and infiltration of T lymphocytes in the livers along with increased expression of proteins involved in endoplasmic reticulum (ER) stress, fibrosis, fatty acid β oxidation and biogenesis, and decreased expression of proteins involved in ATP synthesis, carbohydrate

Alcoholism and liver disease in Mexico: genetic and environmental factors.

PubMed

Roman, Sonia; Zepeda-Carrillo, Eloy Alfonso; Moreno-Luna, Laura Eugenia; Panduro, Arturo

2013-11-28

Alcoholism and cirrhosis, which are two of the most serious health problems worldwide, have a broad spectrum of clinical outcomes. Both diseases are influenced by genetic susceptibility and cultural traits that differ globally but are specific for each population. In contrast to other regions around the world, Mexicans present the highest drinking score and a high mortality rate for alcoholic liver disease with an intermediate category level of per capita alcohol consumption. Mexico has a unique history of alcohol consumption that is linked to profound anthropological and social aspects. The Mexican population has an admixture genome inherited from different races, Caucasian, Amerindian and African, with a heterogeneous distribution within the country. Thus, genes related to alcohol addiction, such as dopamine receptor D2 in the brain, or liver alcohol-metabolizing enzymes, such as alcohol dehydrogenase class I polypeptide B, cytochrome P450 2E1 and aldehyde dehydrogenase class 2, may vary from one individual to another. Furthermore, they may be inherited as risk or non-risk haplogroups that confer susceptibility or resistance either to alcohol addiction or abusive alcohol consumption and possibly liver disease. Thus, in this era of genomics, personalized medicine will benefit patients if it is directed according to individual or population-based data. Additional association studies will be required to establish novel strategies for the prevention, care and treatment of liver disease in Mexico and worldwide.

Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding.

PubMed

Kaphalia, Lata; Boroumand, Nahal; Hyunsu, Ju; Kaphalia, Bhupendra S; Calhoun, William J

2014-06-01

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. Copyright © 2014 Elsevier Inc

Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

PubMed Central

Kaphalia, Lata; Boroumand, Nahal; Ju, Hyunsu; Kaphalia, Bhupendra S.; Calhoun, William J.

2014-01-01

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, compared to <0.2% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 were observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. PMID:24625836

Purification and characterisation of a novel iso-propanol dehydrogenase from Phytomonas sp.

PubMed

Uttaro, A D; Opperdoes, F R

1997-04-01

An alcohol dehydrogenase with two identical subunits and a subunit molecular mass of 40,000 was purified from Phytomonas sp. isolated from the lactiferous tubes of Euphorbia characias. Digitonin titration and subcellular fractionation suggest that the enzyme is present in the mitochondrion. It utilises as substrates, primary and secondary alcohols, is specific for NAD+ as coenzyme and is inhibited by HgCl(2). The pH optimum for the oxidation of ethanol is 9.5, and for the reverse reaction 8.5. The apparent Km values for iso-propanol and ethanol are 40 and 34 microM, respectively and for the reverse reaction, with acetone as substrate, 14 microM. The respective specific activities with iso-propanol and ethanol as substrate, as measured in crude extracts are 300 and 16 mU (milligram of protein)-1. In isoelectric focusing the enzyme showed three major bands with slightly differing isoelectric points that ranged from 6.4 to 6.8. The name, iso-propanol dehydrogenase is proposed for this enzyme.

Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.

PubMed

Liew, Fungmin; Henstra, Anne M; Kӧpke, Michael; Winzer, Klaus; Simpson, Sean D; Minton, Nigel P

2017-03-01

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Asian flushing: genetic and sociocultural factors of alcoholism among East asians.

PubMed

Lee, Haeok; Kim, Sun S; You, Kwang Soo; Park, Wanju; Yang, Jin Hyang; Kim, Minjin; Hayman, Laura L

2014-01-01

Alcohol use can lead to a cascade of problems such as increased chances of risky behavior and negative health consequences, including alcoholic liver disease and upper gastric and liver cancer. Ethanol is metabolized mainly by 2 major enzymes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). Genetic variations of genes encoding the 2 enzymes are very common among East Asians but relatively rare for most other populations. Facial flushing and other physical discomforts after alcohol drinking triggered by accumulation of acetaldehyde through defective genes for ADH and ALDH have been reported. Approximately 40% of East Asians (Chinese, Japanese, and Korean) show facial flushing after drinking alcohol, known as "Asian flush," which is characterized by adverse reactions on alcohol drinking in individuals possessing the fasting metabolizing alleles for ADH, ADH1B*2, and ADH1C*1, and the null allele for ALDH and ALDH2*2. Alcoholism is determined not only by the genetic deficiency but also by behaviors that involve complex interactions between genetic and sociocultural factors. The purpose of this article was to provide nurses with the most current information about genetic and sociocultural influences on alcoholism and alcohol-related health problems specifically for East Asians and implications of this knowledge to nursing practice. The physiological phenomenon of genes and genetics in relation to alcohol metabolism in this special population is emphasized.

Alcohol drinking, mean corpuscular volume of erythrocytes, and alcohol metabolic genotypes in drunk drivers.

PubMed

Pavanello, Sofia; Snenghi, Rossella; Nalesso, Alessandro; Sartore, Daniela; Ferrara, Santo Davide; Montisci, Massimo

2012-02-01

Regular and irregular abuse of alcohol are global health priorities associated with diseases at multiple sites, including cancer. Mechanisms of diseases induced by alcohol are closely related to its metabolism. Among conventional markers of alcohol abuse, the mean corpuscular volume (MCV) of erythrocytes is prognostic of alcohol-related cancer and its predictivity increases when combined with functional polymorphisms of alcohol dehydrogenase (ADH1B [rs1229984] and ADH1C [rs698]) and the mitochondrial aldehyde dehydrogenase (ALDH2 [rs671]). Whether these genetic variants can influence abuse in alcohol drinking and MCV has never been examined in drunk-driving traffic offenders. We examined 149 drunk drivers, diagnosed as alcohol abusers according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition Text Revision (DSM-IV-TR) and enrolled in a probation program, and 257 social drinkers (controls), all Caucasian males. Alcohol intake was assessed according to self-reported drink-units/d and MCV unadjusted and adjusted for age, smoking, and body mass index. Multivariable models were used to compute MCV adjusted means. Genotype analyses were performed by PCR on DNA from blood. The adjusted MCV mean was higher in drunk-driving abusers than in controls (92 vs. 91fL; P<.0001) and increased with the number of drink-units/d in both abusers and controls (P-trend=.0316 and .0089) already at intermediate quantities (0-1 vs. 2-4 drink-units/d: P=.054 and .024). Carriers of the common ADH1B*1/*1 (rs1229984) genotype were more likely to be drunk-driving abusers (P=.008), reported higher drink-units/d (P=.0126), and had larger MCV (P=.035). The rs698 ADH1C and rs671 ALDH2 polymorphisms were not associated with MCV. ADH1B*1/*1 polymorphism is significantly associated with being a drunk-driving abuser, higher alcohol drinking, and MCV enlargement. This suggests that drunk drivers with augmented MCV modulated by the alcohol metabolic ADH1B*1/*1 genotype may be at

Effect of pentachlorophenol and 2,6-dichloro-4-nitrophenol on the activity of cDNA-expressed human alcohol and aldehyde dehydrogenases.

PubMed

Kollock, Ronny; Rost, Katharina; Batke, Monika; Glatt, Hansruedi

2009-12-15

Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), potent inhibitors of phenol sulphotransferases, are frequently used in animal studies to elucidate the role of these enzymes in the biotransformation and toxicity of xenobiotics. An unexpected finding with 1-hydroxymethylpyrene--a strong decrease in the excretion of the corresponding carboxylic acid in rats concurrently treated with PCP-led us to suspect that this sulphotransferase inhibitor may also affect alcohol dehydrogenases (ADHs) and/or aldehyde dehydrogenases (ALDHs). Subsequently we investigated the influence of PCP and DCNP on the activity of cDNA-expressed human ADHs and ALDHs. PCP inhibited all four ADHs studied. The inhibition was strong for ADH3 (K(i) 1.4 microM, K(i)' 5.2 microM, mixed-type) and ADH2 (K(i) 3.7 microM, competitive), but moderate for ADH4 (K(i) 81 microM, competitive) and ADH1C (K(i)' 310 microM, uncompetitive). Activities of ALDH2 and ALDH3A1 were unaffected by PCP (used up to a concentration of 1 mM). In contrast, DCNP primarily inhibited ALDH2 (K(i)=K(i)' 7.4 microM, non-competitive), showed moderate competitive inhibition of ADH2 (K(i) 160 microM) and ADH4 (K(i) 710 microM), but did not affect the remaining enzymes (ADH1C, ADH3 and ALDH3A1). The study demonstrates that caution is required when using putative specific enzyme inhibitors in biotransformation studies.

Downregulation of Cinnamyl-Alcohol Dehydrogenase in Switchgrass by RNA Silencing Results in Enhanced Glucose Release after Cellulase Treatment

PubMed Central

Saathoff, Aaron J.; Sarath, Gautam; Chow, Elaine K.; Dien, Bruce S.; Tobias, Christian M.

2011-01-01

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. “Alamo” with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin. PMID:21298014

Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment.

PubMed

Saathoff, Aaron J; Sarath, Gautam; Chow, Elaine K; Dien, Bruce S; Tobias, Christian M

2011-01-27

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. "Alamo" with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin.

Establishment of the Tree Shrew as an Alcohol-Induced Fatty Liver Model for the Study of Alcoholic Liver Diseases

PubMed Central

Xing, Huijie; Jia, Kun; He, Jun; Shi, Changzheng; Fang, Meixia; Song, Linliang; Zhang, Pu; Zhao, Yue; Fu, Jiangnan; Li, Shoujun

2015-01-01

Currently, the pathogenesis of alcoholic liver diseases (ALDs) is not clear. As a result, there is no effective treatment for ALDs. One limitation is the lack of a suitable animal model for use in studying ALDs. The tree shrew is a lower primate animal, characterized by a high-alcohol diet. This work aimed to establish a fatty liver model using tree shrews and to assess the animals’ suitability for the study of ALDs. Tree shrews were treated with alcohol solutions (10% and 20%) for two weeks. Hemophysiology, blood alcohol concentrations (BACs), oxidative stress factors, alcohol metabolic enzymes and hepatic pathology were checked and assayed with an automatic biochemical analyzer, enzyme-linked immunosorbent assay (ELISA), western blot, hematoxylin-eosin (HE) staining and oil red O staining, and magnetic resonance imaging (MRI). Compared with the normal group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), total cholesterol (TC), triglyceride (TG), reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly enhanced in alcohol-treated tree shrews. However, the activity of reduced glutathione hormone (GSH) and superoxide dismutase (SOD) declined. Notable changes in alcohol dehydrogenase(ADH1), aldehyde dehydrogenase(ALDH2), CYP2E1, UDP-glucuronosyl transferase 1A1 (UGT1A1) and nuclear factor erythroid-related factor 2 (Nrf2) were observed. HE and oil red O staining showed that hepatocyte swelling, hydropic degeneration, and adipohepatic syndrome occurred in the tree shrews. Alcohol can induce fatty liver-like pathological changes and result in alterations in liver function, oxidative stress factors, alcohol metabolism enzymes and Nrf2. Therefore, the established fatty liver model of tree shrews induced by alcohol should be a promising tool for the study of ALDs. PMID:26030870

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-01-01

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

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

PubMed

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

2015-08-01

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

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.).

PubMed

O'malley, D M; Porter, S; Sederoff, R R

1992-04-01

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (K(m) = 1.7 micromolar) compared with sinapaldehyde (K(m) in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the lambdaCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.

Biology, Genetics, and Environment: Underlying Factors Influencing Alcohol Metabolism.

PubMed

Wall, Tamara L; Luczak, Susan E; Hiller-Sturmhöfel, Susanne

2016-01-01

Gene variants encoding several of the alcohol-metabolizing enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), are among the largest genetic associations with risk for alcohol dependence. Certain genetic variants (i.e., alleles)--particularly the ADH1B*2, ADH1B*3, ADH1C*1, and ALDH2*2 alleles--have been associated with lower rates of alcohol dependence. These alleles may lead to an accumulation of acetaldehyde during alcohol metabolism, which can result in heightened subjective and objective effects. The prevalence of these alleles differs among ethnic groups; ADH1B*2 is found frequently in northeast Asians and occasionally Caucasians, ADH1B*3 is found predominantly in people of African ancestry, ADH1C*1 varies substantially across populations, and ALDH2*2 is found almost exclusively in northeast Asians. Differences in the prevalence of these alleles may account at least in part for ethnic differences in alcohol consumption and alcohol use disorder (AUD). However, these alleles do not act in isolation to influence the risk of AUD. For example, the gene effects of ALDH2*2 and ADH1B*2 seem to interact. Moreover, other factors have been found to influence the extent to which these alleles affect a person's alcohol involvement, including developmental stage, individual characteristics (e.g., ethnicity, antisocial behavior, and behavioral undercontrol), and environmental factors (e.g., culture, religion, family environment, and childhood adversity).

Inhibition of human alcohol and aldehyde dehydrogenases by acetaminophen: Assessment of the effects on first-pass metabolism of ethanol.

PubMed

Lee, Yung-Pin; Liao, Jian-Tong; Cheng, Ya-Wen; Wu, Ting-Lun; Lee, Shou-Lun; Liu, Jong-Kang; Yin, Shih-Jiun

2013-11-01

Acetaminophen is one of the most widely used over-the-counter analgesic, antipyretic medications. Use of acetaminophen and alcohol are commonly associated. Previous studies showed that acetaminophen might affect bioavailability of ethanol by inhibiting gastric alcohol dehydrogenase (ADH). However, potential inhibitions by acetaminophen of first-pass metabolism (FPM) of ethanol, catalyzed by the human ADH family and by relevant aldehyde dehydrogenase (ALDH) isozymes, remain undefined. ADH and ALDH both exhibit racially distinct allozymes and tissue-specific distribution of isozymes, and are principal enzymes responsible for ethanol metabolism in humans. In this study, we investigated acetaminophen inhibition of ethanol oxidation with recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and inhibition of acetaldehyde oxidation with recombinant human ALDH1A1 and ALDH2. The investigations were done at near physiological pH 7.5 and with a cytoplasmic coenzyme concentration of 0.5 mM NAD(+). Acetaminophen acted as a noncompetitive inhibitor for ADH enzymes, with the slope inhibition constants (Kis) ranging from 0.90 mM (ADH2) to 20 mM (ADH1A), and the intercept inhibition constants (Kii) ranging from 1.4 mM (ADH1C allozymes) to 19 mM (ADH1A). Acetaminophen exhibited noncompetitive inhibition for ALDH2 (Kis = 3.0 mM and Kii = 2.2 mM), but competitive inhibition for ALDH1A1 (Kis = 0.96 mM). The metabolic interactions between acetaminophen and ethanol/acetaldehyde were assessed by computer simulation using inhibition equations and the determined kinetic constants. At therapeutic to subtoxic plasma levels of acetaminophen (i.e., 0.2-0.5 mM) and physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μm) in target tissues, acetaminophen could inhibit ADH1C allozymes (12-26%) and ADH2 (14-28%) in the liver and small intestine, ADH4 (15-31%) in the stomach, and ALDH1A1 (16-33%) and ALDH2 (8.3-19%) in all 3 tissues. The

Molecular Population Genetics of the Alcohol Dehydrogenase Gene Region of DROSOPHILA MELANOGASTER

PubMed Central

Aquadro, Charles F.; Desse, Susan F.; Bland, Molly M.; Langley, Charles H.; Laurie-Ahlberg, Cathy C.

1986-01-01

Variation in the DNA restriction map of a 13-kb region of chromosome II including the alcohol dehydrogenase structural gene (Adh) was examined in Drosophila melanogaster from natural populations. Detailed analysis of 48 D. melanogaster lines representing four eastern United States populations revealed extensive DNA sequence variation due to base substitutions, insertions and deletions. Cloning of this region from several lines allowed characterization of length variation as due to unique sequence insertions or deletions [nine sizes; 21–200 base pairs (bp)] or transposable element insertions (several sizes, 340 bp to 10.2 kb, representing four different elements). Despite this extensive variation in sequences flanking the Adh gene, only one length polymorphism is clearly associated with altered Adh expression (a copia element approximately 250 bp 5' to the distal transcript start site). Nonetheless, the frequency spectra of transposable elements within and between Drosophila species suggests they are slightly deleterious. Strong nonrandom associations are observed among Adh region sequence variants, ADH allozyme (Fast vs. Slow), ADH enzyme activity and the chromosome inversion ln(2L) t. Phylogenetic analysis of restriction map haplotypes suggest that the major twofold component of ADH activity variation (high vs. low, typical of Fast and Slow allozymes, respectively) is due to sequence variation tightly linked to and possibly distinct from that underlying the allozyme difference. The patterns of nucleotide and haplotype variation for Fast and Slow allozyme lines are consistent with the recent increase in frequency and spread of the Fast haplotype associated with high ADH activity. These data emphasize the important role of evolutionary history and strong nonrandom associations among tightly linked sequence variation as determinants of the patterns of variation observed in natural populations. PMID:3026893

Oxidation of methanol, ethylene glycol, and isopropanol with human alcohol dehydrogenases and the inhibition by ethanol and 4-methylpyrazole.

PubMed

Lee, Shou-Lun; Shih, Hsuan-Ting; Chi, Yu-Chou; Li, Yeung-Pin; Yin, Shih-Jiun

2011-05-30

Human alcohol dehydrogenases (ADHs) include multiple isozymes with broad substrate specificity and ethnic distinct allozymes. ADH catalyzes the rate-limiting step in metabolism of various primary and secondary aliphatic alcohols. The oxidation of common toxic alcohols, that is, methanol, ethylene glycol, and isopropanol by the human ADHs remains poorly understood. Kinetic studies were performed in 0.1M sodium phosphate buffer, at pH 7.5 and 25°C, containing 0.5 mM NAD(+) and varied concentrations of substrate. K(M) values for ethanol with recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2, and class II ADH2 and class IV ADH4 were determined to be in the range of 0.12-57 mM, for methanol to be 2.0-3500 mM, for ethylene glycol to be 4.3-2600mM, and for isopropanol to be 0.73-3400 mM. ADH1B3 appeared to be inactive toward ethylene glycol, and ADH2 and ADH4, inactive with methanol. The variations for V(max) for the toxic alcohols were much less than that of the K(M) across the ADH family. 4-Methylpyrazole (4MP) was a competitive inhibitor with respect to ethanol for ADH1A, ADH1B1, ADH1B2, ADH1C1 and ADH1C2, and a noncompetitive inhibitor for ADH1B3, ADH2 and ADH4, with the slope inhibition constants (K(is)) for the whole family being 0.062-960 μM and the intercept inhibition constants (K(ii)), 33-3000 μM. Computer simulation studies using inhibition equations in the presence of alternate substrate ethanol and of dead-end inhibitor 4MP with the determined corresponding kinetic parameters for ADH family, indicate that the oxidation of the toxic alcohols up to 50mM are largely inhibited by 20 mM ethanol or by 50 μM 4MP with some exceptions. The above findings provide an enzymological basis for clinical treatment of methanol and ethylene glycol poisoning by 4MP or ethanol with pharmacogenetic perspectives. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Identification and functional evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae involved in vanillin resistance.

PubMed

Wang, Xinning; Liang, Zhenzhen; Hou, Jin; Bao, Xiaoming; Shen, Yu

2016-04-01

Vanillin, a type of phenolic released during the pre-treatment of lignocellulosic materials, is toxic to microorganisms and therefore its presence inhibits the fermentation. The vanillin can be reduced to vanillyl alcohol, which is much less toxic, by the ethanol producer Saccharomyces cerevisiae. The reducing capacity of S. cerevisiae and its vanillin resistance are strongly correlated. However, the specific enzymes and their contribution to the vanillin reduction are not extensively studied. In our previous work, an evolved vanillin-resistant strain showed an increased vanillin reduction capacity compared with its parent strain. The transcriptome analysis suggested the reductases and dehydrogenases of this vanillin resistant strain were up-regulated. Using this as a starting point, 11 significantly regulated reductases and dehydrogenases were selected in the present work for further study. The roles of these reductases and dehydrogenases in the vanillin tolerance and detoxification abilities of S. cerevisiae are described. Among the candidate genes, the overexpression of the alcohol dehydrogenase gene ADH6, acetaldehyde dehydrogenase gene ALD6, glucose-6-phosphate 1-dehydrogenase gene ZWF1, NADH-dependent aldehyde reductase gene YNL134C, and aldo-keto reductase gene YJR096W increased 177, 25, 6, 15, and 18 % of the strain μmax in the medium containing 1 g L(-1) vanillin. The in vitro detected vanillin reductase activities of strain overexpressing ADH6, YNL134C and YJR096W were notably higher than control. The vanillin specific reduction rate increased by 8 times in ADH6 overexpressed strain but not in YNL134C and YJR096W overexpressed strain. This suggested that the enzymes encoded by YNL134C and YJR096W might prefer other substrate and/or could not show their effects on vanillin on the high background of Adh6p in vivo. Overexpressing ALD6 and ZWF1 mainly increased the [NADPH]/[NADP(+)] and [GSH]/[GSSG] ratios but not the vanillin reductase activities. Their

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

PubMed

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

2012-07-01

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

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

PubMed

Broadbent, Jeffery R; Gummalla, Sanjay; Hughes, Joanne E; Johnson, Mark E; Rankin, Scott A; Drake, Mary Anne

2004-08-01

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase.

PubMed

Chen, Lei; Auh, Chung-Kyoon; Dowling, Paul; Bell, Jeremey; Chen, Fang; Hopkins, Andrew; Dixon, Richard A; Wang, Zeng-Yu

2003-11-01

Lignification of cell walls during plant development has been identified as the major factor limiting forage digestibility and concomitantly animal productivity. cDNA sequences encoding a key lignin biosynthetic enzyme, cinnamyl alcohol dehydrogenase (CAD), were cloned from the widely grown monocotyledonous forage species tall fescue (Festuca arundinacea Schreb.). Recombinant tall fescue CAD expressed in E. coli exhibited the highest V(max)/K(m) values when coniferaldehyde and sinapaldehyde were used as substrates. Transgenic tall fescue plants carrying either sense or antisense CAD gene constructs were obtained by microprojectile bombardment of single genotype-derived embryogenic suspension cells. Severely reduced levels of mRNA transcripts and significantly reduced CAD enzymatic activities were found in two transgenic plants carrying sense and antisense CAD transgenes, respectively. These CAD down-regulated transgenic lines had significantly decreased lignin content and altered ratios of syringyl (S) to guaiacyl (G), G to p-hydroxyphenyl (H) and S to H units. No significant changes in cellulose, hemicellulose, neutral sugar composition, p-coumaric acid and ferulic acid levels were observed in the transgenic plants. Increases of in vitro dry matter digestibility of 7.2-9.5% were achieved in the CAD down-regulated lines, thus providing a novel germplasm to be used for the development of grass cultivars with improved forage quality.

Alcoholism and liver disease in Mexico: Genetic and environmental factors

PubMed Central

Roman, Sonia; Zepeda-Carrillo, Eloy Alfonso; Moreno-Luna, Laura Eugenia; Panduro, Arturo

2013-01-01

Alcoholism and cirrhosis, which are two of the most serious health problems worldwide, have a broad spectrum of clinical outcomes. Both diseases are influenced by genetic susceptibility and cultural traits that differ globally but are specific for each population. In contrast to other regions around the world, Mexicans present the highest drinking score and a high mortality rate for alcoholic liver disease with an intermediate category level of per capita alcohol consumption. Mexico has a unique history of alcohol consumption that is linked to profound anthropological and social aspects. The Mexican population has an admixture genome inherited from different races, Caucasian, Amerindian and African, with a heterogeneous distribution within the country. Thus, genes related to alcohol addiction, such as dopamine receptor D2 in the brain, or liver alcohol-metabolizing enzymes, such as alcohol dehydrogenase class I polypeptide B, cytochrome P450 2E1 and aldehyde dehydrogenase class 2, may vary from one individual to another. Furthermore, they may be inherited as risk or non-risk haplogroups that confer susceptibility or resistance either to alcohol addiction or abusive alcohol consumption and possibly liver disease. Thus, in this era of genomics, personalized medicine will benefit patients if it is directed according to individual or population-based data. Additional association studies will be required to establish novel strategies for the prevention, care and treatment of liver disease in Mexico and worldwide. PMID:24307790

Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S-transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma.

