Science.gov

Sample records for aldehyde dehydrogenase isozymes

  1. Betaine aldehyde dehydrogenase isozymes of spinach

    SciTech Connect

    Hanson, A.D.; Weretilnyk, E.A.; Weigel, P.

    1986-04-01

    Betaine is synthesized in spinach chloroplasts via the pathway Choline ..-->.. Betaine Aldehyde ..-->.. Betaine; the second step is catalyzed by betaine aldehyde dehydrogenase (BADH). The subcellular distribution of BADH was determined in leaf protoplast lysates; BADH isozymes were separated by 6-9% native PAGE. The chloroplast stromal fraction contains a single BADH isozyme (number1) that accounts for > 80% of the total protoplast activity; the extrachloroplastic fraction has a minor isozyme (number2) which migrates more slowly than number1. Both isozymes appear specific for betaine aldehyde, are more active with NAD than NADP, and show a ca. 3-fold activity increase in salinized leaves. The phenotype of a natural variant of isozyme number1 suggests that the enzyme is a dimer.

  2. Genetics of Aldehyde Dehydrogenase Isozymes in the Mouse: Evidence for Multiple Loci and Localization of Ahd-2 on Chromosome 19

    PubMed Central

    Timms, Glenn P.; Holmes, Roger S.

    1981-01-01

    Electrophoretic and activity variation of the cytoplasmic isozyme of aldehyde dehydrogenase (designated AHD-B4) was observed among inbred strains and Harwell linkage-testing stocks of Mus musculus. The phenotypes are inherited in a normal Mendelian fashion, with two alleles showing co-dominant expression at a single locus (Ahd-2). The locus was shown to segregate independently of Ahd-1 (encoding the mitochondrial AHD-A2 isozymes on chromosome 4; Holmes 1978). Linkage data of Ahd-2 with ep (pale ears), ru (ruby eyes) and bm (brachymorphic) suggest that it is localized near the centromeric end of chromosome 19. Electrophoretic evidence for a third AHD isozyme (designated AHD-Cy), which is predominantly localized in the liver microsomal fraction, is also presented. PMID:7274657

  3. Malate dehydrogenase isozymes in the longnose dace, Rhinichthys cataractae.

    PubMed

    Starzyk, R M; Merritt, R B

    1980-08-01

    The interspecies homology of dace supernatant (A2,AB,B2) and mitochondrial (C2) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.

  4. ALDEHYDE DEHYDROGENASES EXPRESSION DURING POSTNATAL DEVELOPMENT: LIVER VS. LUNG

    EPA Science Inventory

    Aldehydes are highly reactive molecules present in the environment, and can be produced during biotransformation of xenobiotics. Although the lung can be a major target for aldehyde toxicity, development of aldehyde dehydrogenases (ALDHs), which detoxify aldehydes, in lung has be...

  5. Mitochondrial aldehyde dehydrogenase and cardiac diseases

    PubMed Central

    Chen, Che-Hong; Sun, Lihan; Mochly-Rosen, Daria

    2010-01-01

    Numerous conditions promote oxidative stress, leading to the build-up of reactive aldehydes that cause cell damage and contribute to cardiac diseases. Aldehyde dehydrogenases (ALDHs) are important enzymes that eliminate toxic aldehydes by catalysing their oxidation to non-reactive acids. The review will discuss evidence indicating a role for a specific ALDH enzyme, the mitochondrial ALDH2, in combating oxidative stress by reducing the cellular ‘aldehydic load’. Epidemiological studies in humans carrying an inactive ALDH2, genetic models in mice with altered ALDH2 levels, and small molecule activators of ALDH2 all highlight the role of ALDH2 in cardioprotection and suggest a promising new direction in cardiovascular research and the development of new treatments for cardiovascular diseases. PMID:20558439

  6. [Subcellular localization of isozymes of NAD-dependent malate dehydrogenase in sugar beet Beta vulgaris L].

    PubMed

    Iudina, R S; Levites, E V

    2008-12-01

    Subcellular localization of isozymes of NAD-dependent malate dehydrogenase (MDH) in sugar beet was studied. Isozymes ss and 11 controlled by loci Mdh2 and Mdh3, respectively, were shown to locate in mitochondria, whereas isozyme pp controlled by locus Mdh1, in microbodies. All examined samples lack hybrid MDH isozymes, which could testify to the interaction between products of nonallelic Mdh genes. This can be explained by the localization of nonallelic isozymes in various compartments of the cell and organelles