PubMed

Yokoyama, Akira; Kato, Hoichi; Yokoyama, Tetsuji; Tsujinaka, Toshimasa; Muto, Manabu; Omori, Tai; Haneda, Tatsumasa; Kumagai, Yoshiya; Igaki, Hiroyasu; Yokoyama, Masako; Watanabe, Hiroshi; Fukuda, Haruhiko; Yoshimizu, Haruko

2002-11-01

The genetic polymorphisms of aldehyde dehydrogenase-2 (ALDH2), alcohol dehydrogenase-2 (ADH2), ADH3, and glutathione S-transferase M1 (GSTM1) influence the metabolism of alcohol and other carcinogens. The ALDH2*1/2*2 genotype, which encodes inactive ALDH2, and ADH2*1/2*1, which encodes the low-activity form of ADH2, enhance the risk for esophageal cancer in East Asian alcoholics. This case-control study of whether the enzyme-related vulnerability for esophageal cancer can be extended to a general population involved 234 Japanese men with esophageal squamous cell carcinoma and 634 cancer-free Japanese men who received annual health checkups. The GSTM1 genotype was not associated with the risk for this cancer. Light drinkers (1-8.9 units/week) with ALDH2*1/2*2 had an esophageal cancer risk 5.82 times that of light drinkers with ALDH2*1/2*1 (reference category), and their risk was similar to that of moderate drinkers (9-17.9 units/week) with ALDH2*1/2*1 (odds ratio = 5.58). The risk for moderate drinkers with ALDH2*1/2*2 (OR = 55.84) exceeded that for heavy drinkers (18+ units/week) with ALDH2*1/2*1 (OR = 10.38). Similar increased risks were observed for those with ADH2*1/2*1. A multiple logistic model including ALDH2, ADH2, and ADH3 genotypes showed that the ADH3 genotype does not significantly affect the risk for esophageal cancer. For individuals with both ALDH2*1/2*2 and ADH2*1/2*1, the risk of esophageal cancer was enhanced in a multiplicative fashion (OR = 30.12), whereas for those with either ALDH2*1/2*2 or ADH2*1/2*1 alone the ORs were 7.36 and 4.11. In comparison with the estimated population-attributable risks for preference for strong alcoholic beverages (30.7%), smoking (53.6%) and for lower intake of green and yellow vegetables (25.7%) and fruit (37.6%), an extraordinarily high proportion of the excessive risk for esophageal cancer in the Japanese males can be attributed to drinking (90.9%), particularly drinking by persons with inactive heterozygous ALDH

Accentuating effects of nicotine on ethanol response in mice with high genetic predisposition to ethanol-induced locomotor stimulation.

PubMed

Gubner, N R; McKinnon, C S; Reed, C; Phillips, T J

2013-01-01

Co-morbid use of nicotine-containing tobacco products and alcohol is prevalent in alcohol dependent individuals. Common genetic factors could influence initial sensitivity to the independent or interactive effects of these drugs and play a role in their co-abuse. Locomotor sensitivity to nicotine and ethanol, alone and in combination, was assessed in mice bred for high (FAST) and low (SLOW) sensitivity to the locomotor stimulant effects of ethanol and in an inbred strain of mouse (DBA/2J) that has been shown to have extreme sensitivity to ethanol-induced stimulation in comparison to other strains. The effects of nicotine and ethanol, alone and in combination, were dependent on genotype. In FAST and DBA/2J mice that show high sensitivity to ethanol-induced stimulation, nicotine accentuated the locomotor stimulant response to ethanol. This effect was not found in SLOW mice that are not stimulated by ethanol alone. These data indicate that genes underlying differential sensitivity to the stimulant effects of ethanol alone also influence sensitivity to nicotine in combination with ethanol. Sensitivity to the stimulant effects of nicotine alone does not appear to predict the response to the drug combination, as FAST mice are sensitive to nicotine-induced stimulation, whereas SLOW and DBA/2J mice are not. The combination of nicotine and ethanol may have genotype-dependent effects that could impact co-abuse liability. Published by Elsevier Ireland Ltd.

Complex inhibition of autophagy by mitochondrial aldehyde dehydrogenase shortens lifespan and exacerbates cardiac aging.

PubMed

Zhang, Yingmei; Wang, Cong; Zhou, Jingmin; Sun, Aijun; Hueckstaedt, Lindsay K; Ge, Junbo; Ren, Jun

2017-08-01

Autophagy, a conservative degradation process for long-lived and damaged proteins, participates in a cascade of biological processes including aging. A number of autophagy regulators have been identified. Here we demonstrated that mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme with the most common single point mutation in humans, governs cardiac aging through regulation of autophagy. Myocardial mechanical and autophagy properties were examined in young (4months) and old (26-28months) wild-type (WT) and global ALDH2 transgenic mice. ALDH2 overexpression shortened lifespan by 7.7% without affecting aging-associated changes in plasma metabolic profiles. Myocardial function was compromised with aging associated with cardiac hypertrophy, the effects were accentuated by ALDH2. Aging overtly suppressed autophagy and compromised autophagy flux, the effects were exacerbated by ALDH2. Aging dampened phosphorylation of JNK, Bcl-2, IKKβ, AMPK and TSC2 while promoting phosphorylation of mTOR, the effects of which were exaggerated by ALDH2. Co-immunoprecipitation revealed increased dissociation between Bcl-2 and Beclin-1 (result of decreased Bcl-2 phosphorylation) in aging, the effect of which was exacerbated with ALDH2. Chronic treatment of the autophagy inducer rapamycin alleviated aging-induced cardiac dysfunction in both WT and ALDH2 mice. Moreover, activation of JNK and inhibition of either Bcl-2 or IKKβ overtly attenuated ALDH2 activation-induced accentuation of cardiomyocyte aging. Examination of the otherwise elderly individuals revealed a positive correlation between cardiac function/geometry and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may suppress myocardial autophagy possibly through a complex JNK-Bcl-2 and IKKβ-AMPK-dependent mechanism en route to accentuation of myocardial remodeling and contractile dysfunction in aging. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure

Accelerated and accentuated neurocognitive aging in HIV infection.

PubMed

Sheppard, David P; Iudicello, Jennifer E; Morgan, Erin E; Kamat, Rujvi; Clark, Lindsay R; Avci, Gunes; Bondi, Mark W; Woods, Steven Paul

2017-06-01

There is debate as to whether the neurocognitive changes associated with HIV infection represent an acceleration of the typical aging process or more simply reflect a greater accentuated risk for age-related declines. We aimed to determine whether accelerated neurocognitive aging is observable in a sample of older HIV-infected individuals compared to age-matched seronegatives and older old (i.e., aged ≥65) seronegative adults. Participants in a cross-sectional design included 48 HIV-seronegative (O-) and 40 HIV-positive (O+) participants between the ages of 50-65 (mean ages = 55 and 56, respectively) and 40 HIV-seronegative participants aged ≥65 (OO-; mean age = 74) who were comparable for other demographics. All participants were administered a brief neurocognitive battery of attention, episodic memory, speeded executive functions, and confrontation naming (i.e., Boston Naming Test). The O+ group performed more poorly than the O- group (i.e., accentuated aging), but not differently from the OO- on digit span and initial recall of a supraspan word list, consistent with an accelerating aging profile. However, the O+ group's performance was comparable to the O- group on all other neurocognitive tests (ps > 0.05). These data partially support a model of accelerated neurocognitive aging in HIV infection, which was observed in the domain of auditory verbal attention, but not in the areas of memory, language, or speeded executive functions. Future studies should examine whether HIV-infected adults over 65 evidence accelerated aging in downstream neurocognitive domains and subsequent everyday functioning outcomes.

Molecular Basis of Alcohol-Related Gastric and Colon Cancer.

PubMed

Na, Hye-Kyung; Lee, Ja Young

2017-05-24

Many meta-analysis, large cohort studies, and experimental studies suggest that chronic alcohol consumption increases the risk of gastric and colon cancer. Ethanol is metabolized by alcohol dehydrogenases (ADH), catalase or cytochrome P450 2E1 (CYP2E1) to acetaldehyde, which is then further oxidized to acetate by aldehyde dehydrogenase (ALDH). Acetaldehyde has been classified by the International Agency for Research on Cancer (IARC) as a Group 1 carcinogen to humans. The acetaldehyde level in the stomach and colon is locally influenced by gastric colonization by Helicobacter pylori or colonic microbes, as well as polymorphisms in the genes encoding tissue alcohol metabolizing enzymes, especially ALDH2. Alcohol stimulates the uptake of carcinogens and their metabolism and also changes the composition of enteric microbes in a way to enhance the aldehyde level. Alcohol also undergoes chemical coupling to membrane phospholipids and disrupts organization of tight junctions, leading to nuclear translocation of β-catenin and ZONAB, which may contributes to regulation of genes involved in proliferation, invasion and metastasis. Alcohol also generates reactive oxygen species (ROS) by suppressing the expression of antioxidant and cytoprotective enzymes and inducing expression of CYP2E1 which contribute to the metabolic activation of chemical carcinogens. Besides exerting genotoxic effects by directly damaging DNA, ROS can activates signaling molecules involved in inflammation, metastasis and angiogenesis. In addition, alcohol consumption induces folate deficiency, which may result in aberrant DNA methylation profiles, thereby influencing cancer-related gene expression.

Protective effect of heat-treated cucumber (Cucumis sativus L.) juice on alcohol detoxification in experimental rats.

PubMed

Bajpai, Vivek K; Kim, Na-Hyung; Kim, Ji-Eun; Kim, Kangmin; Kang, Sun Chul

2016-05-01

In this study, heat-treated cucumber juice was assessed for its protective effect on blood alcohol levels and hepatic alcohol metabolic enzyme system in experimental rats. Initially, during detoxification of alcohol, all groups were orally dosed to 22% alcohol (6ml/kg body weight) along with different concentrations of heat-treated cucumber juice (10, 100 and 500mg/kg) and commercial goods for hangover-removal on sale (2ml/kg). Cucumber juice was dosed before 30 min, and simultaneously after 30min of alcohol administration, and its hepatoprotective effect on blood alcohol levels and hepatic alcohol metabolic enzyme system in experimental rats was evaluated. As a result, after 7h, remarkable reduction was found in the blood alcohol levels for all concentrations of cucumber juice treatment. Treatment with cucumber juice resulted in increasing dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymatic activities in rat liver at 9h after alcohol administration thereby stimulated blood alcohol metabolism as compared with control group. The effect of heat-treated cucumber juice on alcohol detoxification was observed only in the rats treated before 30min from alcohol administration. These findings indicate that heat-treated cucumber juice has significant protective effect on alcohol detoxification in experimental rats, suggesting its usefulness in the treatment of liver injury caused by alcohol consumption.

Association between alcoholism and the dopamine D4 receptor gene.

PubMed Central

Muramatsu, T; Higuchi, S; Murayama, M; Matsushita, S; Hayashida, M

1996-01-01

A point mutation in the aldehyde dehydrogenase 2 gene (ALDH2(2) allele) is considered to be a genetic deterrent for alcoholism; however, 80 of 655 Japanese alcoholics had the mutant allele. Genotype factors that might increase susceptibility by overriding the deterrent showed a higher frequency of a five repeat allele of the dopamine D4 receptor 48 bp repeat polymorphism in alcoholics with ALDH2(2) than in 100 other alcoholics and 144 controls. Alcoholics with the five repeat allele also abused other drugs more often. These data suggest the involvement of the dopamine system in the development of alcoholism and other addictive behaviour. PMID:8929946

Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol-β-Aryl Ether by Sphingobium sp. Strain SYK-6▿ †

PubMed Central

Sato, Yusuke; Moriuchi, Hideki; Hishiyama, Shojiro; Otsuka, Yuichiro; Oshima, Kenji; Kasai, Daisuke; Nakamura, Masaya; Ohara, Seiji; Katayama, Yoshihiro; Fukuda, Masao; Masai, Eiji

2009-01-01

Degradation of arylglycerol-β-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-β-guaiacyl ether (GGE) to α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (αR,βS)-GGE and (αR,βR)-GGE into (βS)-MPHPV and (βR)-MPHPV, respectively, while LigL and LigN transformed (αS,βR)-GGE and (αS,βS)-GGE to (βR)-MPHPV and (βS)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (αR,βS)-GGE and (αR,βR)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers. PMID:19542348

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks.

PubMed

Yang, Mingfeng; Li, Xuefeng; Bu, Chunya; Wang, Hui; Shi, Guanglu; Yang, Xiushan; Hu, Yong; Wang, Xiaoqin

2014-11-01

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC-MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

Determination of hydride transfer stereospecificity of NADH-dependent alcohol-aldehyde/ketone oxidoreductase from Sulfolobus solfataricus.

PubMed

Trincone, A; Lama, L; Rella, R; D'Auria, S; Raia, C A; Nicolaus, B

1990-10-18

This paper describes the determination of stereospecificity of hydride transfer reaction of an alcohol dehydrogenase isolated from the archaebacterium Sulfolobus solfataricus. The 1H-NMR and EI-MS data indicate that the enzyme transfers the pro-R hydrogen from coenzyme to substrate and is therefore an A-specific dehydrogenase.

DB Dehydrogenase: an online integrated structural database on enzyme dehydrogenase.

PubMed

Nandy, Suman Kumar; Bhuyan, Rajabrata; Seal, Alpana

2012-01-01

Dehydrogenase enzymes are almost inevitable for metabolic processes. Shortage or malfunctioning of dehydrogenases often leads to several acute diseases like cancers, retinal diseases, diabetes mellitus, Alzheimer, hepatitis B & C etc. With advancement in modern-day research, huge amount of sequential, structural and functional data are generated everyday and widens the gap between structural attributes and its functional understanding. DB Dehydrogenase is an effort to relate the functionalities of dehydrogenase with its structures. It is a completely web-based structural database, covering almost all dehydrogenases [~150 enzyme classes, ~1200 entries from ~160 organisms] whose structures are known. It is created by extracting and integrating various online resources to provide the true and reliable data and implemented by MySQL relational database through user friendly web interfaces using CGI Perl. Flexible search options are there for data extraction and exploration. To summarize, sequence, structure, function of all dehydrogenases in one place along with the necessary option of cross-referencing; this database will be utile for researchers to carry out further work in this field. The database is available for free at http://www.bifku.in/DBD/

Aldehyde dehydrogenase polymorphism in North American, South American, and Mexican Indian populations.

PubMed Central

Goedde, H W; Agarwal, D P; Harada, S; Rothhammer, F; Whittaker, J O; Lisker, R

1986-01-01

While about 40% of the South American Indian populations (Atacameños, Mapuche, Shuara) were found to be deficient in aldehyde dehydrogenase isozyme I (ALDH2 or E2), preliminary investigations showed very low incidence of isozyme deficiency among North American natives (Sioux, Navajo) and Mexican Indians (mestizo). Possible implications of such trait differences on cross-cultural behavioral response to alcohol drinking are discussed. PMID:3953578

Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon

PubMed Central

2013-01-01

Background Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production. Results We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants. Conclusion These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency. PMID:23902793

Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts.

PubMed

Guo, Rui; Hu, Nan; Kandadi, Machender R; Ren, Jun

2012-04-01

Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae.

PubMed

Yang, Dong-Dong; de Billerbeck, Gustavo M; Zhang, Jin-Jing; Rosenzweig, Frank; Francois, Jean-Marie

2018-01-01

Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14 , encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5' sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr 73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two

Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae

PubMed Central

de Billerbeck, Gustavo M.; Zhang, Jin-jing; Rosenzweig, Frank

2017-01-01

ABSTRACT Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5′ sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded

Alcohol dehydrogenase and hydrogenase transcript fluctuations during a day-night cycle in Chlamydomonas reinhardtii: the role of anoxia.

PubMed

Whitney, Larisa Angela Swirsky; Loreti, Elena; Alpi, Amedeo; Perata, Pierdomenico

2011-04-01

• The unicellular green alga Chlamydomonas reinhardtii contains two iron (Fe)-hydrogenases which are responsible for hydrogen production under anoxia. In the present work the patterns of expression of alcohol dehydrogenase, a typical anaerobic gene in plants, of the hydrogenases genes (HYD1, HYD2) and of the genes responsible for their maturation (HYDEF, HYDG), were analysed. • The expression patterns were analysed by real-time reverse-transcription polymerase chain reaction in Chlamydomonas cultures during the day-night cycle, as well as in response to oxygen availability. • The results indicated that ADH1, HYD1, HYD2, HYDEF and HYDG were expressed following precise day-night fluctuations. ADH1 and HYD2 were modulated by the day-night cycle. Low oxygen plays an important role for the induction of HYD1, HYDEF and HYDG, while ADH1 and HYD2 expression was relatively insensitive to oxygen availability. • The regulation of the anaerobic gene expression in Chlamydomonas is only partly explained by responses to anoxia. The cell cycle and light-dark cycles are equally important elements in the regulatory network modulating the anaerobic response in Chlamydomonas. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

Purification, Characterization, and Cloning of Cinnamyl Alcohol Dehydrogenase in Loblolly Pine (Pinus taeda L.) 1

PubMed Central

O'Malley, David M.; Porter, Stephanie; Sederoff, Ronald R.

1992-01-01

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1. 195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coniferaldehyde (Km = 1.7 micromolar) compared with sinapaldehyde (Km in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the λCAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme. ImagesFigure 2Figure 3 PMID:16668801

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

PubMed

Lin, Sen; Sun, Shiyong; Wang, Ke; Shen, Kexuan; Ma, Biaobiao; Ren, Yuquan; Fan, Xiaoyu

2018-02-24

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

Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana.

PubMed Central

Porterfield, D M; Matthews, S W; Daugherty, C J; Musgrave, M E

1997-01-01

Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior. PMID:9085569

Spaceflight exposure effects on transcription, activity, and localization of alcohol dehydrogenase in the roots of Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Porterfield, D. M.; Matthews, S. W.; Daugherty, C. J.; Musgrave, M. E.

1997-01-01

Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior.

How and When Accentuation Influences Temporally Selective Attention and Subsequent Semantic Processing during On-Line Spoken Language Comprehension: An ERP Study

ERIC Educational Resources Information Center

Li, Xiao-qing; Ren, Gui-qin

2012-01-01

An event-related brain potentials (ERP) experiment was carried out to investigate how and when accentuation influences temporally selective attention and subsequent semantic processing during on-line spoken language comprehension, and how the effect of accentuation on attention allocation and semantic processing changed with the degree of…

Genetic susceptibility factors for alcohol-induced chronic pancreatitis.

PubMed

Aghdassi, Ali A; Weiss, F Ulrich; Mayerle, Julia; Lerch, Markus M; Simon, Peter

2015-07-01

Chronic pancreatitis is a progressive inflammatory disease of the pancreas and frequently associated with immoderate alcohol consumption. Since only a small proportion of alcoholics eventually develop chronic pancreatitis genetic susceptibility factors have long been suspected to contribute to the pathogenesis of the disease. Smaller studies in ethnically defined populations have found that not only polymorphism in proteins involved in the metabolism of ethanol, such as Alcohol Dehydrogenase and Aldehyde Dehydrogenase, can confer a risk for developing chronic pancreatitis but also mutations that had previously been reported in association with idiopathic pancreatitis, such as SPINK1 mutations. In a much broader approach employing genome wide search strategies the NAPS study found that polymorphisms in the Trypsin locus (PRSS1 rs10273639), and the Claudin 2 locus (CLDN2-RIPPLY1-MORC4 locus rs7057398 and rs12688220) confer an increased risk of developing alcohol-induced pancreatitis. These results from North America have now been confirmed by a European consortium. In another genome wide approach polymorphisms in the genes encoding Fucosyltransferase 2 (FUT2) non-secretor status and blood group B were not only found in association with higher serum lipase levels in healthy volunteers but also to more than double the risk for developing alcohol-associated chronic pancreatitis. These novel genetic associations will allow to investigate the pathophysiological and biochemical basis of alcohol-induced chronic pancreatitis on a cellular level and in much more detail than previously possible. Copyright © 2015 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Overview of the Genetics of Alcohol Use Disorder

PubMed Central

Tawa, Elisabeth A.; Hall, Samuel D.; Lohoff, Falk W.

2016-01-01

Aims Alcohol Use Disorder (AUD) is a chronic psychiatric illness characterized by harmful drinking patterns leading to negative emotional, physical, and social ramifications. While the underlying pathophysiology of AUD is poorly understood, there is substantial evidence for a genetic component; however, identification of universal genetic risk variants for AUD has been difficult. Recent efforts in the search for AUD susceptibility genes will be reviewed in this article. Methods In this review, we provide an overview of genetic studies on AUD, including twin studies, linkage studies, candidate gene studies, and genome-wide association studies (GWAS). Results Several potential genetic susceptibility factors for AUD have been identified, but the genes of alcohol metabolism, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), have been found to be protective against the development of AUD. GWAS have also identified a heterogeneous list of SNPs associated with AUD and alcohol-related phenotypes, emphasizing the complexity and heterogeneity of the disorder. In addition, many of these findings have small effect sizes when compared to alcohol metabolism genes, and biological relevance is often unknown. Conclusions Although studies spanning multiple approaches have suggested a genetic basis for AUD, identification of the genetic risk variants has been challenging. Some promising results are emerging from GWAS studies; however, larger sample sizes are needed to improve GWAS results and resolution. As the field of genetics is rapidly developing, whole genome sequencing could soon become the new standard of interrogation of the genes and neurobiological pathways which contribute to the complex phenotype of AUD. Short summary This review examines the genetic underpinnings of Alcohol Use Disorder (AUD), with an emphasis on GWAS approaches for identifying genetic risk variants. The most promising results associated with AUD and alcohol-related phenotypes have included

Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata.

PubMed

Nair, Smitha; Kukreja, Neetu; Singh, Bhanu Pratap; Arora, Naveen

2011-01-01

Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay. B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera. The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1-P6) and four T cell (P7-P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)(2)GGP(X)(3)KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding. Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.

Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes and Alcoholic Ketosis Are Associated with the Serum Uric Acid Level in Japanese Alcoholic Men.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Mizukami, Takeshi; Matsui, Toshifumi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2016-05-01

To identify determinants of hyperuricemia in alcoholics. The serum uric acid (UA) levels of 1759 Japanese alcoholic men (≥40 years) were measured on their first visit or within 3 days after admission; ADH1B and ALDH2 genotyping on blood DNA samples were performed. Dipstick urinalyses for ketonuria and serum UA measurements were simultaneously performed for 621 men on their first visit. Serum UA levels of >416 μmol/l (7.0 mg/dl) and ≥535 μmol/l (9.0 mg/dl) were observed in 30.4 and 7.8% of the subjects, respectively. Ketonuria was positive in 35.9% of the subjects, and a multivariate analysis revealed that the ketosis level was positively associated with the UA level. The presence of the ADH1B*2 allele and the ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) among subjects with a high UA level of >416 μmol/l (vs. ≤416 μmol/l; 2.04 [1.58-2.65] and 1.48 [1.09-2.01], respectively) and those with a high UA level of ≥535 μmol/l (vs. ≤416 μmol/l; 2.29 [1.42-3.71] and 3.03 [1.51-6.08], respectively). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs (2.86 [1.61-5.10] and 6.21 [1.49-25.88] for a UA level of >416 μmol/l and ≥535 μmol/l, respectively), compared with the ADH1B*1/*1 plus ALDH2*1/*2 combination. The presence of diabetes and the consumption of Japanese sake rather than beer were negatively associated with the UA levels. The faster metabolism of ethanol and acetaldehyde by the ADH1B*2 allele and ALDH2*1/*1 genotype and higher ketosis levels were associated with higher UA levels in alcoholics, while diabetes and the consumption of sake were negative determinants. © The Author 2015. Medical Council on Alcohol and Oxford University Press. All rights reserved.

Estimates of alcohol-related oesophageal cancer burden in Japan: systematic review and meta-analyses

PubMed Central

Shield, Kevin D; Higuchi, Susumu; Yoshimura, Atsushi; Larsen, Elisabeth; Rehm, Maximilien X; Rehm, Jürgen

2015-01-01

Abstract Objective To refine estimates of the burden of alcohol-related oesophageal cancer in Japan. Methods We searched PubMed for published reviews and original studies on alcohol intake, aldehyde dehydrogenase polymorphisms, and risk for oesophageal cancer in Japan, published before 2014. We conducted random-effects meta-analyses, including subgroup analyses by aldehyde dehydrogenase variants. We estimated deaths and loss of disability-adjusted life years (DALYs) from oesophageal cancer using exposure distributions for alcohol based on age, sex and relative risks per unit of exposure. Findings We identified 14 relevant studies. Three cohort studies and four case-control studies had dose–response data. Evidence from cohort studies showed that people who consumed the equivalent of 100 g/day of pure alcohol had an 11.71 fold, (95% confidence interval, CI: 2.67–51.32) risk of oesophageal cancer compared to those who never consumed alcohol. Evidence from case-control studies showed that the increase in risk was 33.11 fold (95% CI: 8.15–134.43) in the population at large. The difference by study design is explained by the 159 fold (95% CI: 27.2–938.2) risk among those with an inactive aldehyde dehydrogenase enzyme variant. Applying these dose–response estimates to the national profile of alcohol intake yielded 5279 oesophageal cancer deaths and 102 988 DALYs lost – almost double the estimates produced by the most recent global burden of disease exercise. Conclusion Use of global dose–response data results in an underestimate of the burden of disease from oesophageal cancer in Japan. Where possible, national burden of disease studies should use results from the population concerned. PMID:26229204

Alcohol dehydrogenase ADH2-1 and ADH2-2 allelic isoforms in the Russian population correlate with type of alcoholic disease.

PubMed

Ogurtsov, Pavel P.; Garmash, Irina V.; Miandina, Galina I.; Guschin, Alexander E.; Itkes, Alexander V.; Moiseev, Valentin S.

2001-09-01

The frequency ADH2-2 allele in the Moscow urban population and a correlation between the ADH2-2 allele, alcoholic dependence without cirrhosis, symptomatic alcoholic cirrhosis and status on hepatitis B and C infection have been studied. One hundred and twenty-three inhabitants of Moscow (50 healthy donors, 36 patients with alcoholic cirrhosis (subdivided into infected and uninfected by HBV and/or HCV) and 37 patients with alcoholic dependence) of a similar age/sex and drinking pattern have been analysed. The frequency of 41% for ADH2-2 allele is characteristic for an urban Moscow population. This value is intermediate between that found for Asian peoples and for Central and Western Europe. There is a negative correlation between the ADH2-2 allele and alcohol misuse (both alcoholic dependence and alcoholic cirrhosis). This correlation is expressed more in alcoholic dependence. In spite of the possession of the ADH2-2 allele (or genotype ADH2-1/2), alcohol misuse increases the risk of cirrhosis. At the same time, positive status for active hepatitis B, C or combined infection B + C (replication markers HBV-DNA or HCV-RNA) increases the risk for symptomatic alcoholic cirrhosis in alcohol abusing patients, independently of ADH2 genotype.

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius.

PubMed

Pennacchio, Angela; Giordano, Assunta; Pucci, Biagio; Rossi, Mosè; Raia, Carlo A

2010-03-01

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75 degrees C and a 30-min half-inactivation temperature of ~90 degrees C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and alpha-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of alpha-methyl and alpha-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.

A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia.

PubMed

Geissler, Marcus; Burghard, Marie; Volk, Jascha; Staniek, Agata; Warzecha, Heribert

2016-03-01

Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites.

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

PubMed

Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

2015-05-01

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA.

PubMed Central

Brogna, S

1999-01-01

From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA. In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus. Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA. Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript. The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript. These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. PMID:10199572

Role of L-alanine for redox self-sufficient amination of alcohols.

PubMed

Klatte, Stephanie; Wendisch, Volker F

2015-01-23

In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578-5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed. The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily

Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients.

PubMed

Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho

2004-01-01

Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

Inducible NAD(H)-linked methylglyoxal oxidoreductase regulates cellular methylglyoxal and pyruvate through enhanced activities of alcohol dehydrogenase and methylglyoxal-oxidizing enzymes in glutathione-depleted Candida albicans.

PubMed

Kwak, Min-Kyu; Ku, MyungHee; Kang, Sa-Ouk

2018-01-01

High methylglyoxal content disrupts cell physiology, but mammals have scavengers to prevent glycolytic and mitochondrial dysfunctions. In yeast, methylglyoxal accumulation triggers methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) activity. While methylglyoxal reductases and glyoxalases have been well studied in prokaryotes and eukaryotes, experimental evidence for methylglyoxal dehydrogenase (Mgd) and other catalytic activities of this enzyme affecting glycolysis and the tricarboxylic acid cycle is lacking. A glycine-rich cytoplasmic Mgd protein, designated as Mgd1/Grp2, was isolated from glutathione-depleted Candida albicans. The effects of Mgd1/Grp2 activities on metabolic pathophysiology were investigated using knockout and overexpression mutants. We measured glutathione-(in)dependent metabolite contents and metabolic effects, including viability, oxygen consumption, ADH1 transcripts, and glutathione reductase and α-ketoglutarate dehydrogenase activities in the mutants. Based on the findings, methylglyoxal-oxidizing proteins were monitored to determine effects of MGD1/GRP2 disruption on methylglyoxal-scavenging traits during glutathione deprivation. Methylglyoxal-oxidizing NAD(H)-linked Mgd1/Grp2 was found solely in glutathione auxotrophs, and it catalyzed the reduction of both methylglyoxal and pyruvate. MGD1/GRP2 disruptants showed growth defects, cell-cycle arrest, and methylglyoxal and pyruvate accumulation with mitochondrial impairment, regardless of ADH1 compensation. Other methylglyoxal-oxidizing enzymes were identified as key glycolytic enzymes with enhanced activity and transcription in MGD1/GRP2 disruptants, irrespective of glutathione content. Failure of methylglyoxal and pyruvate dissimilation by Mgd1/Grp2 deficiency leads to poor glutathione-dependent redox regulation despite compensation by Adh1. This is the first report that multifunctional Mgd activities contribute to scavenging methylglyoxal and pyruvate to maintain metabolic homeostasis

Ethanol metabolism, oxidative stress, and endoplasmic reticulum stress responses in the lungs of hepatic alcohol dehydrogenase deficient deer mice after chronic ethanol feeding

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

Kaphalia, Lata; Boroumand, Nahal; Hyunsu, Ju

Consumption and over-consumption of alcoholic beverages are well-recognized contributors to a variety of pulmonary disorders, even in the absence of intoxication. The mechanisms by which alcohol (ethanol) may produce disease include oxidative stress and prolonged endoplasmic reticulum (ER) stress. Many aspects of these processes remain incompletely understood due to a lack of a suitable animal model. Chronic alcohol over-consumption reduces hepatic alcohol dehydrogenase (ADH), the principal canonical metabolic pathway of ethanol oxidation. We therefore modeled this situation using hepatic ADH-deficient deer mice fed 3.5% ethanol daily for 3 months. Blood ethanol concentration was 180 mg% in ethanol fed mice, comparedmore » to < 1.0% in the controls. Acetaldehyde (oxidative metabolite of ethanol) was minimally, but significantly increased in ethanol-fed vs. pair-fed control mice. Total fatty acid ethyl esters (FAEEs, nonoxidative metabolites of ethanol) were 47.6 μg/g in the lungs of ethanol-fed mice as compared to 1.5 μg/g in pair-fed controls. Histological and immunohistological evaluation showed perivascular and peribronchiolar lymphocytic infiltration, and significant oxidative injury, in the lungs of ethanol-fed mice compared to pair-fed controls. Several fold increases for cytochrome P450 2E1, caspase 8 and caspase 3 found in the lungs of ethanol-fed mice as compared to pair-fed controls suggest role of oxidative stress in ethanol-induced lung injury. ER stress and unfolded protein response signaling were also significantly increased in the lungs of ethanol-fed mice. Surprisingly, no significant activation of inositol-requiring enzyme-1α and spliced XBP1 was observed indicating a lack of activation of corrective mechanisms to reinstate ER homeostasis. The data suggest that oxidative stress and prolonged ER stress, coupled with formation and accumulation of cytotoxic FAEEs may contribute to the pathogenesis of alcoholic lung disease. - Highlights: �

Disuse exaggerates the detrimental effects of alcohol on cortical bone

NASA Technical Reports Server (NTRS)

Hefferan, Theresa E.; Kennedy, Angela M.; Evans, Glenda L.; Turner, Russell T.

2003-01-01

BACKGROUND: Alcohol abuse is associated with an increased risk for osteoporosis. However, comorbidity factors may play an important role in the pathogenesis of alcohol-related bone fractures. Suboptimal mechanical loading of the skeleton, an established risk factor for bone loss, may occur in some alcohol abusers due to reduced physical activity, muscle atrophy, or both. The effect of alcohol consumption and reduced physical activity on bone metabolism has not been well studied. The purpose of this study was to determine whether mechanical disuse alters bone metabolism in a rat model for chronic alcohol abuse. METHODS: Alcohol was administered in the diet (35% caloric intake) of 6-month-old male rats for 4 weeks. Rats were hindlimb-unloaded the final 2 weeks of the experiment to prevent dynamic weight bearing. Afterward, cortical bone histomorphometry was evaluated at the tibia-fibula synostosis. RESULTS: At the periosteal surface of the tibial diaphysis, alcohol and hindlimb unloading independently decreased the mineralizing perimeter, mineral apposition rate, and bone formation rate. In addition, alcohol, but not hindlimb unloading, increased endocortical bone resorption. The respective detrimental effects of alcohol and hindlimb unloading to inhibit bone formation were additive; there was no interaction between the two variables. CONCLUSIONS: Reduced weight bearing accentuates the detrimental effects of alcohol on cortical bone in adult male rats by further inhibiting bone formation. This finding suggests that reduced physical activity may be a comorbidity factor for osteoporosis in alcohol abusers.

Changes in soluble sugar, starch, and alcohol dehydrogenase in Arabidopsis thaliana exposed to N2 diluted atmospheres

NASA Technical Reports Server (NTRS)

Porterfield, D. M.; Crispi, M. L.; Musgrave, M. E.

1997-01-01

Proper exchange of atmospheric gases is important for normal root and shoot metabolism in plants. This study was conducted to determine how restricted air supply affects foliar carbohydrates, while using the marker enzyme alcohol dehydrogenase (ADH) to report on the oxygenation status of the rootzone. Fourteen-day-old Arabidopsis thaliana (L.) Heynh. plants grown singly in 7-ml tubes containing agarified nutrient medium were placed in coupled Magenta vessels and exposed for six days to either ambient air or one of six different air/nitrogen dilutions. Redox potential of the agar medium was measured immediately after harvesting and freezing leaf tissue, and then root systems were quickly extracted from the agar and frozen for subsequent analyses. Redox potential measurements indicated that this series of gas mixtures produced a transition from hypoxia to anoxia in the root zones. Root ADH activity increased at higher rates as the redox potential neared anoxic levels. In contrast, ADH mRNA expression quickly neared its maximum as the medium became hypoxic and showed little further increase as it became anoxic. Foliar carbohydrate levels increased 1.5- to 2-fold with decreased availability of metabolic gases, with starch increasing at higher concentrations of air than soluble carbohydrate. The results serve as a model for plant performance under microgravity conditions, where absence of convective air movement prevents replenishment of metabolic gases.

The accent method of voice therapy: effect of accentuations on FO, SPL, and airflow.

PubMed

Kotby, M N; Shiromoto, O; Hirano, M

1993-12-01

The effect of the increased flow rate (delta U) in response to the Accent Method exercises on fundamental frequency (FO) and sound pressure level (SPL) was studied in three subjects (professionally trained, trained, and untrained in this method). In all the subjects, the rhythmic accentuated exercises produced a variable degree of increase in FO (delta FO) and SPL (delta SPL). The professionally trained subject showed greater delta FO and delta SPL in response to the delta U in the fastest tempo, which requires higher skills. Both trained subjects showed a greater correlation between delta U and both delta SPL and delta FO, as well as between delta FO and delta SPL, as compared to the untrained subject. The effects of the accentuated exercises on FO and SPL in response to the increased airflow rate (delta U) thus appear to demonstrate the treating effectiveness of the Accent Method.

Limonene dehydrogenase hydroxylates the allylic methyl group of cyclic monoterpenes in the anaerobic terpene degradation by Castellaniella defragrans.

PubMed

Puentes-Cala, Edinson; Liebeke, Manuel; Markert, Stephanie; Harder, Jens

2018-05-01

The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprene and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen. The purified limonene:ferrocenium oxidoreductase activity hydroxylated the methyl group of limonene (1-methyl-4-(1-methylethenyl)-cyclohex-1-ene) yielding perillyl alcohol ([4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methanol). The enzyme had a dithiothreitol:perillyl alcohol oxidoreductase activity yielding limonene. Mass spectrometry and molecular size determinations revealed a heterodimeric enzyme comprising CtmA and CtmB. Recently the two proteins had been identified by transposon mutagenesis and proteomics as part of the cyclic terpene metabolism ( ctm ) in Castellaniella defragrans and were annotated as FAD-dependent oxidoreductases of the protein domain family phytoene dehydrogenases and related proteins (COG1233). CtmAB is the first heterodimeric enzyme in this protein superfamily. Flavins in the purified CtmAB are oxidized by ferrocenium and are reduced by limonene. Heterologous expression of CtmA, CtmB and CtmAB in E. coli demonstrated that limonene dehydrogenase activity required both subunits carrying each a flavin cofactor. Native CtmAB oxidized a wide range of monocyclic monoterpenes containing the allylic methyl group motif (1-methyl-cyclohex-1-ene). In conclusion, we have identified CtmAB as a hydroxylating limonene dehydrogenase and the first heteromer in a family of FAD-dependent dehydrogenases acting on allylic methylene or methyl CH-bonds. We suggest a placement in EC 1

Asymmetric reduction of ketones and β-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum.

PubMed

Dudzik, A; Snoch, W; Borowiecki, P; Opalinska-Piskorz, J; Witko, M; Heider, J; Szaleniec, M

2015-06-01

Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

PubMed

Chen, Xiaoyan; Xu, Jingliang; Yang, Liu; Yuan, Zhenhong; Xiao, Shiyuan; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

2015-11-01

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 μmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

PubMed

Jantama, Kaemwich; Polyiam, Pattharasedthi; Khunnonkwao, Panwana; Chan, Sitha; Sangproo, Maytawadee; Khor, Kirin; Jantama, Sirima Suvarnakuta; Kanchanatawee, Sunthorn

2015-07-01

Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis.

PubMed

Kärkönen, Anna; Fry, Stephen C

2006-03-01

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K (m) (for UDP-glucose) 0.5-1.0 mM; there was also a minor activity with an unusually high K (m) of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K (m) values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E(L), E(M) and E(H) respectively). E(M) was the single major contributor to extractable UDPGDH activity when assayed at 0.6-9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E(L)-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E(H) activity is not due to ADH. At 30 microM UDP-glucose, 20-150 microM UDP-xylose inhibited UDPGDH activity, whereas 5-15 microM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

DOE PAGES

Hon, Shuen; Lanahan, Anthony; Tian, Liang; ...

2016-04-22

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (bothmore » wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.« less

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

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

Hon, Shuen; Lanahan, Anthony; Tian, Liang

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (bothmore » wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.« less

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs).

PubMed

Hon, Shuen; Lanahan, Anthony A; Tian, Liang; Giannone, Richard J; Hettich, Robert L; Olson, Daniel G; Lynd, Lee R

2016-12-01

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE . To explore the effects of overexpressing wild-type, mutant, and exogenous adhE s, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum . As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

Protective effect of Flos puerariae extract following acute alcohol intoxication in mice.

PubMed

Chen, Xiao; Cai, Fei; Guo, Shuang; Ding, Fang; He, Yi; Wu, Jiliang; Liu, Chao

2014-07-01

The effect of Flos Puerariae extract (FPE) on alcohol metabolism, hepatic injury, and memory impairment was assessed following acute ethanol (EtOH) intoxication in mice. The model of acute EtOH intoxication was established by intragastric administration with 8 g/kg EtOH in mice. FPE was orally administrated (gavage) once a day for 7 consecutive days. Mice were randomly divided into 4 groups: control group, model group, and FPE groups (100, 200 mg/kg). Alcohol tolerance and intoxication time, blood alcohol concentration, the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in liver, aspartate amino transferase (AST) and alanine amino transferase (ALT) in serum, superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase and the formation of malondialdehyde (MDA) in both liver and brain, as well as memory ability were determined after acute alcohol exposure. Compared with model group, pretreatment with FPE significantly prolonged alcohol tolerance time and shortened intoxication time, which is accompanied by decreased blood alcohol concentration and elevated activities of ADH and ALDH in liver. Moreover, the index of hepatic injury, ALT, and AST activities in serum was markedly decreased by pretreatment with FPE. Additionally, decreased MDA level, enhanced GSH-px and catalase activities in liver, as well as enhanced SOD and catalase activities in brain were found in FPE pretreated mice after acute exposure to EtOH. Furthermore, FPE pretreated mice showed markedly relieved memory disruption following acute EtOH intoxication. This study suggests that FPE pretreatment could enhance alcohol metabolism, prevent hepatic injury, and relieve memory impairment after acute alcohol intoxication and that this effect is likely related to its modulation on the alcohol metabolizing and antioxidant enzymes. Copyright © 2014 by the Research Society on Alcoholism.

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase.

PubMed

Miyazaki, Kentaro

2005-05-27

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.

The functional divergence of short-chain dehydrogenases involved in tropinone reduction.

PubMed

Brock, Andrea; Brandt, Wolfgang; Dräger, Birgit

2008-05-01

Tropane alkaloids typically occur in the Solanaceae and are also found in Cochlearia officinalis, a member of the Brassicaceae. Tropinone reductases are key enzymes of tropane alkaloid metabolism. Two different tropinone reductases form one stereoisomeric product each, either tropine for esterified alkaloids or pseudotropine that is converted to calystegines. A cDNA sequence with similarity to known tropinone reductases (TR) was cloned from C. officinalis. The protein was expressed in Escherichia coli, and found to catalyze the reduction of tropinone. The enzyme is a member of the short-chain dehydrogenase enzyme family and shows broad substrate specificity. Several synthetic ketones were accepted as substrates, with higher affinity and faster enzymatic turnover than observed for tropinone. C. officinalis TR produced both the isomeric alcohols tropine and pseudotropine from tropinone using NADPH + H(+) as co-substrate. Tropinone reductases of the Solanaceae, in contrast, are strictly stereospecific and form one tropane alcohol only. The Arabidopsis thaliana homologue of C. officinalis TR showed high sequence similarity, but did not reduce tropinone. A tyrosine residue was identified in the active site of C. officinalis TR that appeared responsible for binding and orientation of tropinone. Mutagenesis of the tyrosine residue yielded an active reductase, but with complete loss of TR activity. Thus C. officinalis TR presents an example of an enzyme with relaxed substrate specificity, like short-chain dehydrogenases, that provides favorable preconditions for the evolution of novel functions in biosynthetic sequences.

Association between ADH1C and ALDH2 polymorphisms and alcoholism in a Turkish sample.

PubMed

Ayhan, Yavuz; Gürel, Şeref Can; Karaca, Özgür; Zoto, Teuta; Hayran, Mutlu; Babaoğlu, Melih; Yaşar, Ümit; Bozkurt, Atilla; Dilbaz, Nesrin; Uluğ, Berna Diclenur; Demir, Başaran

2015-04-01

Polymorphisms in the genes encoding alcohol metabolizing enzymes are associated with alcohol dependence. To evaluate the association between the alcohol dehydrogenase 1C (ADH1C) Ile350Val and aldehyde dehydrogenase 2 (ALDH2) Glu504Lys polymorphisms and alcohol dependence in a Turkish sample. 235 individuals (115 alcohol-dependent patients and 120 controls) were genotyped for ADH1C and ALDH2 with PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). Association between the polymorphisms and family history, daily and maximum amount of alcohol consumed was investigated. The associations between alcohol dependence, severity of consumption and family history and the polymorphisms were analyzed by chi-square or Fisher's exact test where necessary. Relationship between genotypes and dependence related features was evaluated using analysis of variance (ANOVA). The -350Val allele for ADH1C (ADH1C*2) was increased in alcohol-dependent patients (P = 0.05). In individuals with a positive family history, the genotype distribution differed significantly (P = 0.031) and more patients carried the Val allele compared with controls (P = 0.025). Genotyping of 162 participants did not reveal the -504Lys allele in ALDH2. These findings suggest that ADH1C*2 is associated with alcohol dependence in the Turkish population displaying a dominant inheritance model. ADH1C*2 allele may contribute to the variance in heritability of alcohol dependence. The ALDH2 -504Lys/Lys or Glu/Lys genotypes were not present in alcohol-dependent patients, similar to that seen in European populations and in contrast to the findings in the Asian populations.

How and when predictability interacts with accentuation in temporally selective attention during speech comprehension.

PubMed

Li, Xiaoqing; Lu, Yong; Zhao, Haiyan

2014-11-01

The present study used EEG to investigate how and when top-down prediction interacts with bottom-up acoustic signals in temporally selective attention during speech comprehension. Mandarin Chinese spoken sentences were used as stimuli. We systematically manipulated the predictability and de/accentuation of the critical words in the sentence context. Meanwhile, a linguistic attention probe 'ba' was presented concurrently with the critical words or not. The results showed that, first, words with a linguistic attention probe elicited a larger N1 than those without a probe. The latency of this N1 effect was shortened for accented or lowly predictable words, indicating more attentional resources allocated to these words. Importantly, prediction and accentuation showed a complementary interplay on the latency of this N1 effect, demonstrating that when the words had already attracted attention due to low predictability or due to the presence of pitch accent, the other factor did not modulate attention allocation anymore. Second, relative to the lowly predictable words, the highly predictable words elicited a reduced N400 and enhanced gamma-band power increases, especially under the accented conditions; moreover, under the accented conditions, shorter N1 peak-latency was found to correlate with larger gamma-band power enhancement, which indicates that a close relationship might exist between early selective attention and later semantic integration. Finally, the interaction between top-down selective attention (driven by prediction) and bottom-up selective attention (driven by accentuation) occurred before lexical-semantic processing, namely before the N400 effect evoked by predictability, which was discussed with regard to the language comprehension models. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Alcohol intake--a two-edged sword. Part 1: metabolism and pathogenic effects of alcohol].

PubMed

Ströhle, Alexander; Wolters, Maike; Hahn, Andreas

2012-08-01

From the biomedical point of view alcohol is a Janus-faced dietary component with a dose-dependent effect varying from cardiovascular protection to cytotoxicity. Alcohol is absorbed in the upper gastrointestinal tract by passive diffusion, is quickly distributed throughout body water and is mostly eliminated through oxidation. The enzymatically-catalyzed oxidative degradation to acetaldehyde and further to acetate is primarily localized in the liver. In case of a low blood alcohol concentration (<0.5 per thousand) alcohol is predominantely metabolized by the enzyme aldehyde dehydrogenase; higher blood concentrations (>0.5 per thousand) are increasingly oxidized by the microsomal ethanoloxidizing system (MEOS). Alcohol consumption induces several metabolic reactions as well as acute effects on the central nervous system. Chronic alcohol consumption to some extent irreparably damages nearly every organ with the liver being particularly concerned. There are three stages of alcohol-induced liver disease (fatty liver, alcohol hepatitis, liver cirrhosis) and the liver damages mainly result from reaction products of alcohol degradation (acetaldehyde, NADH and reactive oxygen species). An especially dreaded clinical complication of the alcohol-induced liver disease is the hepatic encephalopathy. Its pathogenesis is a multifactorial and self-perpetuating process with the swelling of astrocytes being a crucial point. Swollen astrocytes induce several reactions such as oxidative/nitrosative stress, impaired signal transduction, protein modifications and a modified gene expression profile. The swelling of astrocytes and the change in neuronal activity are attributed to several neurotoxins, especially ammonia and aromatic amino acids. In alcohol addicted subjects multiple micronutrient deficiencies are common. The status of folic acid, thiamine, pyridoxine and zinc is especially critical.

Association between alcohol-induced erythrocyte membrane alterations and hemolysis in chronic alcoholics

PubMed Central

Bulle, Saradamma; Reddy, Vaddi Damodara; Padmavathi, Pannuru; Maturu, Paramahamsa; Puvvada, Pavan Kumar; Nallanchakravarthula, Varadacharyulu

2017-01-01

The present study aimed to understand the association between erythrocyte membrane alterations and hemolysis in chronic alcoholics. Study was conducted on human male volunteers aged between 35–45 years with a drinking history of 8–10 years. Results showed that plasma marker enzymes AST, ALT, ALP and γGT were increased in alcoholic subjects. Plasma and erythrocyte membrane lipid peroxidation, erythrocyte lysate nitric oxide (NOx) levels were also increased significantly in alcoholics. Furthermore, erythrocyte membrane protein carbonyls, total cholesterol, phospholipid and cholesterol/phospholipid (C/P) ratio were increased in alcoholics. SDS-PAGE analysis of erythrocyte membrane proteins revealed that increased density of band 3, protein 4.2, 4.9, actin and glycophorins, whereas glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glycophorin A showed slight increase, however, decreased ankyrin with no change in spectrins (α and β) and protein 4.1 densities were observed in alcoholics. Moreover, alcoholics red blood cells showed altered morphology with decreased resistance to osmotic hemolysis. Increased hemolysis showed strong positive association with lipid peroxidation (r = 0.703, p<0.05), protein carbonyls (r = 0.754, p<0.05), lysate NOx (r = 0.654, p<0.05) and weak association with C/P ratio (r = 0.240, p<0.05). Bottom line, increased lipid and protein oxidation, altered membrane C/P ratio and membrane cytoskeletal protein profile might be responsible for the increased hemolysis in alcoholics. PMID:28163384

Association between alcohol-induced erythrocyte membrane alterations and hemolysis in chronic alcoholics.

PubMed

Bulle, Saradamma; Reddy, Vaddi Damodara; Padmavathi, Pannuru; Maturu, Paramahamsa; Puvvada, Pavan Kumar; Nallanchakravarthula, Varadacharyulu

2017-01-01

The present study aimed to understand the association between erythrocyte membrane alterations and hemolysis in chronic alcoholics. Study was conducted on human male volunteers aged between 35-45 years with a drinking history of 8-10 years. Results showed that plasma marker enzymes AST, ALT, ALP and γGT were increased in alcoholic subjects. Plasma and erythrocyte membrane lipid peroxidation, erythrocyte lysate nitric oxide (NOx) levels were also increased significantly in alcoholics. Furthermore, erythrocyte membrane protein carbonyls, total cholesterol, phospholipid and cholesterol/phospholipid (C/P) ratio were increased in alcoholics. SDS-PAGE analysis of erythrocyte membrane proteins revealed that increased density of band 3, protein 4.2, 4.9, actin and glycophorins, whereas glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glycophorin A showed slight increase, however, decreased ankyrin with no change in spectrins (α and β) and protein 4.1 densities were observed in alcoholics. Moreover, alcoholics red blood cells showed altered morphology with decreased resistance to osmotic hemolysis. Increased hemolysis showed strong positive association with lipid peroxidation ( r  = 0.703, p <0.05), protein carbonyls ( r  = 0.754, p <0.05), lysate NOx ( r  = 0.654, p <0.05) and weak association with C/P ratio ( r  = 0.240, p <0.05). Bottom line, increased lipid and protein oxidation, altered membrane C/P ratio and membrane cytoskeletal protein profile might be responsible for the increased hemolysis in alcoholics.

The Genetics of a Small Autosomal Region of DROSOPHILA MELANOGASTER Containing the Structural Gene for Alcohol Dehydrogenase. I. Characterization of Deficiencies and Mapping of ADH and Visible Mutations

PubMed Central

Woodruff, R. C.; Ashburner, M.

1979-01-01

The position of the structural gene coding for alcohol dehydrogenase (ADH) in Drosophila melanogaster has been shown to be within polytene chromosome bands 35B1 and 35B3, most probably within 35B2. The genetic and cytological properties of twelve deficiencies in polytene chromosome region 34–35 have been characterized, eleven of which include Adh. Also mapped cytogenetically are seven other recessive visible mutant loci. Flies heterozygous for overlapping deficiencies that include both the Adh locus and that for the outspread mutant (osp: a recessive wing phenotype) are homozygous viable and show a complete ADH negative phenotype and strong osp phenotype. These deficiencies probably include two polytene chromosome bands, 35B2 and 35B3. PMID:115743

Gender differences in the effects of ADH1B and ALDH2 polymorphisms on alcoholism.

PubMed

Kimura, Mitsuru; Miyakawa, Tomohiro; Matsushita, Sachio; So, Mirai; Higuchi, Susumu

2011-11-01

Gender differences are known to exist in the prevalence, characteristics, and course of alcohol dependence. Elucidating gender differences in the characteristics of alcohol dependence is important in gender-based medicine and may improve treatment outcomes. Many studies have shown that genetic factors are associated with the risk of alcohol dependence in both genders. Polymorphisms of alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) are strong genetic determinants of alcohol dependence. This study aimed to clarify gender differences in the effects of ADH1B and ALDH2 polymorphism on the development of alcohol dependence. Subjects were 200 female alcoholics and 415 male alcoholics hospitalized in Kurihama Alcoholism Center. Clinical information and background data were obtained by chart review. ALDH2 and ADH1B genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method. The onset age of female alcoholics with inactive ALDH2 genotype was significantly lower than those with active ALDH2 genotype, but the onset age did not differ between the inactive and active ALDH2 group in male alcoholics. The difference in onset age between the ADH1B genotype groups did not reach significant levels. The prevalence of comorbid psychiatric disorders, including major depression, eating disorder, panic disorder, and borderline personality disorder, was significantly higher in female alcoholics with inactive ALDH2 or superactive ADH1B than in those with active ALDH2 or normal ADH1B. ALDH2 polymorphism appears to have contrasting effects on the development of alcoholism in women and men. One possible reason for this gender difference may be the high prevalence of psychiatric comorbidities in female alcoholics with inactive ALDH2. Copyright © 2011 by the Research Society on Alcoholism.

Allelic variants of ADH, ALDH and the five factor model of personality in alcohol dependence syndrome

PubMed Central

Salujha, S. K.; Chaudhury, S.; Menon, P. K.; Srivastava, K.; Gupta, A.

2014-01-01

Background: The etiology of alcohol dependence is a complex interplay of biopsychosocial factors. The genes for alcohol-metabolizing enzymes: Alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) exhibit functional polymorphisms. Vulnerability of alcohol dependence may also be in part due to heritable personality traits. Aim: To determine whether any association exists between polymorphisms of ADH2, ADH3 and ALDH2 and alcohol dependence syndrome in a group of Asian Indians. In addition, the personality of these patients was assessed to identify traits predisposing to alcoholism. Materials and Methods: In this study, 100 consecutive males with alcohol dependence syndrome attending the psychiatric outpatient department of a tertiary care service hospital and an equal number of matched healthy controls were included with their consent. Blood samples of all the study cases and controls were collected and genotyped for the ADH2, ADH3 and ALDH2 loci. Personality was evaluated using the neuroticism, extraversion, openness (NEO) personality inventory and sensation seeking scale. Results: Allele frequencies of ADH2*2 (0.50), ADH3*1 (0.67) and ALSH2*2 (0.09) were significantly low in the alcohol dependent subjects. Personality traits of NEO personality inventory and sensation seeking were significantly higher when compared to controls. Conclusions: The functional polymorphisms of genes coding for alcohol metabolizing enzymes and personality traits of NEO and sensation seeking may affect the propensity to develop dependence. PMID:25535445

Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.

PubMed

Shirasu, Naoto; Kuroki, Masahide

2014-01-01

We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp(®) DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.

Crystallization and preliminary X-ray analysis of binary and ternary complexes of Haloferax mediterranei glucose dehydrogenase

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

Esclapez, Julia; Britton, K. Linda; Baker, Patrick J.

2005-08-01

Single crystals of binary and ternary complexes of wild-type and D38C mutant H. mediterranei glucose dehydrogenase have been obtained by the hanging-drop vapour-diffusion method. Haloferax mediterranei glucose dehydrogenase (EC 1.1.1.47) belongs to the medium-chain alcohol dehydrogenase superfamily and requires zinc for catalysis. In the majority of these family members, the catalytic zinc is tetrahedrally coordinated by the side chains of a cysteine, a histidine, a cysteine or glutamate and a water molecule. In H. mediterranei glucose dehydrogenase, sequence analysis indicates that the zinc coordination is different, with the invariant cysteine replaced by an aspartate residue. In order to analyse themore » significance of this replacement and to contribute to an understanding of the role of the metal ion in catalysis, a range of binary and ternary complexes of the wild-type and a D38C mutant protein have been crystallized. For most of the complexes, crystals belonging to space group I222 were obtained using sodium/potassium citrate as a precipitant. However, for the binary and non-productive ternary complexes with NADPH/Zn, it was necessary to replace the citrate with 2-methyl-2,4-pentanediol. Despite the radical change in conditions, the crystals thus formed were isomorphous.« less

The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns

PubMed Central

Jin, Yazhong; Zhang, Chong; Liu, Wei; Tang, Yufan; Qi, Hongyan; Chen, Hao; Cao, Songxiao

2016-01-01

Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and –insensitive groups, and the functions of CmADHs were discussed. PMID:27242871

Prognostic value of alcohol dehydrogenase mRNA expression in gastric cancer.

PubMed

Guo, Erna; Wei, Haotang; Liao, Xiwen; Xu, Yang; Li, Shu; Zeng, Xiaoyun

2018-04-01

Previous studies have reported that alcohol dehydrogenase (ADH) isoenzymes possess diagnostic value in gastric cancer (GC). However, the prognostic value of ADH isoenzymes in GC remains unclear. The aim of the present study was to identify the prognostic value of ADH genes in patients with GC. The prognostic value of ADH genes was investigated in patients with GC using the Kaplan-Meier plotter tool. Kaplan-Meier plots were used to assess the difference between groups of patients with GC with different prognoses. Hazard ratios (HR) and 95% confidence intervals (CI) were used to assess the relative risk of GC survival. Overall, 593 patients with GC and 7 ADH genes were included in the survival analysis. High expression of ADH 1A (class 1), α polypeptide ( ADH1A; log-rank P=0.043; HR=0.79; 95% CI: 0.64-0.99), ADH 1B (class 1), β polypeptide ( ADH1B ; log-rank P=1.9×10 -05 ; HR=0.65; 95% CI: 0.53-0.79) and ADH 5 (class III), χ polypeptide ( ADH5 ; log-rank P=0.0011; HR=0.73; 95% CI: 0.6-0.88) resulted in a significantly decreased risk of mortality in all patients with GC compared with patients with low expression of those genes. Furthermore, protective effects may additionally be observed in patients with intestinal-type GC with high expression of ADH1B (log-rank P=0.031; HR=0.64; 95% CI: 0.43-0.96) and patients with diffuse-type GC with high expression of ADH1A (log-rank P=0.014; HR=0.51; 95% CI: 0.3-0.88), ADH1B (log-rank P=0.04; HR=0.53; 95% CI: 0.29-0.98), ADH 4 (class II), π polypeptide (log-rank P=0.033; HR=0.58; 95% CI: 0.35-0.96) and ADH 6 (class V) (log-rank P=0.037; HR=0.59; 95% CI: 0.35-0.97) resulting in a significantly decreased risk of mortality compared with patients with low expression of those genes. In contrast, patients with diffuse-type GC with high expression of ADH5 (log-rank P=0.044; HR=1.66; 95% CI: 1.01-2.74) were significantly correlated with a poor prognosis. The results of the present study suggest that ADH1A and ADH1B may be potential

Prognostic value of alcohol dehydrogenase mRNA expression in gastric cancer

PubMed Central

Guo, Erna; Wei, Haotang; Liao, Xiwen; Xu, Yang; Li, Shu; Zeng, Xiaoyun

2018-01-01

Previous studies have reported that alcohol dehydrogenase (ADH) isoenzymes possess diagnostic value in gastric cancer (GC). However, the prognostic value of ADH isoenzymes in GC remains unclear. The aim of the present study was to identify the prognostic value of ADH genes in patients with GC. The prognostic value of ADH genes was investigated in patients with GC using the Kaplan-Meier plotter tool. Kaplan-Meier plots were used to assess the difference between groups of patients with GC with different prognoses. Hazard ratios (HR) and 95% confidence intervals (CI) were used to assess the relative risk of GC survival. Overall, 593 patients with GC and 7 ADH genes were included in the survival analysis. High expression of ADH 1A (class 1), α polypeptide (ADH1A; log-rank P=0.043; HR=0.79; 95% CI: 0.64–0.99), ADH 1B (class 1), β polypeptide (ADH1B; log-rank P=1.9×10−05; HR=0.65; 95% CI: 0.53–0.79) and ADH 5 (class III), χ polypeptide (ADH5; log-rank P=0.0011; HR=0.73; 95% CI: 0.6–0.88) resulted in a significantly decreased risk of mortality in all patients with GC compared with patients with low expression of those genes. Furthermore, protective effects may additionally be observed in patients with intestinal-type GC with high expression of ADH1B (log-rank P=0.031; HR=0.64; 95% CI: 0.43–0.96) and patients with diffuse-type GC with high expression of ADH1A (log-rank P=0.014; HR=0.51; 95% CI: 0.3–0.88), ADH1B (log-rank P=0.04; HR=0.53; 95% CI: 0.29–0.98), ADH 4 (class II), π polypeptide (log-rank P=0.033; HR=0.58; 95% CI: 0.35–0.96) and ADH 6 (class V) (log-rank P=0.037; HR=0.59; 95% CI: 0.35–0.97) resulting in a significantly decreased risk of mortality compared with patients with low expression of those genes. In contrast, patients with diffuse-type GC with high expression of ADH5 (log-rank P=0.044; HR=1.66; 95% CI: 1.01–2.74) were significantly correlated with a poor prognosis. The results of the present study suggest that ADH1A and ADH1B may

Glucose-6-phosphate dehydrogenase

MedlinePlus

... page: //medlineplus.gov/ency/article/003671.htm Glucose-6-phosphate dehydrogenase test To use the sharing features on this page, please enable JavaScript. Glucose-6-phosphate dehydrogenase (G6PD) is a protein that helps ...

High efficiency preparation and characterization of intact poly(vinyl alcohol) dehydrogenase from Sphingopyxis sp.113P3 in Escherichia coli by inclusion bodies renaturation.

PubMed

Jia, Dongxu; Yang, Yu; Peng, Zhengcong; Zhang, Dongxu; Li, Jianghua; Liu, Long; Du, Guocheng; Chen, Jian

2014-03-01

Poly(vinyl alcohol) dehydrogenase (PVADH, EC 1.1.99.23) is an enzyme which has potential application in textile industry to degrade the poly(vinyl alcohol) (PVA) in waste water. Previously, a 1,965-bp fragment encoding a PVADH from Sphingopyxis sp. 113P3 was synthesized based on the replacement of the rare codons in Escherichia coli (E. coli). In this work, the deduced mature PVADH (mPVADH) gene of 1,887 bp was amplified by polymerase chain reaction (PCR) and inserted into the site between NcoI and HindIII in pET-32a(+). The constructed recombinant plasmid was transformed into E. coli Rosetta (DE3). In shake flask, the fusion protein of thioredoxin (Trx)-mPVADH was expressed precisely; however, Trx-mPVADH was found to accumulate mainly as inclusion bodies. After isolating, dissolving in buffer containing urea, purification, dialysis renaturation, and digesting with recombinant enterokinase/His (rEK/His), the bioactive mPVADH fragments were obtained with protein concentration of 0.56 g/L and enzymatic activity of 194 U/mL. The K m and V max values for PVA 1799 were 2.33 mg/mL and 15.7 nmol/(min·mg protein), respectively. (1)H-NMR and infrared (IR) spectrum demonstrated that its biological function was oxidizing hydroxyl groups of PVA 1799 to form diketone, and PVA 1799 could be degraded completely by successive treatment with mPVADH and oxidized PVA hydrolase (OPH).

Dynamic stimuli: accentuating aesthetic preference biases.

PubMed

Friedrich, Trista E; Harms, Victoria L; Elias, Lorin J

2014-01-01

Despite humans' preference for symmetry, artwork often portrays asymmetrical characteristics that influence the viewer's aesthetic preference for the image. When presented with asymmetrical images, aesthetic preference is often given to images whose content flows from left-to-right and whose mass is located on the right of the image. Cerebral lateralization has been suggested to account for the left-to-right directionality bias; however, the influence of cultural factors, such as scanning habits, on aesthetic preference biases is debated. The current research investigates aesthetic preference for mobile objects and landscapes, as previous research has found contrasting preference for the two image types. Additionally, the current experiment examines the effects of dynamic movement on directionality preference to test the assumption that static images are perceived as aesthetically equivalent to dynamic images. After viewing mirror-imaged pairs of pictures and videos, right-to-left readers failed to show a preference bias, whereas left-to-right readers preferred stimuli with left-to-right directionality regardless of the location of the mass. The directionality bias in both reading groups was accentuated by the videos, but the bias was significantly stronger in left-to-right readers. The findings suggest that scanning habits moderate the leftward bias resulting from hemispheric specialization and that dynamic stimuli further fluent visual processing.

Diversity and Evolutionary Analysis of Iron-Containing (Type-III) Alcohol Dehydrogenases in Eukaryotes

PubMed Central

Gaona-López, Carlos; Julián-Sánchez, Adriana

2016-01-01

Background Alcohol dehydrogenase (ADH) activity is widely distributed in the three domains of life. Currently, there are three non-homologous NAD(P)+-dependent ADH families reported: Type I ADH comprises Zn-dependent ADHs; type II ADH comprises short-chain ADHs described first in Drosophila; and, type III ADH comprises iron-containing ADHs (FeADHs). These three families arose independently throughout evolution and possess different structures and mechanisms of reaction. While types I and II ADHs have been extensively studied, analyses about the evolution and diversity of (type III) FeADHs have not been published yet. Therefore in this work, a phylogenetic analysis of FeADHs was performed to get insights into the evolution of this protein family, as well as explore the diversity of FeADHs in eukaryotes. Principal Findings Results showed that FeADHs from eukaryotes are distributed in thirteen protein subfamilies, eight of them possessing protein sequences distributed in the three domains of life. Interestingly, none of these protein subfamilies possess protein sequences found simultaneously in animals, plants and fungi. Many FeADHs are activated by or contain Fe2+, but many others bind to a variety of metals, or even lack of metal cofactor. Animal FeADHs are found in just one protein subfamily, the hydroxyacid-oxoacid transhydrogenase (HOT) subfamily, which includes protein sequences widely distributed in fungi, but not in plants), and in several taxa from lower eukaryotes, bacteria and archaea. Fungi FeADHs are found mainly in two subfamilies: HOT and maleylacetate reductase (MAR), but some can be found also in other three different protein subfamilies. Plant FeADHs are found only in chlorophyta but not in higher plants, and are distributed in three different protein subfamilies. Conclusions/Significance FeADHs are a diverse and ancient protein family that shares a common 3D scaffold with a patchy distribution in eukaryotes. The majority of sequenced FeADHs from

Manipulating cinnamyl alcohol dehydrogenase (CAD) expression in flax affects fibre composition and properties

PubMed Central

2014-01-01

Background In recent decades cultivation of flax and its application have dramatically decreased. One of the reasons for this is unpredictable quality and properties of flax fibre, because they depend on environmental factors, retting duration and growing conditions. These factors have contribution to the fibre composition, which consists of cellulose, hemicelluloses, lignin and pectin. By far, it is largely established that in flax, lignin reduces an accessibility of enzymes either to pectin, hemicelluloses or cellulose (during retting or in biofuel synthesis and paper production). Therefore, in this study we evaluated composition and properties of flax fibre from plants with silenced CAD (cinnamyl alcohol dehydrogenase) gene, which is key in the lignin biosynthesis. There is evidence that CAD is a useful tool to improve lignin digestibility and/or to lower the lignin levels in plants. Results Two studied lines responded differentially to the introduced modification due to the efficiency of the CAD silencing. Phylogenetic analysis revealed that flax CAD belongs to the “bona-fide” CAD family. CAD down-regulation had an effect in the reduced lignin amount in the flax fibre cell wall and as FT-IR results suggests, disturbed lignin composition and structure. Moreover introduced modification activated a compensatory mechanism which was manifested in the accumulation of cellulose and/or pectin. These changes had putative correlation with observed improved fiber’s tensile strength. Moreover, CAD down-regulation did not disturb at all or has only slight effect on flax plants’ development in vivo, however, the resistance against flax major pathogen Fusarium oxysporum decreased slightly. The modification positively affected fibre possessing; it resulted in more uniform retting. Conclusion The major finding of our paper is that the modification targeted directly to block lignin synthesis caused not only reduced lignin level in fibre, but also affected amount and

Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis

PubMed Central

Leitsch, David; Drinić, Mirjana; Kolarich, Daniel; Duchêne, Michael

2012-01-01

The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem. In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE). Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde. PMID:22449940

How long-term memory and accentuation interact during spoken language comprehension.

PubMed

Li, Xiaoqing; Yang, Yufang

2013-04-01

Spoken language comprehension requires immediate integration of different information types, such as semantics, syntax, and prosody. Meanwhile, both the information derived from speech signals and the information retrieved from long-term memory exert their influence on language comprehension immediately. Using EEG (electroencephalogram), the present study investigated how the information retrieved from long-term memory interacts with accentuation during spoken language comprehension. Mini Chinese discourses were used as stimuli, with an interrogative or assertive context sentence preceding the target sentence. The target sentence included one critical word conveying new information. The critical word was either highly expected or lowly expected given the information retrieved from long-term memory. Moreover, the critical word was either consistently accented or inconsistently de-accented. The results revealed that for lowly expected new information, inconsistently de-accented words elicited a larger N400 and larger theta power increases (4-6 Hz) than consistently accented words. In contrast, for the highly expected new information, consistently accented words elicited a larger N400 and larger alpha power decreases (8-14 Hz) than inconsistently de-accented words. The results suggest that, during spoken language comprehension, the effect of accentuation interacted with the information retrieved from long-term memory immediately. Moreover, our results also have important consequences for our understanding of the processing nature of the N400. The N400 amplitude is not only enhanced for incorrect information (new and de-accented word) but also enhanced for correct information (new and accented words). Copyright © 2013 Elsevier Ltd. All rights reserved.

Alcoholic Ketosis: Prevalence, Determinants, and Ketohepatitis in Japanese Alcoholic Men.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Mizukami, Takeshi; Matsui, Toshifumi; Shiraishi, Koichi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2014-11-01

Alcoholic ketosis and ketoacidosis are metabolic abnormalities often diagnosed in alcoholics in emergency departments. We attempted to identify determinants or factors associated with alcoholic ketosis. The subjects of this cross-sectional survey were 1588 Japanese alcoholic men (≥40 years) who came to an addiction center within 14 days of their last drink. The results of the dipstick urinalyses revealed a prevalence of ketosis of 34.0% (±, 21.5%; +, 8.9%; and 2+/3+; 3.6%) in the alcoholics. Higher urine ketone levels were associated with higher serum total bilirubin, aspartate transaminase (AST), alanine transaminase and gamma-glutamyl transpeptidase levels. A multivariate analysis by the proportional odds model showed that the odds ratio (95% confidence interval) for an increase in ketosis by one category was 0.94 (0.84-1.06) per 10-year increase in age, 0.93 (0.89-0.97) per 1-day increase in interval since the last drink, 1.78 (1.41-2.26) in the presence of slow-metabolizing alcohol dehydrogenase-1B (ADH1B*1/*1), 1.61 (1.10-2.36) and 1.30 (1.03-1.65) when the beverage of choice was whiskey and shochu, respectively (distilled no-carbohydrate beverages vs. the other beverages), 2.05 (1.27-3.32) in the presence of hypoglycemia <80 mg/dl, 0.91 (0.88-0.94) per 1-kg/m(2) increase in body mass index (BMI), 1.09 (1.00-1.18) per +10 cigarettes smoked, and 2.78 (2.05-3.75) when the serum total bilirubin level was ≥2.0 mg/dl, and 1.97 (1.47-2.66) when the serum AST level was ≥200 IU/l. Ketosis was a very common complication and frequently accompanied by alcoholic liver injury in our Japanese male alcoholic population, in which ADH1B*1/*1 genotype, consumption of whiskey or shochu, hypoglycemia, lower BMI and smoking were significant determinants of the development of ketosis. © The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.

S-adenosylmethionine decreases the peak blood alcohol levels 3 h after an acute bolus of ethanol by inducing alcohol metabolizing enzymes in the liver.

PubMed

Bardag-Gorce, Fawzia; Oliva, Joan; Wong, Wesley; Fong, Stephanie; Li, Jun; French, Barbara A; French, Samuel W

2010-12-01

An alcohol bolus causes the blood alcohol level (BAL) to peak at 1-2 h post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3 h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking. Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3 h or 12 h after the bolus. RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3 h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12 h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3 h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3 h post bolus. These results correlated with the low BAL found at 3 h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2. Copyright © 2010 Elsevier Inc. All rights reserved.

Cyclic ethanol metabolism in hypophysectomized rats continuously infused alcohol-containing diets

USDA-ARS?s Scientific Manuscript database

Chronic ethanol (EtOH) intake induces hepatic alcohol dehydrogenase (ADH) expression via disruption of insulin signaling in liver (JBC 2006; 281:1126-34). Total enteral nutrition (TEN) is a method of slow and continuous (approx. 23/day) feeding patients through an intragastric tube. Rats fed EtOH-co...

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

PubMed

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

2002-12-06

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

Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

PubMed Central

Hara, A; Hayashibara, M; Nakayama, T; Hasebe, K; Usui, S; Sawada, H

1985-01-01

We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase. PMID:2983661

Alcohol and Airways Function in Health and Disease

PubMed Central

Sisson, Joseph H.

2007-01-01

The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The impact of alcohol on lung airway functions is dependent on the concentration, duration and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation and probably attenuates the airway inflammation and injury observed in asthma and COPD. Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management and likely worsens outcomes including lung function and mortality in COPD patients. Non-alcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase type 2 (ALDH2). The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation and the interaction with other airway exposure agents, such as cigarette smoke, represent opportunities for future investigation. PMID:17764883

Accentuated antagonism in vagal heart rate control mediated through muscarinic potassium channels.

PubMed

Mizuno, Masaki; Kamiya, Atsunori; Kawada, Toru; Miyamoto, Tadayoshi; Shimizu, Shuji; Shishido, Toshiaki; Sugimachi, Masaru

2008-12-01

Although muscarinic K(+) (K(ACh)) channels contribute to a rapid heart rate (HR) response to vagal stimulation, whether background sympathetic tone affects the HR control via the K(ACh)channels remains to be elucidated. In seven anesthetized rabbits with sinoaortic denervation and vagotomy, we estimated the dynamic transfer function of the HR response by using random binary vagal stimulation (0-10 Hz). Tertiapin, a selective K(ACh) channel blocker, decreased the dynamic gain (to 2.3+/- 0.9 beats.min(-1).Hz(-1), from 4.6+/- 1.1, P < 0.01, mean+/- SD) and the corner frequency (to 0.05+/- 0.01 Hz, from 0.26+/- 0.04, P < 0.01). Under 5 Hz tonic cardiac sympathetic stimulation (CSS), tertiapin decreased the dynamic gain (to 3.6+/- 1.0 beats.min(-1).Hz(-1), from 7.3+/- 1.1, P < 0.01) and the corner frequency (to 0.06+/- 0.02 Hz, from 0.23+/- 0.06, P < 0.01). Two-way analysis of variance indicated significant interaction between the tertiapin and CSS effects on the dynamic gain. In contrast, no significant interactions were observed between the tertiapin and CSS effects on the corner frequency and the lag time. In conclusion, although a cyclic AMP-dependent mechanism has been well established, an accentuated antagonism also occurred in the direct effect of ACh via the K(ACh) channels. The rapidity of the HR response obtained by the K(ACh) channel pathway was robust during the accentuated antagonism.

Interactions Between Alcohol Metabolism Genes and Religious Involvement in Association With Maximum Drinks and Alcohol Dependence Symptoms

PubMed Central

Chartier, Karen G.; Dick, Danielle M.; Almasy, Laura; Chan, Grace; Aliev, Fazil; Schuckit, Marc A.; Scott, Denise M.; Kramer, John; Bucholz, Kathleen K.; Bierut, Laura J.; Nurnberger, John; Porjesz, Bernice; Hesselbrock, Victor M.

2016-01-01

Objective: Variations in the genes encoding alcohol dehydrogenase (ADH) enzymes are associated with both alcohol consumption and dependence in multiple populations. Additionally, some environmental factors have been recognized as modifiers of these relationships. This study examined the modifying effect of religious involvement on relationships between ADH gene variants and alcohol consumption–related phenotypes. Method: Subjects were African American, European American, and Hispanic American adults with lifetime exposure to alcohol (N = 7,716; 53% female) from the Collaborative Study on the Genetics of Alcoholism. Genetic markers included ADH1B-rs1229984, ADH1B-rs2066702, ADH1C-rs698, ADH4-rs1042364, and ADH4-rs1800759. Phenotypes were maximum drinks consumed in a 24-hour period and total number of alcohol dependence symptoms according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Religious involvement was defined by self-reported religious services attendance. Results: Both religious involvement and ADH1B-rs1229984 were negatively associated with the number of maximum drinks consumed and the number of lifetime alcohol dependence symptoms endorsed. The interactions of religious involvement with ADH1B-rs2066702, ADH1C-rs698, and ADH4-rs1042364 were significantly associated with maximum drinks and alcohol dependence symptoms. Risk variants had weaker associations with maximum drinks and alcohol dependence symptoms as a function of increasing religious involvement. Conclusions: This study provided initial evidence of a modifying effect for religious involvement on relationships between ADH variants and maximum drinks and alcohol dependence symptoms. PMID:27172571

cis-Chlorobenzene Dihydrodiol Dehydrogenase (TcbB) from Pseudomonas sp. Strain P51, Expressed in Escherichia coli DH5α(pTCB149), Catalyzes Enantioselective Dehydrogenase Reactions

PubMed Central

Raschke, Henning; Fleischmann, Thomas; Van Der Meer, Jan Roelof; Kohler, Hans-Peter E.

1999-01-01

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5α(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (−)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (−)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols. PMID:10583971

cis-chlorobenzene dihydrodiol dehydrogenase (TcbB) from Pseudomonas sp. strain P51, expressed in Escherichia coli DH5alpha(pTCB149), catalyzes enantioselective dehydrogenase reactions.

PubMed

Raschke, H; Fleischmann, T; Van Der Meer, J R; Kohler, H P

1999-12-01

cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5alpha(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3, 4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1, 2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.

The effect of altered lignin composition on mechanical properties of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) deficient poplars.

PubMed

Özparpucu, Merve; Gierlinger, Notburga; Burgert, Ingo; Van Acker, Rebecca; Vanholme, Ruben; Boerjan, Wout; Pilate, Gilles; Déjardin, Annabelle; Rüggeberg, Markus

2018-04-01

CAD-deficient poplars enabled studying the influence of altered lignin composition on mechanical properties. Severe alterations in lignin composition did not influence the mechanical properties. Wood represents a hierarchical fiber-composite material with excellent mechanical properties. Despite its wide use and versatility, its mechanical behavior has not been entirely understood. It has especially been challenging to unravel the mechanical function of the cell wall matrix. Lignin engineering has been a useful tool to increase the knowledge on the mechanical function of lignin as it allows for modifications of lignin content and composition and the subsequent studying of the mechanical properties of these transgenics. Hereby, in most cases, both lignin composition and content are altered and the specific influence of lignin composition has hardly been revealed. Here, we have performed a comprehensive micromechanical, structural, and spectroscopic analysis on xylem strips of transgenic poplar plants, which are downregulated for cinnamyl alcohol dehydrogenase (CAD) by a hairpin-RNA-mediated silencing approach. All parameters were evaluated on the same samples. Raman microscopy revealed that the lignin of the hpCAD poplars was significantly enriched in aldehydes and reduced in the (relative) amount of G-units. FTIR spectra indicated pronounced changes in lignin composition, whereas lignin content was not significantly changed between WT and the hpCAD poplars. Microfibril angles were in the range of 18°-24° and were not significantly different between WT and transgenics. No significant changes were observed in mechanical properties, such as tensile stiffness, ultimate stress, and yield stress. The specific findings on hpCAD poplar allowed studying the specific influence of lignin composition on mechanics. It can be concluded that the changes in lignin composition in hpCAD poplars did not affect the micromechanical tensile properties.

Alpha-ketoglutarate reduces ethanol toxicity in Drosophila melanogaster by enhancing alcohol dehydrogenase activity and antioxidant capacity.

PubMed

Bayliak, Maria M; Shmihel, Halyna V; Lylyk, Maria P; Storey, Kenneth B; Lushchak, Volodymyr I

2016-09-01

Ethanol at low concentrations (<4%) can serve as a food source for fruit fly Drosophila melanogaster, whereas at higher concentrations it may be toxic. In this work, protective effects of dietary alpha-ketoglutarate (AKG) against ethanol toxicity were studied. Food supplementation with 10-mM AKG alleviated toxic effects of 8% ethanol added to food, and improved fly development. Two-day-old adult flies, reared on diet containing both AKG and ethanol, possessed higher alcohol dehydrogenase (ADH) activity as compared with those reared on control diet or diet with ethanol only. Native gel electrophoresis data suggested that this combination diet might promote post-translational modifications of ADH protein with the formation of a highly active ADH form. The ethanol-containing diet led to significantly higher levels of triacylglycerides stored in adult flies, and this parameter was not altered by AKG supplement. The influence of diet on antioxidant defenses was also assessed. In ethanol-fed flies, catalase activity was higher in males and the levels of low molecular mass thiols were unchanged in both sexes compared to control values. Feeding on a mixture of AKG and ethanol did not affect catalase activity but caused a higher level of low molecular mass thiols compared to ethanol-fed flies. It can be concluded that both a stimulation of some components of antioxidant defense and the increase in ADH activity may be responsible for the protective effects of AKG diet supplementation in combination with ethanol. The results suggest that AKG might be useful as a treatment option to neutralize toxic effects of excessive ethanol intake and to improve the physiological state of D. melanogaster and other animals, potentially including humans. Copyright © 2016 Elsevier Inc. All rights reserved.

The sap of Acer okamotoanum decreases serum alcohol levels after acute ethanol ingestion in rats.

PubMed

Yoo, Yeong-Min; Jung, Eui-Man; Kang, Ha-Young; Choi, In-Gyu; Choi, Kyung-Chul; Jeung, Eui-Bae

2011-10-01

In the present study, we examined whether Acer okamotoanum (A. okamotoanum) sap decreased the serum alcohol and acetaldehyde levels after acute ethanol treatment in a rat model. Male rats were orally administered 25, 50 or 100% A. okamotoanum sap 30 min prior to oral challenge with 3 ml of ethanol (15 ml/kg of a 20% ethanol solution in water), and the blood concentrations of alcohol and acetaldehyde were analyzed up to 7 h after the treatment. Pre-treatment with the sap significantly decreased the blood ethanol and acetaldehyde concentrations after 5 h when compared with ethanol treatment alone (a negative control). The expression levels of liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) mRNA were increased significantly in animals pre-treated with A. okamotoanum sap when compared with negative and positive controls. The data suggest that sap pre-treatment enhanced the alcohol metabolism rate in the rat liver. To investigate the involvement of mitochondrial regulation in the ethanol-induced hepatocyte apoptosis, we carried out an immunohistochemical analysis of Bax and Bcl-2. Pre-treatment with sap significantly decreased Bax expression and increased Bcl-2 expression 7 h after ethanol administration when compared with the negative control. The data suggest that A. okamotoanum sap pre-treatment may reduce the alcohol-induced oxidative stress in the rat liver.

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

PubMed Central

Baron, S F; Franklund, C V; Hylemon, P B

1991-01-01

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7 alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gt11, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7 alpha-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7 alpha-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5 alpha transformant harboring pBH51 had approximately 30-fold greater levels of 7 alpha-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7 alpha-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reached with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding. Images PMID:1856160

Responses to Alcohol and Cigarette Use During Ecologically Assessed Drinking Episodes

PubMed Central

Piasecki, Thomas M.; Wood, Phillip K.; Shiffman, Saul; Sher, Kenneth J.; Heath, Andrew C.

2012-01-01

Rationale Tobacco and alcohol are frequently used together, and this may be partly explained by a distinct profile of subjective effects associated with co-administration. Ecological Momentary Assessment studies have examined effects of naturally occurring co-use, but, to date, have not assessed differing effects as alcohol levels rise and fall. Objectives To describe subjective states and appraisals of cigarette and alcohol effects reported during the entirety of real-world drinking episodes. Methods Currently-smoking frequent drinkers (N = 255) carried electronic diaries for 21 days. Analyses focused on reports made during 2,046 drinking episodes. Signaled prompts intensively oversampled moments in the hours following consumption of the first drink in an episode. Multilevel regression analyses were used to predict ratings of buzz, dizziness, excitement, and sluggishness as a function of person-level and contextual covariates, estimated blood alcohol concentration (eBAC) level, ascending vs. descending eBAC, smoking, and their interactions. Appraisals of cigarette and alcohol effects were also examined within this framework. Results Buzz, excitement, and pleasure from alcohol and cigarettes were prominent features of real-world drinking episodes. Smoking was associated with enhanced buzz and excitement when eBAC was high and descending. Smoking slightly accentuated the relation between eBAC and ratings of drinking pleasure among women, but this relation was somewhat weakened by smoking among men. Conclusions Smoking during drinking episodes may be partly be explained by a persistence of stimulant alcohol effects beyond the BAC peak. Acute effects of nicotine and tobacco use on the descending limb deserve further scrutiny in experimental alcohol challenge research. PMID:22538731

Alcohol and airways function in health and disease.

PubMed

Sisson, Joseph H

2007-08-01

The volatility of alcohol promotes the movement of alcohol from the bronchial circulation across the airway epithelium and into the conducting airways of the lung. The exposure of the airways through this route likely accounts for many of the biologic effects of alcohol on lung airway functions. The effect of alcohol on lung airway functions is dependent on the concentration, duration, and route of exposure. Brief exposure to mild concentrations of alcohol may enhance mucociliary clearance, stimulates bronchodilation, and probably attenuates the airway inflammation and injury observed in asthma and chronic obstructive pulmonary disease (COPD). Prolonged and heavy exposure to alcohol impairs mucociliary clearance, may complicate asthma management, and likely worsens outcomes including lung function and mortality in COPD patients. Nonalcohol congeners and alcohol metabolites act as triggers for airway disease exacerbations especially in atopic asthmatics and in Asian populations who have a reduced capacity to metabolize alcohol. Research focused on the mechanisms of alcohol-mediated changes in airway functions has identified specific mechanisms that mediate alcohol effects within the lung airways. These include prominent roles for the second messengers calcium and nitric oxide, regulatory kinases including PKG and PKA, alcohol- and acetaldehyde-metabolizing enzymes such as aldehyde dehydrogenase 2. The role alcohol may play in the pathobiology of airway mucus, bronchial blood flow, airway smooth muscle regulation, and the interaction with other airway exposure agents, such as cigarette smoke, represents opportunities for future investigation.

Trends in gastrectomy and ADH1B and ALDH2 genotypes in Japanese alcoholic men and their gene-gastrectomy, gene-gene and gene-age interactions for risk of alcoholism.

PubMed

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2013-01-01

The life-time drinking profiles of Japanese alcoholics have shown that gastrectomy increases susceptibility to alcoholism. We investigated the trends in gastrectomy and alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) genotypes and their interactions in alcoholics. This survey was conducted on 4879 Japanese alcoholic men 40 years of age or older who underwent routine gastrointestinal endoscopic screening during the period 1996-2010. ADH1B/ALDH2 genotyping was performed in 3702 patients. A history of gastrectomy was found in 508 (10.4%) patients. The reason for the gastrectomy was peptic ulcer in 317 patients and gastric cancer in 187 patients. The frequency of gastrectomy had gradually decreased from 13.3% in 1996-2000 to 10.5% in 2001-2005 and to 7.8% in 2006-2010 (P < 0.0001). ADH1B*1/*1 was less frequent in the gastrectomy group than in the non-gastrectomy group (age-adjusted prevalence: 20.4 vs. 27.6%, P = 0.006). ALDH2 genotype distribution did not differ between the two groups. The frequency of inactive ALDH2*1/*2 heterozygotes increased slightly from 13.0% in 1996-2000 to 14.0% in 2001-2005 and to 15.4% in 2006-2010 (P < 0.08). Two alcoholism-susceptibility genotypes, ADH1B*1/*1 and ALDH2*1/*1, modestly but significantly tended not to occur in the same individual (P = 0.026). The frequency of ADH1B*1/*1 decreased with ascending age groups. The high frequency of history of gastrectomy suggested that gastrectomy is still a risk factor for alcoholism, although the percentage decreased during the period. The alcoholism-susceptibility genotype ADH1B*1/*1 was less frequent in the gastrectomy group, suggesting a competitive gene-gastrectomy interaction for alcoholism. A gene-gene interaction and gene-age interactions regarding the ADH1B genotype were observed.

[Character accentuations as a criterion for psychological risks in the professional activity of the builders of main gas pipelines in the conditions of arctic].

PubMed

Korneeva, Ia A; Simonova, N N

2015-01-01

The article is devoted to the study of character accentuations as a criterion for psychological risks in the professional activity of builders of main gas pipelines in the conditions of Arctic. to study the severity of character accentuations in rotation-employed builders of main gas pipelines, stipulated by their professional activities, as well as personal resources to overcome these destructions. The study involved 70 rotation-employed builders of trunk pipelines, working in the Tyumen Region (duration of the shift-in--52 days), aged from 23 to 59 (mean age 34,9 ± 8.1) years, with the experience of work from 0.5 years to 14 years (the average length of 4.42 ± 3.1). Methods of the study: questionnaires, psychological testing, participant observation. One-Sample t-test of Student, multiple regression analysis, incremental analysis. In the work there were revealed differences of expression of character accentuations in builders of trunk pipelines with experience in work on rotation less and more than five years. There was determined that builders of the main gas pipelines, working on the rotation in Arctic, with more pronounced accentuation ofthe character use mainly psychological defenses of compensation, substitution and denial, and have an average level of expression of flexibility as the regulatory process.

Polymorphism of ethanol-metabolism genes and alcoholism: correlation of allelic variations with the pharmacokinetic and pharmacodynamic consequences.

PubMed

Chen, Yi-Chyan; Peng, Giia-Sheun; Wang, Ming-Fang; Tsao, Tien-Ping; Yin, Shih-Jiun

2009-03-16

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for metabolism of ethanol. Both ADH and ALDH exhibit genetic polymorphisms among racial populations. Functional variant alleles ADH1B*2 and ALDH2*2 have been consistently replicated to show protection against developing alcohol dependence. Multiple logistic regression analyses suggest that ADH1B*2 and ALDH2*2 may independently influence the risk for alcoholism. It has been well documented that homozygosity of ALDH2*2 almost fully protects against developing alcoholism and that the heterozygosity only affords a partial protection to varying degrees. Correlations of blood ethanol and acetaldehyde concentrations, cardiovascular hemodynamic responses, and subjective perceptions have been investigated in men with different combinatorial ADH1B and ALDH2 genotypes following challenge with ethanol for a period of 130 min. The pharmacokinetic and pharmacodynamic consequences indicate that acetaldehyde, rather than ethanol, is primarily responsible for the observed alcohol sensitivity reactions, suggesting that the full protection by ALDH2*2/*2 can be ascribed to the intense unpleasant physiological and psychological reactions caused by persistently elevated blood acetaldehyde after ingesting a small amount of alcohol and that the partial protection by ALDH2*1/*2 can be attributed to a faster elimination of acetaldehyde and the lower accumulation in circulation. ADH1B polymorphism does not significantly contribute to buildup of the blood acetaldehyde. Physiological tolerance or innate insensitivity to acetaldehyde may be crucial for development of alcohol dependence in alcoholics carrying ALDH2*2.

Alcohol sales and risk of serious assault.

PubMed

Ray, Joel G; Moineddin, Rahim; Bell, Chaim M; Thiruchelvam, Deva; Creatore, Maria Isabella; Gozdyra, Piotr; Cusimano, Michael; Redelmeier, Donald A

2008-05-13

Alcohol is a contributing cause of unintentional injuries, such as motor vehicle crashes. Prior research on the association between alcohol use and violent injury was limited to survey-based data, and the inclusion of cases from a single trauma centre, without adequate controls. Beyond these limitations was the inability of prior researchers to comprehensively capture most alcohol sales. In Ontario, most alcohol is sold through retail outlets run by the provincial government, and hospitals are financed under a provincial health care system. We assessed the risk of being hospitalized due to assault in association with retail alcohol sales across Ontario. We performed a population-based case-crossover analysis of all persons aged 13 years and older hospitalized for assault in Ontario from 1 April 2002 to 1 December 2004. On the day prior to each assault case's hospitalization, the volume of alcohol sold at the store in closest proximity to the victim's home was compared to the volume of alcohol sold at the same store 7 d earlier. Conditional logistic regression analysis was used to determine the associated relative risk (RR) of assault per 1,000 l higher daily sales of alcohol. Of the 3,212 persons admitted to hospital for assault, nearly 25% were between the ages of 13 and 20 y, and 83% were male. A total of 1,150 assaults (36%) involved the use of a sharp or blunt weapon, and 1,532 (48%) arose during an unarmed brawl or fight. For every 1,000 l more of alcohol sold per store per day, the relative risk of being hospitalized for assault was 1.13 (95% confidence interval [CI] 1.02-1.26). The risk was accentuated for males (1.18, 95% CI 1.05-1.33), youth aged 13 to 20 y (1.21, 95% CI 0.99-1.46), and those in urban areas (1.19, 95% CI 1.06-1.35). The risk of being a victim of serious assault increases with alcohol sales, especially among young urban men. Akin to reducing the risk of driving while impaired, consideration should be given to novel methods of preventing

Alcohol Sales and Risk of Serious Assault

PubMed Central

Ray, Joel G; Moineddin, Rahim; Bell, Chaim M; Thiruchelvam, Deva; Creatore, Maria Isabella; Gozdyra, Piotr; Cusimano, Michael; Redelmeier, Donald A

2008-01-01

Background Alcohol is a contributing cause of unintentional injuries, such as motor vehicle crashes. Prior research on the association between alcohol use and violent injury was limited to survey-based data, and the inclusion of cases from a single trauma centre, without adequate controls. Beyond these limitations was the inability of prior researchers to comprehensively capture most alcohol sales. In Ontario, most alcohol is sold through retail outlets run by the provincial government, and hospitals are financed under a provincial health care system. We assessed the risk of being hospitalized due to assault in association with retail alcohol sales across Ontario. Methods and Findings We performed a population-based case-crossover analysis of all persons aged 13 years and older hospitalized for assault in Ontario from 1 April 2002 to 1 December 2004. On the day prior to each assault case's hospitalization, the volume of alcohol sold at the store in closest proximity to the victim's home was compared to the volume of alcohol sold at the same store 7 d earlier. Conditional logistic regression analysis was used to determine the associated relative risk (RR) of assault per 1,000 l higher daily sales of alcohol. Of the 3,212 persons admitted to hospital for assault, nearly 25% were between the ages of 13 and 20 y, and 83% were male. A total of 1,150 assaults (36%) involved the use of a sharp or blunt weapon, and 1,532 (48%) arose during an unarmed brawl or fight. For every 1,000 l more of alcohol sold per store per day, the relative risk of being hospitalized for assault was 1.13 (95% confidence interval [CI] 1.02–1.26). The risk was accentuated for males (1.18, 95% CI 1.05–1.33), youth aged 13 to 20 y (1.21, 95% CI 0.99–1.46), and those in urban areas (1.19, 95% CI 1.06–1.35). Conclusions The risk of being a victim of serious assault increases with alcohol sales, especially among young urban men. Akin to reducing the risk of driving while impaired

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

PubMed

Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

2010-12-01

Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. © 2010 Wiley Periodicals, Inc.

Distribution of Silicified Microstructures, Regulation of Cinnamyl Alcohol Dehydrogenase and Lodging Resistance in Silicon and Paclobutrazol Mediated Oryza sativa

PubMed Central

Dorairaj, Deivaseeno; Ismail, Mohd Razi

2017-01-01

Lodging is a phenomenon that affects most of the cereal crops including rice, Oryza sativa. This is due to the fragile nature of herbaceous plants whose stems are non-woody, thus affecting its ability to grow upright. Silicon (Si), a beneficial nutrient is often used to toughen and protect plants from biotic and abiotic stresses. Deposition of Si in plant tissues enhances the rigidity and stiffness of the plant as a whole. Silicified cells provide the much needed strength to the culm to resist breaking. Lignin plays important roles in cell wall structural integrity, stem strength, transport, mechanical support, and plant pathogen defense. The aim of this study is to resolve effects of Si on formation of microstructure and regulation of cinnamyl alcohol dehydrogenase (CAD), a key gene responsible for lignin biosynthesis. Besides evaluating silicon, paclobutrazol (PBZ) a plant growth retartdant that reduces internode elongation is also incorporated in this study. Hardness, brittleness and stiffness were improved in presence of silicon thus reducing lodging. Scanning electron micrographs with the aid of energy dispersive x-ray (EDX) was used to map silicon distribution. Presence of trichomes, silica cells, and silica bodies were detected in silicon treated plants. Transcripts of CAD gene was also upregulated in these plants. Besides, phloroglucinol staining showed presence of lignified vascular bundles and sclerenchyma band. In conclusion, silicon treated rice plants showed an increase in lignin content, silicon content, and formation of silicified microstructures. PMID:28747889

Reconstitution of the Escherichia coli pyruvate dehydrogenase complex.

PubMed Central

Reed, L J; Pettit, F H; Eley, M H; Hamilton, L; Collins, J H; Oliver, R M

1975-01-01

The binding of pyruvate dehydrogenase and dihydrolipoyl dehydrogenase (flavoprotein) to dihydrolipoyl transacetylase, the core enzyme of the E. coli pyruvate dehydrogenase complex [EC 1.2.4.1:pyruvate:lipoate oxidoreductase (decaryboxylating and acceptor-acetylating)], has been studied using sedimentation equilibrium analysis and radioactive enzymes in conjunction with gel filtration chromatography. The results show that the transacetylase, which consists of 24 apparently identical polypeptide chains organized into a cube-like structure, has the potential to bind 24 pyruvate dehydrogenase dimers in the absence of flavoprotein and 24 flavoprotein dimers in the absence of pyruvate dehydrogenase. The results of reconstitution experiments, utilizing binding and activity measurements, indicate that the transacetylase can accommodate a total of only about 12 pyruvate dehydrogenase dimers and six flavoprotein dimers and that this stoichiometry, which is the same as that of the native pyruvate dehydrogenase complex, produces maximum activity. It appears that steric hindrance between the relatively bulky pyruvate dehydrogenase and flavoprotein molecules prevents the transacetylase from binding 24 molecules of each ligand. A structural model for the native and reconstituted pyruvate dehydrogenase complexes is proposed in which the 12 pyruvate dehydrogenase dimers are distributed symmetrically on the 12 edges of the transacetylase cube and the six flavoprotein dimers are distributed in the six faces of the cube. Images PMID:1103138

Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma.

PubMed

Kipp, Benjamin R; Voss, Jesse S; Kerr, Sarah E; Barr Fritcher, Emily G; Graham, Rondell P; Zhang, Lizhi; Highsmith, W Edward; Zhang, Jun; Roberts, Lewis R; Gores, Gregory J; Halling, Kevin C

2012-10-01

Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases. Copyright © 2012 Elsevier Inc. All rights reserved.

Direct evidence that genetic variation in glycerol-3-phosphate and malate dehydrogenase genes (Gpdh and Mdh1) affects adult ethanol tolerance in Drosophila melanogaster.

PubMed

Eanes, Walter F; Merritt, Thomas J S; Flowers, Jonathan M; Kumagai, Seiji; Zhu, Chen-Tseh

2009-02-01

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

21 CFR 862.1670 - Sorbitol dehydrogenase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sorbitol dehydrogenase test system. 862.1670... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum...

[Carbohydrates metabolism disturbances when simulating prenatal alcohol intoxication].

PubMed

Kurch, N M; Vysokogorskiĭ, V E

2013-01-01

The influence of prenatal alcohol intoxication on carbohydrate metabolism markers has been investigated at different terms of postnatal offspring development (15, 30 and 60 days). Plasma glucose decreased as compared with the same in control group was detected. In the liver homogenates an increase of phosphorylase activity and a decrease of glucose-6-phosphatase, aldolase and glucose-6-phosphate dehydrogenase activities were found. These changes were accompanied by the incease in the lactate/pyruvate index attributed to increased lactate content in the liver tissue. The obtained data indicate essential disturbances of carbohydrate metabolism markers in prenatal alcoholized offspring, which include stable hypoglycemia, suppression of glycolytic and pentosephosphate pathways of glucose metabolism and lactate accumulation in the liver.

Acute alcohol intoxication-induced microvascular leakage.

PubMed

Doggett, Travis M; Breslin, Jerome W

2014-09-01

Alcohol intoxication can increase inflammation and worsen injury, yet the mechanisms involved are not clear. We investigated whether acute alcohol intoxication increases microvascular permeability and investigated potential signaling mechanisms in endothelial cells that may be involved. Conscious rats received a 2.5 g/kg alcohol bolus via gastric catheters to produce acute intoxication. Microvascular leakage of intravenously administered fluorescein isothiocyanate (FITC)-conjugated albumin (FITC-albumin) from the mesenteric microcirculation was assessed by intravital microscopy. Endothelial-specific mechanisms were studied using cultured endothelial cell monolayers. Transendothelial electrical resistance (TER) served as an index of barrier function, before and after treatment with alcohol or its metabolite acetaldehyde. Pharmacologic agents were used to test the roles of alcohol metabolism, oxidative stress, p38 mitogen-activated protein kinase (MAPK), myosin light-chain kinase (MLCK), rho kinase (ROCK), and exchange protein activated by cAMP (Epac). VE-cadherin localization was investigated to assess junctional integrity. Rac1 and RhoA activation was assessed by ELISA assays. Alcohol significantly increased FITC-albumin extravasation from the mesenteric microcirculation. Alcohol also significantly decreased TER and disrupted VE-cadherin organization at junctions. Acetaldehyde significantly decreased TER, but inhibition of alcohol dehydrogenase or application of a superoxide dismutase mimetic failed to prevent alcohol-induced decreases in TER. Inhibition of p38 MAPK, but not MLCK or ROCK, significantly attenuated the alcohol-induced barrier dysfunction. Alcohol rapidly decreased GTP-bound Rac1 but not RhoA during the drop in TER. Activation of Epac increased TER, but did not prevent alcohol from decreasing TER. However, activation of Epac after initiation of alcohol-induced barrier dysfunction quickly resolved TER to baseline levels. Our results suggest that

Methanol-Water Aqueous-Phase Reforming with the Assistance of Dehydrogenases at Near-Room Temperature.

PubMed

Shen, Yangbin; Zhan, Yulu; Li, Shuping; Ning, Fandi; Du, Ying; Huang, Yunjie; He, Ting; Zhou, Xiaochun

2018-03-09

As an excellent hydrogen-storage medium, methanol has many advantages, such as high hydrogen content (12.6 wt %), low cost, and availability from biomass or photocatalysis. However, conventional methanol-water reforming usually proceeds at high temperatures. In this research, we successfully designed a new effective strategy to generate hydrogen from methanol at near-room temperature. The strategy involved two main processes: CH 3 OH→HCOOH→H 2 and NADH→HCOOH→H 2 . The first process (CH 3 OH→HCOOH→H 2 ) was performed by an alcohol dehydrogenase (ADH), an aldehyde dehydrogenase (ALDH), and an Ir catalyst. The second procedure (NADH→HCOOH→H 2 ) was performed by formate dehydrogenase (FDH) and the Ir catalyst. The Ir catalyst used was a previously reported polymer complex catalyst [Cp*IrCl 2 (ppy); Cp*=pentamethylcyclopentadienyl, ppy=polypyrrole] with high catalytic activity for the decomposition of formic acid at room temperature and is compatible with enzymes, coenzymes, and poisoning chemicals. Our results revealed that the optimum hydrogen generation rate could reach up to 17.8 μmol h -1  g cat -1 under weak basic conditions at 30 °C. This will have high impact on hydrogen storage, production, and applications and should also provide new inspiration for hydrogen generation from methanol. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzymatic characterization of a novel bovine liver dihydrodiol dehydrogenase--reaction mechanism and bile acid dehydrogenase activity.

PubMed

Nanjo, H; Adachi, H; Morihana, S; Mizoguchi, T; Nishihara, T; Terada, T

1995-05-11

Bovine liver cytosolic dihydrodiol dehydrogenase (DD3) has been characterized by its unique dihydrodiol dehydrogenase activity for trans-benzenedihydrodiol (trans-1,2-dihydrobenzene-1,2-diol) with the highest affinity and the greatest velocity among three multiple forms of dihydrodiol dehydrogenases (DD1-DD3). It is the first time that DD3 has shown a significant dehydrogenase activity for (S)-(+)-1-indanol with low Km value (0.33 +/- 0.022 mM) and high K(cat) value (25 +/- 0.79 min-1). The investigation of the product inhibition of (S)-(+)-1-indanol with NADP+ versus 1-indanone and NADPH clearly showed that the enzymatic reaction of DD3 may follow a typical ordered Bi Bi mechanism similar to many aldo/keto reductases. Additionally, DD3 was shown to catalyze the dehydrogenation of bile acids (lithocholic acid, taurolithocholic acid and taurochenodeoxycholic acid) having no 12-hydroxy groups with low Km values (17 +/- 0.65, 33 +/- 1.9 and 890 +/- 73 microM, respectively). In contrast, DD1, 3 alpha-hydroxysteroid dehydrogenase, shows a broad substrate specificity for many bile acids with higher affinity than those of DD3. Competitive inhibition of DD3 with androsterone against dehydrogenase activity for (S)-(+)-1-indanol, trans-benzenedihydrodiol or lithocholic acid suggests that these three substrates bind to the same substrate binding site of DD3, different from the case of human liver bile acid binder/dihydrodiol dehydrogenase (Takikawa, H., Stolz, A., Sugiyama, Y., Yoshida, H., Yamamoto, M. and Kaplowitz, N. (1990) J. Biol. Chem. 265, 2132-2136). Considering the reaction mechanism, DD3 may also play an important role in bile acids metabolism as well as the detoxication of aromatic hydrocarbons.

Differential Effect of Initiating Moderate Red Wine Consumption on 24-h Blood Pressure by Alcohol Dehydrogenase Genotypes: Randomized Trial in Type 2 Diabetes.

PubMed

Gepner, Yftach; Henkin, Yaakov; Schwarzfuchs, Dan; Golan, Rachel; Durst, Ronen; Shelef, Ilan; Harman-Boehm, Ilana; Spitzen, Shosana; Witkow, Shula; Novack, Lena; Friger, Michael; Tangi-Rosental, Osnat; Sefarty, Dana; Bril, Nitzan; Rein, Michal; Cohen, Noa; Chassidim, Yoash; Sarusi, Benny; Wolak, Talia; Stampfer, Meir J; Rudich, Assaf; Shai, Iris

2016-04-01

Observational studies report inconsistent associations between moderate alcohol intake and blood pressure (BP). In a sub-study of a larger randomized controlled trial, we assessed the effect of initiating moderate red wine consumption on 24-h BP recordings and the effect of a common genetic variant of alcohol dehydrogenases (ADH) among patients with type 2 diabetes. Fifty-four type 2 diabetes, alcohol abstainers were randomized to consume 150 ml/dinner dry red wine or mineral water. Both groups were guided to adhere to a Mediterranean diet, without caloric restriction. We measured 24-h ambulatory BP monitoring (ABPM) at baseline and after 6 months. Participants (age = 57 years; 85% men; mean 24-h BP = 129/77 mm Hg) had 92% 6-month retention. After 6 months of intervention, the average 24-h BP did not differ between the wine and water groups. A transient decrease in BP was observed in the red wine group at midnight (3-4 hours after wine intake: systolic BP: red wine = -10.6mm Hg vs. mineral water = +2.3 mm Hg; P = 0.031) and the following morning at 7-9 am (red wine: -6.2mm Hg vs. mineral water: +5.6mm Hg; P = 0.014). In a second post hoc sub-analysis among the red wine consumers, individuals who were homozygous for the gene encoding ADH1B*2 variant (Arg48His; rs1229984, TT, fast ethanol metabolizers), exhibited a reduction in mean 24-h systolic BP (-8.0mm Hg vs. +3.7 mm Hg; P = 0.002) and pulse pressure (-3.8 mm Hg vs. +1.2 mm Hg; P = 0.032) compared to heterozygotes and those homozygous for the ADH1B*1 variant (CC, slow metabolizers). Initiating moderate red wine consumption at dinner among type 2 diabetes patients does not have a discernable effect on mean 24-h BP. Yet, a modest temporal BP reduction could be documented, and a more pronounced BP-lowering effect is suggested among fast ethanol metabolizers. ClinicalTrials.gov Identifier: NCT00784433. © American Journal of Hypertension, Ltd 2015. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Differential Effect of Initiating Moderate Red Wine Consumption on 24-h Blood Pressure by Alcohol Dehydrogenase Genotypes: Randomized Trial in Type 2 Diabetes

PubMed Central

Gepner, Yftach; Henkin, Yaakov; Schwarzfuchs, Dan; Golan, Rachel; Durst, Ronen; Shelef, Ilan; Harman-Boehm, Ilana; Spitzen, Shosana; Witkow, Shula; Novack, Lena; Friger, Michael; Tangi-Rosental, Osnat; Sefarty, Dana; Bril, Nitzan; Rein, Michal; Cohen, Noa; Chassidim, Yoash; Sarusi, Benny; Wolak, Talia; Stampfer, Meir J.; Rudich, Assaf

2016-01-01

AIMS Observational studies report inconsistent associations between moderate alcohol intake and blood pressure (BP). In a sub-study of a larger randomized controlled trial, we assessed the effect of initiating moderate red wine consumption on 24-h BP recordings and the effect of a common genetic variant of alcohol dehydrogenases (ADH) among patients with type 2 diabetes. METHODS Fifty-four type 2 diabetes, alcohol abstainers were randomized to consume 150ml/dinner dry red wine or mineral water. Both groups were guided to adhere to a Mediterranean diet, without caloric restriction. We measured 24-h ambulatory BP monitoring (ABPM) at baseline and after 6 months. RESULTS Participants (age = 57 years; 85% men; mean 24-h BP = 129/77mm Hg) had 92% 6-month retention. After 6 months of intervention, the average 24-h BP did not differ between the wine and water groups. A transient decrease in BP was observed in the red wine group at midnight (3–4 hours after wine intake: systolic BP: red wine = −10.6mm Hg vs. mineral water = +2.3mm Hg; P = 0.031) and the following morning at 7–9 am (red wine: −6.2mm Hg vs. mineral water: +5.6mm Hg; P = 0.014). In a second post hoc sub-analysis among the red wine consumers, individuals who were homozygous for the gene encoding ADH1B*2 variant (Arg48His; rs1229984, TT, fast ethanol metabolizers), exhibited a reduction in mean 24-h systolic BP (−8.0mm Hg vs. +3.7mm Hg; P = 0.002) and pulse pressure (−3.8mm Hg vs. +1.2mm Hg; P = 0.032) compared to heterozygotes and those homozygous for the ADH1B*1 variant (CC, slow metabolizers). CONCLUSIONS Initiating moderate red wine consumption at dinner among type 2 diabetes patients does not have a discernable effect on mean 24-h BP. Yet, a modest temporal BP reduction could be documented, and a more pronounced BP-lowering effect is suggested among fast ethanol metabolizers. CLINICAL TRIALS REGISTRATION ClinicalTrials.gov Identifier: NCT00784433. PMID:26232779

Acamprosate attenuates the handling induced convulsions during alcohol withdrawal in Swiss Webster mice

PubMed Central

Farook, Justin M.; Krazem, Ali; Littleton, John M.; Barron, Susan

2008-01-01

In the present study, we examined the effects of acamprosate for its ability to reduce handling induced convulsions (HICs) during alcohol withdrawal. Diazepam was used as a positive control. Swiss Webster male mice received three daily IP injections of alcohol (2.5 g/kg) or alcohol (2.5 g/kg) + methylpyrazole (4-MP) (9 mg/kg). (4-MP, being an alcohol dehydrogenase inhibitor slows down the breakdown of alcohol. 4-MP in combination with alcohol exhibits a dramatic increase in blood alcohol level compared to alcohol alone). Ten hours following the last alcohol injection, the mice were picked up by the tail and examined for their seizure susceptibility (HICs). Diazepam, a benzodiazepine known to reduce seizures during alcohol withdrawal, significantly reduced these HICs at doses of 0.25, 0.5 and 1mg/kg (p’s < 0.001). Acamprosate, an anti-relapse compound used clinically in newly abstinent alcoholics, also reduced these HICs at doses of 100, 200 and 300mg/kg (p’s < 0.05). This study supports the use of acamprosate during periods of alcohol withdrawal as well as during abstinence. PMID:18577392

Role of Alcohol Metabolism in Chronic Pancreatitis

PubMed Central

Vonlaufen, Alain; Wilson, Jeremy S.; Pirola, Romano C.; Apte, Minoti V.

2007-01-01

Alcohol abuse is the major cause of chronic inflammation of the pancreas (i.e., chronic pancreatitis). Although it has long been thought that alcoholic pancreatitis is a chronic disease from the outset, evidence is accumulating to indicate that chronic damage in the pancreas may result from repeated attacks of acute tissue inflammation and death (i.e., necroinflammation). Initially, research into the pathogenesis of alcoholic pancreatitis was related to ductular and sphincteric abnormalities. In recent years, the focus has shifted to the type of pancreas cell that produces digestive juices (i.e., acinar cell). Alcohol now is known to exert a number of toxic effects on acinar cells. Notably, acinar cells have been shown to metabolize alcohol (i.e., ethanol) via both oxidative (i.e., involving oxygen) and nonoxidative pathways. The isolation and study of pancreatic stellate cells (PSCs)—the key effectors in the development of connective tissue fibers (i.e., fibrogenesis) in the pancreas—has greatly enhanced our understanding of the pathogenesis of chronic pancreatitis. Pancreatic stellate cells become activated in response to ethanol and acetaldehyde, a toxic byproduct of alcohol metabolism. In addition, PSCs have the capacity to metabolize alcohol via alcohol dehydrogenase (the major oxidizing enzyme for ethanol). The fact that only a small percentage of heavy alcoholics develop chronic pancreatitis has led to the search for precipitating factors of the disease. Several studies have investigated whether variations in ethanol-metabolizing enzymes may be a trigger factor for chronic pancreatitis, but no definite relationship has been established so far. PMID:17718401

Hederagenin Supplementation Alleviates the Pro-Inflammatory and Apoptotic Response to Alcohol in Rats.

PubMed

Kim, Gyeong-Ji; Song, Da Hye; Yoo, Han Seok; Chung, Kang-Hyun; Lee, Kwon Jai; An, Jeung Hee

2017-01-06

In this study, we determined the effects of hederagenin isolated from Akebia quinata fruit on alcohol-induced hepatotoxicity in rats. Specifically, we investigated the hepatoprotective, anti-inflammatory, and anti-apoptotic effects of hederagenin, as well as the role of AKT and mitogen-activated protein kinase (MAPK) signaling pathways in ethanol-induced liver injury. Experimental animals were randomly divided into three groups: normal (sham), 25% ethanol, and 25% ethanol + hederagenin (50 mg/kg/day). Each group was orally administered the respective treatments once per day for 21 days. Acetaldehyde dehydrogenase-2 mRNA expression was higher and alcohol dehydrogenase mRNA expression was lower in the ethanol + hederagenin group than those in the ethanol group. Pro-inflammatory cytokines, including TNF-α, IL-6, and cyclooxygenase-2, significantly increased in the ethanol group, but these increases were attenuated by hederagenin. Moreover, Western blot analysis showed increased expression of the apoptosis-associated protein, Bcl-2, and decreased expression of Bax and p53 after treatment with hederagenin. Hederagenin treatment attenuated ethanol-induced increases in activated p38 MAPK and increased the levels of phosphorylated AKT and ERK. Hederagenin alleviated ethanol-induced liver damage through anti-inflammatory and anti-apoptotic activities. These results suggest that hederagenin is a potential candidate for preventing alcoholic liver injury.

Increased alcohol consumption as a cause of alcoholism, without similar evidence for depression: a Mendelian randomization study.

PubMed

Wium-Andersen, Marie Kim; Ørsted, David Dynnes; Tolstrup, Janne Schurmann; Nordestgaard, Børge Grønne

2015-04-01

Increased alcohol consumption has been associated with depression and alcoholism, but whether these associations are causal remains unclear. We tested whether alcohol consumption is causally associated with depression and alcoholism. We included 78,154 men and women aged 20-100 years randomly selected in 1991-2010 from the general population of Copenhagen, Denmark, and genotyped 68,486 participants for two genetic variants in two alcohol dehydrogenase (ADH) genes, ADH-1B (rs1229984) and ADH-1C (rs698). We performed observational and causal analyses using a Mendelian randomization design with antidepressant medication use and hospitalization/death, with depression and alcoholism as outcomes. In prospective analyses, the multifactorially adjusted hazard ratio for participants reporting >6 drinks/day vs participants reporting 0.1-1 drinks/day was 1.28 (95% confidence interval, 1.00-1.65) for prescription antidepressant use, with a corresponding hazard ratio of 0.80 (0.45-1.45) for hospitalization/death with depression and of 11.7 (8.77-15.6) for hospitalization/death with alcoholism. For hospitalization/death with alcoholism, instrumental variable analysis yielded a causal odds ratio of 28.6 (95 % confidence interval 6.47-126) for an increase of 1 drink/day estimated from the combined genotype combination, whereas the corresponding multifactorially adjusted observational odds ratio was 1.28 (1.25-1.31). Corresponding odds ratios were 1.11 (0.67-1.83) causal and 1.04 (1.03-1.06) observational for prescription antidepressant use, and 4.52 (0.99-20.5) causal and 0.98 (0.94-1.03) observational for hospitalization/death with depression. These data indicate that the association between increased alcohol consumption and alcoholism is causal, without similar strong evidence for depression. © The Author 2014; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.

Hangover symptoms in Asian Americans with variations in the aldehyde dehydrogenase (ALDH2) gene.

PubMed

Wall, T L; Horn, S M; Johnson, M L; Smith, T L; Carr, L G

2000-01-01

Hangovers are not experienced by all people and whether they contribute to the development of alcoholism is unclear. One population that might provide some insight into the role of hangover in the etiology of alcohol use disorders is that of individuals of Asian heritage. Certain Asians have lower rates of alcohol use and alcoholism, findings associated with a mutation in the aldehyde dehydrogenase (ALDH2) gene. Asians with ALDH2*2 alleles drink less and are less likely to be alcoholic than Asians without this mutation. Following alcohol ingestion, they exhibit more intense reactions to alcohol and generate higher levels of the metabolite acetaldehyde. This study evaluated hangover symptoms in Asian Americans with variations in the ALDH2 gene. Men and women of Chinese, Japanese and Korean heritage (N = 140) were asked about their drinking history and a blood sample was collected for genotyping at the ALDH2 locus. Subjects used a Likert-type scale to estimate their severity of hangover and completed a 13-item hangover scale assessing the frequency of hangover symptoms during the previous 6 months. With abstainers (n = 17) excluded and with the effects of gender and recent drinking history controlled, ALDH2 genotype accounted for a significant amount of additional variability in the estimated severity of hangover score with a similar, but nonsignificant, trend for a five-item subscale score derived from the hangover scale. These results suggest that Asian Americans with ALDH2*2 alleles may experience more severe hangovers that may contribute, in part, to protection against the development of excessive or problematic drinking in this population.

Origin and dispersal of atypical aldehyde dehydrogenase ALDH2487Lys.

PubMed

Luo, Huai-Rong; Wu, Gui-Sheng; Pakstis, Andrew J; Tong, Li; Oota, Hiroki; Kidd, Kenneth K; Zhang, Ya-Ping

2009-04-15

The East Asian respond with a marked facial flushing and mild to moderate symptoms of intoxication after drinking the amounts of alcohol that has no detectable effect on European. The alcohol sensitivity in Orientals is due to a delayed oxidation of acetaldehyde by an atypical aldehyde dehydrogenase ALDH2487Lys, which is resulted from a structural mutation in gene ALDH2. The atypical ALDH2487Lys allele has been associated with various phenotypic statuses, such as protective against alcohol dependence and the risk of alcohol-related digestive tract cancers. Here, we have examined this SNP, adjacent four non-coding SNPs, and one downstream STRP on ALDH2 gene, in total of 1072 unrelated healthy individuals from 14 Chinese populations and 130 Indian individuals. Five major haplotypes based on five SNPs across the ALDH2 gene 40 kb were found in all East Asian populations. The frequencies of the ancestral haplotype GCCTG and the East Asian special haplotype GCCTA containing the atypical ALDH2487Lys allele were 44.8% and 14.9%, respectively. The frequency of the atypical ALDH2487Lys allele or the East Asian specific haplotype GCCTA is high in Yunnan, South coastal, east coastal of China, and decreased gradually toward inland China, West, Northwest and North China. Combined with demographic history in East Asian, our results showed that the presence of ALDH2487Lys allele in peripheral regions of China might be the results of historical migration events from China to these regions. The origin of ALDH2487Lys could be possibly traced back to ancient Pai-Yuei tribe in South China.

Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency.

PubMed

Li, Kunpeng; Zhang, Rongzhen; Xu, Yan; Wu, Zhimeng; Li, Jing; Zhou, Xiaotian; Jiang, Jiawei; Liu, Haiyan; Xiao, Rong

2017-06-08

(S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular "stapler" for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat /K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (T m ) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved T m s and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.

The Future of Qualitative Research in Psychology: Accentuating the Positive.

PubMed

Gough, Brendan; Lyons, Antonia

2016-06-01

In this paper we reflect on current trends and anticipate future prospects regarding qualitative research in Psychology. We highlight various institutional and disciplinary obstacles to qualitative research diversity, complexity and quality. At the same time, we note some causes for optimism, including publication breakthroughs and vitality within the field. The paper is structured into three main sections which consider: 1) the positioning of qualitative research within Psychology; 2) celebrating the different kinds of knowledge produced by qualitative research; and 3) implementing high quality qualitative research. In general we accentuate the positive, recognising and illustrating innovative qualitative research practices which generate new insights and propel the field forward. We conclude by emphasising the importance of research training: for qualitative research to flourish within Psychology (and beyond), students and early career researchers require more sophisticated, in-depth instruction than is currently offered.

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6

PubMed Central

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-01-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P212121, with unit-cell parameters a = 88.35, b = 128.73, c = 131.03 Å. PMID:24100567

Crystallization and preliminary X-ray study of a (2R,3R)-2,3-butanediol dehydrogenase from Bacillus coagulans 2-6.

PubMed

Miao, Xiangzhi; Huang, Xianhui; Zhang, Guofang; Zhao, Xiufang; Zhu, Xianming; Dong, Hui

2013-10-01

(2R,3R)-2,3-Butanediol dehydrogenase (R,R-BDH) from Bacillus coagulans 2-6 is a zinc-dependent medium-chain alcohol dehydrogenase. Recombinant R,R-BDH with a His6 tag at the C-terminus was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni2+-chelating affinity and size-exclusion chromatography. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K. The crystallization condition consisted of 8%(v/v) Tacsimate pH 4.6, 18%(w/v) polyethylene glycol 3350. The crystal diffracted to 2.8 Å resolution in the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=88.35, b=128.73, c=131.03 Å.

Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

PubMed Central

Ohmura, M; Hara, A; Nakagawa, M; Sawada, H

1990-01-01

NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase. Images Fig. 1. Fig. 2. PMID:2317205

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

PubMed

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

Chronic Hypoxia Accentuates Dysanaptic Lung Growth.

PubMed

Llapur, Conrado J; Martínez, Myriam R; Grassino, Pedro T; Stok, Ana; Altieri, Héctor H; Bonilla, Federico; Caram, María M; Krowchuk, Natasha M; Kirby, Miranda; Coxson, Harvey O; Tepper, Robert S

2016-08-01

Adults born and raised at high altitudes have larger lung volumes and greater pulmonary diffusion capacity compared with adults at low altitude; however, it remains unclear whether the air and tissue volumes have comparable increases and whether there is a difference in airway size. To assess the effect of chronic hypoxia on lung growth using in vivo high-resolution computed tomography measurements. Healthy adults born and raised at moderate altitude (2,000 m above sea level; n = 19) and at low altitude (400 m above sea level; n = 23) underwent high-resolution computed tomography. Differences in total lung, air, and tissue volume, mean lung density, as well as airway lumen and wall areas in anatomically matched airways were compared between groups. No significant differences for age, sex, weight, or height were found between the two groups (P > 0.05). In a multivariate regression model, altitude was a significant contributor for total lung volume (P = 0.02), air volume (P = 0.03), and tissue volume (P = 0.03), whereby the volumes were greater for the moderate- versus the low-altitude group. However, altitude was not a significant contributor for mean lung density (P = 0.35) or lumen and wall areas in anatomically matched segmental, subsegmental, and subsubsegmental airways. Our findings suggest that the adult lung did not increase lung volume later in life by expansion of an existing number of alveoli, but rather from increased alveolarization early in life. In addition, chronic hypoxia accentuates dysanaptic lung growth by increasing the lung parenchyma but not the airways.

Yeast surface display of dehydrogenases in microbial fuel-cells.

PubMed

Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

2016-12-01

Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. Copyright © 2016 Elsevier B.V. All rights reserved.

True-3D Accentuating of Grids and Streets in Urban Topographic Maps Enhances Human Object Location Memory

PubMed Central

Edler, Dennis; Bestgen, Anne-Kathrin; Kuchinke, Lars; Dickmann, Frank

2015-01-01

Cognitive representations of learned map information are subject to systematic distortion errors. Map elements that divide a map surface into regions, such as content-related linear symbols (e.g. streets, rivers, railway systems) or additional artificial layers (coordinate grids), provide an orientation pattern that can help users to reduce distortions in their mental representations. In recent years, the television industry has started to establish True-3D (autostereoscopic) displays as mass media. These modern displays make it possible to watch dynamic and static images including depth illusions without additional devices, such as 3D glasses. In these images, visual details can be distributed over different positions along the depth axis. Some empirical studies of vision research provided first evidence that 3D stereoscopic content attracts higher attention and is processed faster. So far, the impact of True-3D accentuating has not yet been explored concerning spatial memory tasks and cartography. This paper reports the results of two empirical studies that focus on investigations whether True-3D accentuating of artificial, regular overlaying line features (i.e. grids) and content-related, irregular line features (i.e. highways and main streets) in official urban topographic maps (scale 1/10,000) further improves human object location memory performance. The memory performance is measured as both the percentage of correctly recalled object locations (hit rate) and the mean distances of correctly recalled objects (spatial accuracy). It is shown that the True-3D accentuating of grids (depth offset: 5 cm) significantly enhances the spatial accuracy of recalled map object locations, whereas the True-3D emphasis of streets significantly improves the hit rate of recalled map object locations. These results show the potential of True-3D displays for an improvement of the cognitive representation of learned cartographic information. PMID:25679208

Genetics Home Reference: lactate dehydrogenase deficiency

MedlinePlus

... dehydrogenase-B pieces (subunits) of the lactate dehydrogenase enzyme. This enzyme is found throughout the body and is important ... cells. There are five different forms of this enzyme, each made up of four protein subunits. Various ...

Association between ALDH2 and ADH1B polymorphisms, alcohol drinking and gastric cancer: a replication and mediation analysis.

PubMed

Ishioka, Kuka; Masaoka, Hiroyuki; Ito, Hidemi; Oze, Isao; Ito, Seiji; Tajika, Masahiro; Shimizu, Yasuhiro; Niwa, Yasumasa; Nakamura, Shigeo; Matsuo, Keitaro

2018-04-03

Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms have a strong impact on carcinogenic acetaldehyde accumulation after alcohol drinking. To date, however, evidence for a significant ALDH2-alcohol drinking interaction and a mediation effect of ALDH2/ADH1B through alcohol drinking on gastric cancer have remained unclear. We conducted two case-control studies to validate the interaction and to estimate the mediation effect on gastric cancer. We calculated odds ratios (OR) and 95% confidence intervals (CI) for ALDH2/ADH1B genotypes and alcohol drinking using conditional logistic regression models after adjustment for potential confounding in the HERPACC-2 (697 cases and 1372 controls) and HERPACC-3 studies (678 cases and 678 controls). We also conducted a mediation analysis of the combination of the two studies to assess whether the effects of these polymorphisms operated through alcohol drinking or through other pathways. ALDH2 Lys alleles had a higher risk with increased alcohol consumption compared with ALDH2 Glu/Glu (OR for heavy drinking, 3.57; 95% CI 2.04-6.27; P for trend = 0.007), indicating a significant ALDH2-alcohol drinking interaction (P interaction  = 0.024). The mediation analysis indicated a significant positive direct effect (OR 1.67; 95% CI 1.38-2.03) and a protective indirect effect (OR 0.84; 95% CI 0.76-0.92) of the ALDH2 Lys alleles with the ALDH2-alcohol drinking interaction. No significant association of ADH1B with gastric cancer was observed. The observed ALDH2-alcohol drinking interaction and the direct effect of ALDH2 Lys alleles may suggest the involvement of acetaldehyde in the development of gastric cancer.

COMPARISON OF IN VIVO AND IN VITRO METHODS FOR ASSESSING EFFECTS OF ALLYL ALCOHOL ON THE LIVER

EPA Science Inventory

Allyl alcohol was administered to female Fischer 344 rats at doses of 0, 3, 10 and 30 mg/kg daily for 7 days. Plasma sorbitol dehydrogenase was minimally elevated. No dose related changes were observed in hexobarbital oxidation, aniline hydroxylation, or ethylmorphine demethylati...

Candidate genes for alcohol dependence: A genetic association study from India.

PubMed

Malhotra, Savita; Basu, Debasish; Khullar, Madhu; Ghosh, Abhishek; Chugh, Neera

2016-11-01

Search for candidate genes for alcohol dependence (AD) has been inconsistent and inconclusive. Moreover, most of the research has been confined to a few specific ethnic groups. Hence, the aim of our study was to explore specific candidate genes for AD in north Indian male population. In this clinic-based genetic association study, 210 males with AD and 200 controls matched for age, gender and ethnicity were recruited from the clinic and the general population, respectively. Cases were diagnosed with Semi-structured Assessment for Genetics of Alcoholism-II (SSAGA-II). Single-nucleotide polymorphism genotyping was done by real-time quantitative-polymerase chain reaction (PCR) using Taq Man assay (ABI 7500) fast real-time PCR system. Both at the genotypic level and at allelic frequency, Met158 variant of catechol-O-methyl transferase (COMT) showed significant increase in cases as compared to controls. The frequency of heterozygous genotype (A/G) of gamma-aminobutyric acid receptor A1 (GABRA1) was significantly lower in cases as compared to controls. Likewise, for GABRA2, the frequency of homozygous recessive genotype (G/G) was significantly higher in the control group. With respect to the 5-hydroxytryptamine (5HT) transporter long promoter region (5HTTLPR), cholinergic receptor muscarinic (CHRM2) and alcohol dehydrogenase 1B (ADH1B) genes, there was no significant difference between the cases and the controls. Aldehyde dehydrogenase (ALDH2) gene was found to be monomorphic in our study population. Our study findings showed COMT polymorphism conferring risk and GABRA polymorphism as a protective genotype for Indian male with AD. Genes for alcohol metabolism, serotonin transporter and cholinergic receptor gene polymorphism were perhaps not contributory to AD for Indian population.

21 CFR 862.1440 - Lactate dehydrogenase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Systems § 862.1440 Lactate dehydrogenase test system. (a) Identification. A lactate dehydrogenase test system is a device intended to measure the activity of the enzyme lactate dehydrogenase in serum. Lactate... hepatitis, cirrhosis, and metastatic carcinoma of the liver, cardiac diseases such as myocardial infarction...

21 CFR 862.1420 - Isocitric dehydrogenase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Systems § 862.1420 Isocitric dehydrogenase test system. (a) Identification. An isocitric dehydrogenase test system is a device intended to measure the activity of the enzyme isocitric dehydrogenase in serum... disease such as viral hepatitis, cirrhosis, or acute inflammation of the biliary tract; pulmonary disease...

Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

DOE PAGES

Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee; ...

2016-08-24

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

Cofactor engineering of ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD) improves the fusel alcohol yield in algal protein anaerobic fermentation

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

Wu, Weihua; Tran-Gyamfi, Mary Bao; Jaryenneh, James Dekontee

Recently the feasibility of conversion of algal protein to mixed alcohols has been demonstrated with an engineered E.coli strain, enabling comprehensive utilization of the biomass for biofuel applications. However, the yield and titers of mixed alcohol production must be improved for market adoption. A major limiting factor for achieving the necessary yield and titer improvements is cofactor imbalance during the fermentation of algal protein. To resolve this problem, a directed evolution approach was applied to modify the cofactor specificity of two key enzymes (IlvC and YqhD) from NADPH to NADH in the mixed alcohol metabolic pathway. Using high throughput screening,more » more than 20 YqhD mutants were identified to show activity on NADH as a cofactor. Of these 20 mutants, the top five of YqhD mutants were selected for combination with two IlvC mutants with NADH as a cofactor for the modification of the protein conversion strain. The combination of the IlvC and YqhD mutants yielded a refined E.coli strain, subtype AY3, with increased fusel alcohol yield of ~60% compared to wild type under anaerobic fermentation on amino acid mixtures. When applied to real algal protein hydrolysates, the strain AY3 produced 100% and 38% more total mixed alcohols than the wild type strain on two different algal hydrolysates, respectively. The results indicate that cofactor engineering is a promising approach to improve the feasibility of bioconversion of algal protein into mixed alcohols as advanced biofuels.« less

[Dose effect of alcohol on sex differences in blood alcohol metabolism--cases where healthy subjects with ALDH2*1/1 genotype drunk beer with meal].

PubMed

Oshima, Shunji; Haseba, Takeshi; Masuda, Chiaki; Kakimi, Ema; Kitagawa, Yasushi; Ohno, Youkichi

2013-06-01

It is said that blood alcohol concentrations (BAG) are higher in female than in male due to the smaller distribution volume of alcohol in female, whereas the rate of alcohol metabolism is faster in female than in males due to a higher activity of liver alcohol dehydrogenase (ADH) in female. However, it is also known that alcohol metabolism varies depending on drinking conditions. In this study, we evaluated the dose effect of alcohol on sex differences in alcohol metabolism in daily drinking conditions, where young adults (16 males, 15 females) with ALDH2*1/1 genotype drunk beer at a dose of 0.32g or 1.0g ethanol/kg body weight with a test meal (460kcal). This study was conducted using a randomized cross-over design. In the considerable drinking condition (1.0g/kg), BAG was significantly higher in females than in males, whereas the rate of alcohol metabolism (beta) was higher in female than in male. In the moderate drinking condition (0.32g/kg), however, no sex differences in alcohol metabolism including BAG were seen. These results suggest that an increased first pass metabolism through liver ADH in female, which may be caused by the reduction of gastric emptying rate due to the meal intake, contribute to the vanishing of sex difference in BAC in the moderate drinking condition.

Ethanol Extract of Pinus koraiensis Leaf Ameliorates Alcoholic Fatty Liver via the Activation of LKB1-AMPK Signaling In Vitro and In Vivo.

PubMed

Hong, Sang-Hyuk; Lee, Hyemin; Lee, Hyo-Jung; Kim, Bonglee; Nam, Min-Ho; Shim, Bum-Sang; Kim, Sung-Hoon

2017-05-01

Although Pinus koraiensis leaf (PKL) was reported for its anti-diabetes, anti-obesity and anticancer effects as a folk remedy, the inhibitory effect of PKL on alcoholic fatty liver has never been elucidated yet. This study investigated the molecular mechanisms of PKL on alcoholic fatty liver in HepG2 cells, Sprague Dawley (SD) rats and Imprinting Control Region (ICR) mice. Pinus koraiensis leaf increased phosphorylation of liver kinase B1 (LKB1)/AMP-activated protein kinase signaling, low-density lipoprotein receptor and decreased fatty acid biosynthesis-related proteins such as sterol regulatory element-binding protein 1c, fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase in HepG2 cells. In SD rats with 25% alcohol-induced fatty liver, PKL suppressed the levels of aspartate aminotransferase and triglyceride and also enhanced the activities of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and glutathione s-transferase compared with untreated control. Furthermore, PKL increased serum alcohol dehydrogenase and serum aldehyde dehydrogenase, but decreased serum alcohol concentration in ICR mice after alcohol administration. Consistently, histochemical analysis revealed that PKL attenuated alcohol-induced fatty liver in SD rats. Overall, these findings suggest that PKL ameliorates alcohol-induced fatty liver via activation of LKB1-AMP-activated protein kinase and modulation of proteins related to lipogenesis synthesis, cholesterol synthesis and fatty acid oxidation. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Picosecond-Resolved Fluorescent Probes at Functionally Distinct Tryptophans within a Thermophilic Alcohol Dehydrogenase: Relationship of Temperature-Dependent Changes in Fluorescence to Catalysis

PubMed Central

2015-01-01

Two single-tryptophan variants were generated in a thermophilic alcohol dehydrogenase with the goal of correlating temperature-dependent changes in local fluorescence with the previously demonstrated catalytic break at ca. 30 °C (Kohen et al., Nature1999, 399, 496). One tryptophan variant, W87in, resides at the active site within van der Waals contact of bound alcohol substrate; the other variant, W167in, is a remote-site surface reporter located >25 Å from the active site. Picosecond-resolved fluorescence measurements were used to analyze fluorescence lifetimes, time-dependent Stokes shifts, and the extent of collisional quenching at Trp87 and Trp167 as a function of temperature. A subnanosecond fluorescence decay rate constant has been detected for W87in that is ascribed to the proximity of the active site Zn2+ and shows a break in behavior at 30 °C. For the remainder of the reported lifetime measurements, there is no detectable break between 10 and 50 °C, in contrast with previously reported hydrogen/deuterium exchange experiments that revealed a temperature-dependent break analogous to catalysis (Liang et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556). We conclude that the motions that lead to the rigidification of ht-ADH below 30 °C are likely to be dominated by global processes slower than the picosecond to nanosecond motions measured herein. In the case of collisional quenching of fluorescence by acrylamide, W87in and W167in behave in a similar manner that resembles free tryptophan in water. Stokes shift measurements, by contrast, show distinctive behaviors in which the active-site tryptophan relaxation is highly temperature-dependent, whereas the solvent-exposed tryptophan’s dynamics are temperature-independent. These data are concluded to reflect a significantly constrained environment surrounding the active site Trp87 that both increases the magnitude of the Stokes shift and its temperature-dependence. The results are discussed in the context

Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays

PubMed Central

Davis, Mindy I.; Shen, Min; Simeonov, Anton

2016-01-01

Abstract Dehydrogenases are an important target for the development of cancer therapeutics. Dehydrogenases either produce or consume NAD(P)H, which is fluorescent but at a wavelength where many compounds found in chemical libraries are also fluorescent. By coupling dehydrogenases to diaphorase, which utilizes NAD(P)H to produce the fluorescent molecule resorufin from resazurin, the assay can be red-shifted into a spectral region that reduces interference from compound libraries. Dehydrogenases that produce NAD(P)H, such as isocitrate dehydrogenase 1 (IDH1), can be read in kinetic mode. Dehydrogenases that consume NAD(P)H, such as mutant IDH1 R132H, can be read in endpoint mode. Here, we report protocols for robust and miniaturized 1,536-well assays for WT IDH1 and IDH1 R132H coupled to diaphorase, and the counterassays used to further detect compound interference with the coupling reagents. This coupling technique is applicable to dehydrogenases that either produce or consume NAD(P)H, and the examples provided here can act as guidelines for the development of high-throughput screens against this enzyme class. PMID:27078681

Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

PubMed Central

Tramonti, Angela; Lanini, Claudio; Cialfi, Samantha; De Biase, Daniela; Falcone, Claudio

2012-01-01

In the yeast Kluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH in K. lactis led us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+ bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activity in vivo. The appearance of a new G6PDH-specific activity band, following incubation of Saccharomyces cerevisiae and human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes. PMID:23064253

21 CFR 862.1670 - Sorbitol dehydrogenase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Systems § 862.1670 Sorbitol dehydrogenase test system. (a) Identification. A sorbitol dehydrogenase test system is a device intended to measure the activity of the enzyme sorbitol dehydrogenase in serum... cirrhosis or acute hepatitis. (b) Classification. Class I (general controls). The device is exempt from the...

Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons.

PubMed

Kim, Jae-Ick; Ganesan, Subhashree; Luo, Sarah X; Wu, Yu-Wei; Park, Esther; Huang, Eric J; Chen, Lu; Ding, Jun B

2015-10-02

Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction. Copyright © 2015, American Association for the Advancement of Science.

Cellulase and alcohol dehydrogenase immobilized in Langmuir and Langmuir-Blodgett films and their molecular-level effects upon contact with cellulose and ethanol.

PubMed

Rodrigues, Dilmer; Camilo, Fernanda Ferraz; Caseli, Luciano

2014-02-25

The key challenges for producing devices based on nanostructured films with control over the molecular architecture are to preserve the catalytic activity of the immobilized biomolecules and to provide a reliable method for determining the intermolecular interactions and the accommodation of molecules at very small scales. In this work, the enzymes cellulase and alcohol dehydrogenase (ADH) were coimmobilized with dipalmitoylphosphatidylcholine (DPPC) as Langmuir-Blodgett (LB) films, and their biological activities were assayed by accommodating the structure formed in contact with cellulose. For this purpose, the polysaccharide was dissolved in an ionic liquid, 1-buthyl-3-methylimidazolium chloride (BMImCl), and dropped on the top of the hybrid cellulase-ADH-DPPC LB film. The interactions between cellulose and ethanol, which are the catalytic substrates of the enzymes as well as important elements in the production of second-generation fuels, were then investigated using polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS). Investigation of the secondary structures of the enzymes was performed using PM-IRRAS, through which the presence of ethanol and cellulose was observed to highly affect the structures of ADH and cellulase, respectively. The detection of products formed from the catalyzed reactions as well as the changes of secondary structure of the enzymes immobilization could be carried out, which opens the possibility to produce a means for producing second-generation ethanol using nanoscale arrangements.

Exploring the reversal of enantioselectivity on a zinc-dependent alcohol dehydrogenase† †Electronic supplementary information (ESI) available. See DOI: 10.1039/C7OB00482F Click here for additional data file.

PubMed Central

Maria-Solano, Miguel A.; Romero-Rivera, Adrian

2017-01-01

Alcohol Dehydrogenase (ADH) enzymes catalyse the reversible reduction of prochiral ketones to the corresponding alcohols. These enzymes present two differently shaped active site pockets, which dictate their substrate scope and selectivity. In this study, we computationally evaluate the effect of two commonly reported active site mutations (I86A, and W110T) on a secondary alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) through Molecular Dynamics simulations. Our results indicate that the introduced mutations induce dramatic changes in the shape of the active site, but most importantly they impact the substrate–enzyme interactions. We demonstrate that the combination of Molecular Dynamics simulations with the tools POVME and NCIplot corresponds to a powerful strategy for rationalising and engineering the stereoselectivity of ADH variants. PMID:28436515

Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

PubMed Central

Teng, Sophie X; Katz, Paige S; Maxi, John K; Mayeux, Jacques P; Gilpin, Nicholas W; Molina, Patricia E

2014-01-01

Traumatic brain injury (TBI) represents a leading cause of morbidity and mortality among young individuals. Alcohol abuse is a risk factor associated with increased TBI incidence. In addition, up to 26% of TBI patients engage in alcohol consumption after TBI. Limited preclinical studies have examined the impact of post-injury alcohol exposure on TBI recovery. The aim of this study was to determine the isolated and combined effects of TBI and alcohol on cognitive, behavioral, and physical recovery, as well as on associated neuroinflammatory changes. Male Sprague-Dawley rats (~300 g) were subjected to a mild focal TBI by lateral fluid percussion (~30 PSI, ~25 ms) under isoflurane anesthesia. On day 4 after TBI, animals were exposed to either sub-chronic intermittent alcohol vapor (95% ethanol 14h on /10h off; BAL~200 mg/dL) or room air for 10 days. TBI induced neurological dysfunction reflected by an increased neurological severity score (NSS) showed progressive improvement in injured animals exposed to room air (TBI/air). In contrast, TBI animals exposed to alcohol vapor (TBI/alcohol) showed impaired NSS recovery throughout the 10-day period of alcohol exposure. Open-field exploration test revealed an increased anxiety-like behavior in TBI/alcohol group compared to TBI/air group. Additionally, alcohol-exposed animals showed decreased locomotion and impaired novel object recognition. Immunofluorescence showed enhanced reactive astrocytes, microglial activation, and HMGB1 expression localized to the injured cortex of TBI/alcohol as compared to TBI/air animals. The expression of neuroinflammatory markers showed significant positive correlation with NSS. These findings indicated a close relationship between accentuated neuroinflammation and impaired neurological recovery from post-TBI alcohol exposure. The clinical implications of long-term consequences in TBI patients exposed to alcohol during recovery warrant further investigation. PMID:25489880

Glutamate Dehydrogenase from Apodachlya (Oomycetes) 1

PubMed Central

Price, Jeffrey S.; Gleason, Frank H.

1972-01-01

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

Contribution of ALDH2 polymorphism to alcoholism-associated hypertension.

PubMed

Hu, Nan; Zhang, Yingmei; Nair, Sreejayan; Culver, Bruce W; Ren, Jun

2014-01-01

Chronic alcohol intake is considered as an independent lifestyle factor that may influence the risk of a number of cardiovascular anomalies including hypertension. In healthy adults, binge drinking and chronic alcohol ingestion lead to the onset and development of hypertension although the precise mechanism(s) remains obscure. Although oxidative stress and endothelial injury have been postulated to play a major contributing role to alcoholism-induced hypertension, recent evidence depicted a rather unique role for the genotype of the acetaldehyde-metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), which is mainly responsible for detoxifying ethanol consumed, in alcoholism-induced elevation of blood pressure. Genetic polymorphism of ALDH2 in human results in altered ethanol pharmacokinetic properties and ethanol metabolism, leading to accumulation of the ethanol metabolite acetaldehyde following alcohol intake. The unfavorable consequence of the ALDH2 variants is believed to be governed by the accumulation of the ethanol metabolite acetaldehyde. Presence of the mutant or inactive ALDH2*2 gene often results in an increased risk of hypertension in human. Such association between blood pressure and ALDH2 enzymatic activity may be affected by the interplay between gene and environment, such as life style and ethnicity. The aim of this mini-review is to summarize the possible contribution of ALDH2 genetic polymorphism in the onset and development of alcoholism-related development of hypertension. Furthermore, the double-edged sword of ALDH2 gene and genetic polymorphism in alcoholism and alcoholic tissue damage and relevant patents will be discussed.

Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from 'Aromatoleum aromaticum' EbN1.

PubMed

Büsing, Imke; Höffken, H Wolfgang; Breuer, Michael; Wöhlbrand, Lars; Hauer, Bernhard; Rabus, Ralf

2015-01-01

The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium 'Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the 'classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD(+)-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe(187)), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr(93)) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93]. © 2015 S. Karger AG, Basel.

SAXS fingerprints of aldehyde dehydrogenase oligomers.

PubMed

Tanner, John J

2015-12-01

Enzymes of the aldehyde dehydrogenase (ALDH) superfamily catalyze the nicotinamide adenine dinucleotide-dependent oxidation of aldehydes to carboxylic acids. ALDHs are important in detoxification of aldehydes, amino acid metabolism, embryogenesis and development, neurotransmission, oxidative stress, and cancer. Mutations in genes encoding ALDHs cause metabolic disorders, including alcohol flush reaction (ALDH2), Sjögren-Larsson syndrome (ALDH3A2), hyperprolinemia type II (ALDH4A1), γ-hydroxybutyric aciduria (ALDH5A1), methylmalonic aciduria (ALDH6A1), pyridoxine dependent epilepsy (ALDH7A1), and hyperammonemia (ALDH18A1). We previously reported crystal structures and small-angle X-ray scattering (SAXS) analyses of ALDHs exhibiting dimeric, tetrameric, and hexameric oligomeric states (Luo et al., Biochemistry 54 (2015) 5513-5522; Luo et al., J. Mol. Biol. 425 (2013) 3106-3120). Herein I provide the SAXS curves, radii of gyration, and distance distribution functions for the three types of ALDH oligomer. The SAXS curves and associated analysis provide diagnostic fingerprints that allow rapid identification of the type of ALDH oligomer that is present in solution. The data sets provided here serve as a benchmark for characterizing oligomerization of ALDHs.

The Natural History of Class I Primate Alcohol Dehydrogenases Includes Gene Duplication, Gene Loss, and Gene Conversion

PubMed Central

Carrigan, Matthew A.; Uryasev, Oleg; Davis, Ross P.; Zhai, LanMin; Hurley, Thomas D.; Benner, Steven A.

2012-01-01

Background Gene duplication is a source of molecular innovation throughout evolution. However, even with massive amounts of genome sequence data, correlating gene duplication with speciation and other events in natural history can be difficult. This is especially true in its most interesting cases, where rapid and multiple duplications are likely to reflect adaptation to rapidly changing environments and life styles. This may be so for Class I of alcohol dehydrogenases (ADH1s), where multiple duplications occurred in primate lineages in Old and New World monkeys (OWMs and NWMs) and hominoids. Methodology/Principal Findings To build a preferred model for the natural history of ADH1s, we determined the sequences of nine new ADH1 genes, finding for the first time multiple paralogs in various prosimians (lemurs, strepsirhines). Database mining then identified novel ADH1 paralogs in both macaque (an OWM) and marmoset (a NWM). These were used with the previously identified human paralogs to resolve controversies relating to dates of duplication and gene conversion in the ADH1 family. Central to these controversies are differences in the topologies of trees generated from exonic (coding) sequences and intronic sequences. Conclusions/Significance We provide evidence that gene conversions are the primary source of difference, using molecular clock dating of duplications and analyses of microinsertions and deletions (micro-indels). The tree topology inferred from intron sequences appear to more correctly represent the natural history of ADH1s, with the ADH1 paralogs in platyrrhines (NWMs) and catarrhines (OWMs and hominoids) having arisen by duplications shortly predating the divergence of OWMs and NWMs. We also conclude that paralogs in lemurs arose independently. Finally, we identify errors in database interpretation as the source of controversies concerning gene conversion. These analyses provide a model for the natural history of ADH1s that posits four ADH1 paralogs in

21 CFR 866.5560 - Lactic dehydrogenase immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5560 Lactic dehydrogenase immunological test system. (a) Identification. A lactic dehydrogenase... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lactic dehydrogenase immunological test system...

GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE-S, A SPERM-SPECIFIC GLYCOLYTIC ENZYME, IS REQUIRED FOR SPERM MOTILITY AND MALE FERTILITY

EPA Science Inventory

While glycolysis is highly conserved, it is remarkable that several novel isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like it...

Alcohol and sexual risk behavior among migrant female sex workers and male workers in districts with high in-migration from four high HIV prevalence states in India.

PubMed

Verma, Ravi K; Saggurti, Niranjan; Singh, Ajay K; Swain, Suvakanta N

2010-08-01

This paper examines the association between alcohol use and sexual risk in two critical migrant populations living within the same geographical areas--migrant men and female sex-workers (FSWs). Data are drawn from two independent surveys of migrant FSWs and male workers in 14 districts of four high HIV prevalent Indian states. In the paper we have examined the independent effects of degree of mobility and alcohol use prior to sex on HIV risk behaviors. Nearly two-thirds of FSWs and a similar proportion of male migrant workers, as well as nine out of ten clients of FSWs consume alcohol. More than half of the FSWs and their clients consumed alcohol prior to sex. The practice of alcohol use prior to sex among both FSWs and their clients has a significant association with inconsistent condom use during paid as well as unpaid sex, and these effects are independent of degree of mobility. The results suggest a need for developing an in-depth understanding of the role of alcohol in accentuating HIV risk particularly among migrant populations who move frequently from one place another.

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance

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

Kim, Sun -Ki; Groom, Joseph; Chung, Daehwan

Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrationsmore » found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Lastly, expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.« less

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus 39E in Clostridium thermocellum 1313 results in increased hydroxymethylfurfural resistance

DOE PAGES

Kim, Sun -Ki; Groom, Joseph; Chung, Daehwan; ...

2017-03-15

Resistance to deconstruction is a major limitation to the use of lignocellulosic biomass as a substrate for the production of fuels and chemicals. Consolidated bioprocessing (CBP), the use of microbes for the simultaneous hydrolysis of lignocellulose into soluble sugars and fermentation of the resulting sugars to products of interest, is a potential solution to this obstacle. The pretreatment of plant biomass, however, releases compounds that are inhibitory to the growth of microbes used for CBP. Heterologous expression of the Thermoanaerobacter pseudethanolicus 39E bdhA gene, that encodes an alcohol dehydrogenase, in Clostridium thermocellum significantly increased resistance to furan derivatives at concentrationsmore » found in acid-pretreated biomass. The mechanism of detoxification of hydroxymethylfurfural was shown to be primarily reduction using NADPH as the cofactor. In addition, we report the construction of new expression vectors for homologous and heterologous expression in C. thermocellum. These vectors use regulatory signals from both C. bescii (the S-layer promoter) and C. thermocellum (the enolase promoter) shown to efficiently drive expression of the BdhA enzyme. Toxic compounds present in lignocellulose hydrolysates that inhibit cell growth and product formation are obstacles to the commercialization of fuels and chemicals from biomass. Lastly, expression of genes that reduce the effect of these inhibitors, such as furan derivatives, will serve to enable commercial processes using plant biomass for the production of fuels and chemicals.« less

Alcohol affects brain functional connectivity and its coupling with behavior: greater effects in male heavy drinkers.

PubMed

Shokri-Kojori, E; Tomasi, D; Wiers, C E; Wang, G-J; Volkow, N D

2017-08-01

Acute and chronic alcohol exposure significantly affect behavior but the underlying neurobiological mechanisms are still poorly understood. Here, we used functional connectivity density (FCD) mapping to study alcohol-related changes in resting brain activity and their association with behavior. Heavy drinkers (HD, N=16, 16 males) and normal controls (NM, N=24, 14 males) were tested after placebo and after acute alcohol administration. Group comparisons showed that NM had higher FCD in visual and prefrontal cortices, default mode network regions and thalamus, while HD had higher FCD in cerebellum. Acute alcohol significantly increased FCD within the thalamus, impaired cognitive and motor functions, and affected self-reports of mood/drug effects in both groups. Partial least squares regression showed that alcohol-induced changes in mood/drug effects were associated with changes in thalamic FCD in both groups. Disruptions in motor function were associated with increases in cerebellar FCD in NM and thalamus FCD in HD. Alcohol-induced declines in cognitive performance were associated with connectivity increases in visual cortex and thalamus in NM, but in HD, increases in precuneus FCD were associated with improved cognitive performance. Acute alcohol reduced 'neurocognitive coupling', the association between behavioral performance and FCD (indexing brain activity), an effect that was accentuated in HD compared with NM. Findings suggest that reduced cortical connectivity in HD contribute to decline in cognitive abilities associated with heavy alcohol consumption, whereas increased cerebellar connectivity in HD may have compensatory effects on behavioral performance. The results reveal how drinking history alters the association between brain FCD and individual differences in behavioral performance.

A Bacillus subtilis malate dehydrogenase gene.

PubMed Central

Jin, S; De Jesús-Berríos, M; Sonenshein, A L

1996-01-01

A Bacillus subtilis gene for malate dehydrogenase (citH) was found downstream of genes for citrate synthase and isocitrate dehydrogenase. Disruption of citH caused partial auxotrophy for aspartate and a requirement for aspartate during sporulation. In the absence of aspartate, citH mutant cells were blocked at a late stage of spore formation. PMID:8550482

β-Catenin is Essential for Ethanol Metabolism and Protection Against Alcohol-mediated Liver Steatosis in Mice

PubMed Central

Liu, Shiguang; Yeh, Tzu-Hsuan; Singh, Vijay P.; Shiva, Sruti; Krauland, Lindsay; Li, Huanan; Zhang, Pili; Kharbanda, Kusum; Ritov, Vladimir; Monga, Satdarshan P. S.; Scott, Donald K.; Eagon, Patricia K.; Behari, Jaideep

2011-01-01

The liver plays a central role in ethanol metabolism and oxidative stress is implicated in alcohol-mediated liver injury. β-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific β-catenin knockout (KO) mice and wild type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pair-wise fashion. Liver histology, biochemistry, and gene expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and five to six-fold higher serum ALT and AST levels. KO mice had modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD-2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N-Acetyl cysteine (NAC) did not prevent ethanol-induced mortality in KO mice. In WT livers, β-catenin was found to co-precipitate with FoxO3, the upstream regulator of SOD-2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were upregulated in ethanol-fed WT mice but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. Conclusion β-catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by β-catenin is critical for adaptation to ethanol ingestion in vivo. PMID:22031168

Protective effect of Xingnaojia formulation on rats with brain and liver damage caused by chronic alcoholism.

PubMed

Li, Shuang; Wang, S U; Guo, Zhi-Gang; Huang, Ning; Zhao, Fan-Rong; Zhu, Mo-Li; Ma, Li-Juan; Liang, Jin-Ying; Zhang, Yu-Lin; Huang, Zhong-Lin; Wan, Guang-Rui

2015-11-01

The aim of this study was to observe the effect of a formulation of traditional Chinese medicine extracts known as Xingnaojia (XNJ) on the liver function, learning ability and memory of rats with chronic alcoholism and to verify the mechanism by which it protects the brain and liver. A rat model of chronic alcoholism was used in the study. The spatial learning ability and memory of the rats were tested. The rats were then sacrificed and their brains and hepatic tissues were isolated. The activity of superoxide dismutase (SOD) and levels of glutamate (Glu), N-methyl D-aspartate receptor subtype 2B (NR2B), cyclin-dependent kinase 5 (CDK5) and cannabinoid receptor 1 (CB1) in the hippocampus were analyzed. The ultrastructure of the hepatic tissue was observed by electron microscopy. In addition, the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in serum were tested and the levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG) and total cholesterol (TCHOL) were analyzed. XNJ enhanced the learning and memory of rats with chronic alcoholism. Treatment with XNJ increased the activity of SOD, and decreased the expression levels of NR2B mRNA and NR2B, CB1 and CDK5 proteins in the brain tissues compared with those in the model rats. It also increased the activity of ALDH in the serum and liver, decreased the serum levels of LDL, TG and TCHOL and increased the serum level of HDL. These results indicate that XNJ exhibited a protective effect against brain and liver damage in rats with chronic alcoholism.

Maternal folate, alcohol and energy metabolism-related gene polymorphisms and the risk of recurrent pregnancy loss.

PubMed

Sata, F; Yamada, H; Kishi, R; Minakami, H

2012-10-01

Epidemiological studies have suggested that the condition of recurrent pregnancy loss (RPL) may be multifactorial, with both genetic predisposition and environmental factors potentially involved in its pathogenesis. The aim of this study is to elucidate the associations between maternal folate, alcohol and energy metabolism-related gene polymorphisms and the risk of RPL. This case-control study, which involved 116 cases with two or more instances of RPL and 306 fertile controls, was performed in the city of Sapporo, Japan. The associations between eight single nucleotide polymorphisms of folate, alcohol and energy metabolism-related genes [methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), alcohol dehydrogenase 1B (ADH1B), aldehyde dehydrogenase 2 (ALDH2), beta-3-adrenergic receptor (ADRB3) and peroxisome proliferator-activated receptor gamma (PPARG)], and RPL were assessed. Without consideration of cigarette smoking or alcohol use, the risk of RPL significantly decreased in women with the MTHFR rs1801133 TT, MTR rs1805087 AG or ALDH2 rs671 AA genotype (P < 0.05). The risk of RPL associated with cigarette smoking and alcohol use decreased significantly in women carrying the MTHFR rs1801133 T allele [odds ratio (OR), 0.51; 95% confidence interval (CI), 0.27-0.95]. Similarly, the risk of RPL significantly decreased in women carrying the MTR rs1805087 G allele (OR, 0.44; 95% CI, 0.23-0.85). Our findings suggest that maternal gene polymorphisms related to folate metabolism may decrease the risk of RPL. Molecular epidemiological studies are needed to unequivocally elucidate the multifactorial effects of both genetic and environmental factors on human fecundity.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed

Sakoda, H; Imanaka, T

1992-02-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

PubMed Central

Sakoda, H; Imanaka, T

1992-01-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH. Images PMID:1735726

Marine collagen peptides protect against early alcoholic liver injury in rats.

PubMed

Lin, Bing; Zhang, Feng; Yu, Yongchao; Jiang, Qinghao; Zhang, Zhaofeng; Wang, Junbo; Li, Yong

2012-04-01

Marine collagen peptides (MCP) have been reported to exhibit antioxidative activity, which is the common property of numerous hepatoprotective agents. Previous studies have shown that MCP have biological functions including anti-hypertension, anti-ulcer, anti-skin ageing and extending the life span. However, its role in alcoholic liver injury remains unknown. The present study aimed to investigate the effects of MCP on early alcoholic liver injury in rats. Rats were administered with alcohol at a dose of 6 g/kg body weight intragastrically per d to induce early liver injury, which was then evaluated by serum markers and histopathological examination. Treatment with MCP could reverse the increased level of serum aminotransferase and reduce hepatic histological damage. In addition, MCP attenuated the alteration in serum superoxide dismutase and malondialdehyde levels. MCP also counteracted the increased levels of total cholesterol and TAG. However, no significant difference was observed in the contents of alcohol dehydrogenase both in liver and serum protein of rats. These findings suggest that MCP have a protective effect on early alcoholic liver injury in rats by their antioxidative activity and improving lipid metabolism.

Flow-guided sketch and accentuated-tone adjusting for pencil drawing generation

NASA Astrophysics Data System (ADS)

Li, Ruirui; Yang, Lei; Hu, Wei

2017-07-01

Pencil drawing is an important artistic style which is widely used in draft sketch and finished rendering. It is non-trivial if we can automatically generate the pencil drawing more precisely in order to ease the human efforts. In this paper, we propose flow-guided sketch and accentuated-tone adjusting techniques to transform the pencil drawing from natural images. This work is extended from the existing pencil drawing framework where authors combine sketch and tone together for penciling purpose. Through an edge tangent flow guided classification and convolution, we can obtain more coherent and smooth strokes, especially for portraits. In addition, we also introduce a hierarchical approach to generate strokes. According to saliency map, we can adjust the tone of images automatically. The experimental results indicate the generated toned pencil drawings by our approach are of fewer artifacts and could even solve the images with dark background

Molecular cloning of a novel widely expressed human 80 kDa 17 beta-hydroxysteroid dehydrogenase IV.

PubMed Central

Adamski, J; Normand, T; Leenders, F; Monté, D; Begue, A; Stéhelin, D; Jungblut, P W; de Launoit, Y

1995-01-01

Reactions of oestrogens and androgens at position C-17 are catalysed by 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). Cloning of the cDNA of a novel human 17 beta-HSD IV and expression of its mRNA are described. A probe derived from the recently discovered porcine 17 beta-oestradiol dehydrogenase (17 beta-EDH) was used to isolate a 2.6 kb human cDNA encoding a continuous protein of 736 amino acids of high (84%) similarity to the porcine 17 beta-EDH. The calculated molecular mass of the human enzyme is 79,595 Da. Other sequence similarities shared by the two enzymes are: an N-terminal sequence which is similar to that of members of the short-chain alcohol dehydrogenase family; amino acids 343-607 which are similar to the C-terminal domains of a trifunctional Candida tropicalis enzyme and the FOX2 gene product of Saccharomyces cerevisiae; amino acids 596-736 which are similar to human sterol carrier protein 2. The previously cloned human 17 beta-HSD I, II and III are less than 25% identical with 17 beta-HSD IV. mRNA for HSD IV is a single species of 3.0 kb, present in many tissues with highest concentrations in liver, heart, prostate and testes. When over-expressed in mammalian cells, the human 17 beta-HSD IV enzyme displays a specific unidirectional oxidative 17 beta-HSD activity. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7487879

Liver proteomics in progressive alcoholic steatosis

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

Fernando, Harshica; Wiktorowicz, John E.; Soman, Kizhake V.

2013-02-01

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved inmore » alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1

The investigation of plasma glucose-6-phosphate dehydrogenase, 6-phoshogluconate dehydrogenase, glutathione reductase in premenauposal patients with iron deficiency anemia.

PubMed

Ozcicek, Fatih; Aktas, Mehmet; Türkmen, Kultigin; Coban, T Abdulkadir; Cankaya, Murat

2014-07-01

Iron is an essential element that is necessary for all cells in the body. Iron deficiency anemia (IDA) is one of the most common nutritional disorders in both developed and developing countries. The glutathione pathway is paramount to antioxidant defense and glucose-6-phosphate dehydrogenase (G6PD)-deficient cells do not cope well with oxidative damage. The goal of this study was to check the activities of G6PD, 6-phosphogluconate dehydrogenase, glutathione reductase in patients with IDA. We analyzed the plasma samples of 102 premenopausal women with IDA and 88 healthy control subjects. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity as compared to the reduction of NADP +, glutathione reductase activity was performed based on the oxidation of NADPH. 2 ml of plasma were used in all analyzes. SPSS program was used for all of the statistical analysis. Diagnosis of iron deficiency in patients belonging to the analysis of blood were ferritin 3.60 ± 2.7 ng / mL, hemoglobin 9.4 ± 1.5 mg / dl and hematocrit 30.7 ± 4.1% ratio; in healthy subjects ferritin 53.5 ± 41.7 ng/ml, hemoglobin level 13.9 ± 1.3 mg / dl and hematocrit ratio 42 ± 3.53%. When compared to healthy subjects the glutathione reductase level (P<0.001) was found to be significantly higher in patients with IDA. IDA patients with moderate and severe anemia had lower GR activity when compared to IDA patients with mild anemia. But the plasma levels of glucose-6-phosphate dehydrogenase (P<0,600) and 6-phosphogluconate dehydrogenase (P<0,671) did not show any differences between healthy subjects and in patients with IDA. It was shown that Glucose-6-Phosphate Dehydrogenase and 6-Phosphogluconate Dehydrogenase have no effect on iron-deficiency anemia in patients. The plasma GR levels of premenopausal women with IDA were found to be higher compared to healthy subjects, which could be secondary to erythrocyte protection against oxidative stress being commonly seen in IDA.

Biochemical and structural characterization of Cryptosporidium parvum Lactate dehydrogenase.

PubMed

Cook, William J; Senkovich, Olga; Hernandez, Agustin; Speed, Haley; Chattopadhyay, Debasish

2015-03-01

The protozoan parasite Cryptosporidium parvum causes waterborne diseases worldwide. There is no effective therapy for C. parvum infection. The parasite depends mainly on glycolysis for energy production. Lactate dehydrogenase is a major regulator of glycolysis. This paper describes the biochemical characterization of C. parvum lactate dehydrogenase and high resolution crystal structures of the apo-enzyme and four ternary complexes. The ternary complexes capture the enzyme bound to NAD/NADH or its 3-acetylpyridine analog in the cofactor binding pocket, while the substrate binding site is occupied by one of the following ligands: lactate, pyruvate or oxamate. The results reveal distinctive features of the parasitic enzyme. For example, C. parvum lactate dehydrogenase prefers the acetylpyridine analog of NADH as a cofactor. Moreover, it is slightly less sensitive to gossypol inhibition compared with mammalian lactate dehydrogenases and not inhibited by excess pyruvate. The active site loop and the antigenic loop in C. parvum lactate dehydrogenase are considerably different from those in the human counterpart. Structural features and enzymatic properties of C. parvum lactate dehydrogenase are similar to enzymes from related parasites. Structural comparison with malate dehydrogenase supports a common ancestry for the two genes. Copyright © 2014 Elsevier B.V. All rights reserved.

Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

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

Boernke, W. E.; Millard, C. S.; Stevens, P. W.

1995-09-10

Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the nativemore » enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

PubMed Central

ter Schure, Eelko G.; Flikweert, Marcel T.; van Dijken, Johannes P.; Pronk, Jack T.; Verrips, C. Theo

1998-01-01

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production. PMID:9546164

Genetics Home Reference: isobutyryl-CoA dehydrogenase deficiency

MedlinePlus

... dehydrogenase deficiency Orphanet: Isobutyryl-CoA dehydrogenase deficiency Screening, Technology and Research in Genetics Patient Support and Advocacy Resources (3 links) Children's Cardiomyopathy Foundation CLIMB (Children Living with Inherited Metabolic ...

Alcohol discrimination and preferences in two species of nectar-feeding primate

PubMed Central

Brown, Michael B.; Dominy, Nathaniel J.

2016-01-01

Recent reports suggest that dietary ethanol, or alcohol, is a supplemental source of calories for some primates. For example, slow lorises (Nycticebus coucang) consume fermented nectars with a mean alcohol concentration of 0.6% (range: 0.0–3.8%). A similar behaviour is hypothesized for aye-ayes (Daubentonia madagascariensis) based on a single point mutation (A294V) in the gene that encodes alcohol dehydrogenase class IV (ADH4), the first enzyme to catabolize alcohol during digestion. The mutation increases catalytic efficiency 40-fold and may confer a selective advantage to aye-ayes that consume the nectar of Ravenala madagascariensis. It is uncertain, however, whether alcohol exists in this nectar or whether alcohol is preferred or merely tolerated by nectarivorous primates. Here, we report the results of a multiple-choice food preference experiment with two aye-ayes and a slow loris. We conducted observer-blind trials with randomized, serial dilutions of ethanol (0–5%) in a standard array of nectar-simulating sucrose solutions. We found that both species can discriminate varying concentrations of alcohol; and further, that both species prefer the highest available concentrations. These results bolster the hypothesized adaptive function of the A294V mutation in ADH4, and a connection with fermented foods, both in aye-ayes and the last common ancestor of African apes and humans. PMID:27493777

Ginsenoside-free molecules from steam-dried ginseng berry promote ethanol metabolism: an alternative choice for an alcohol hangover.

PubMed

Lee, Do Ik; Kim, Seung Tae; Lee, Dong Hoon; Yu, Jung Min; Jang, Su Kil; Joo, Seong Soo

2014-07-01

Ethanol metabolism produces harmful compounds that contribute to liver damage and cause an alcohol hangover. The intermediate metabolite acetaldehyde is responsible for alcohol hangover and CYP2E1-induced reactive oxygen species damage liver tissues. In this study, we examined whether ginsenoside-free molecules (GFMs) from steam-dried ginseng berries promote ethanol metabolism and scavenge free radicals by stimulating primary enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, CYP2E1, and catalase) and antioxidant effects using in vitro and in vivo models. The results revealed that GFM effectively scavenged 2,2-diphenyl-1-picrylhydrazyl hydrate radicals and hydroxyl radicals. Notably, GFM significantly enhanced the expression of primary enzymes within 2 h in HepG2 cells. GFM clearly removed the consumed ethanol and significantly reduced the level of acetaldehyde as well as enhancement of primary gene expression in BALB/c mice. Moreover, GFM successfully protected HepG2 cells from ethanol attack. Of the major components identified in GFM, it was believed that linoleic acid was the most active ingredient. Based on these findings, we conclude that GFM holds promise for use as a new candidate for ethanol metabolism and as an antihangover agent. © 2014 Institute of Food Technologists®

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

PubMed Central

Anderson, Nickolas A.; Tobimatsu, Yuki; Ciesielski, Peter N.; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S.; Ladisch, Michael; Chapple, Clint

2015-01-01

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. PMID:26265762

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure.

PubMed

Anderson, Nickolas A; Tobimatsu, Yuki; Ciesielski, Peter N; Ximenes, Eduardo; Ralph, John; Donohoe, Bryon S; Ladisch, Michael; Chapple, Clint

2015-08-01

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content. © 2015 American Society of Plant Biologists. All rights reserved.

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice

PubMed Central

Han, Derick; Ybanez, Maria D.; Johnson, Heather S.; McDonald, Jeniece N.; Mesropyan, Lusine; Sancheti, Harsh; Martin, Gary; Martin, Alanna; Lim, Atalie M; Dara, Lily; Cadenas, Enrique; Tsukamoto, Hidekazu; Kaplowitz, Neil

2012-01-01

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD+ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD+, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver. PMID:23086958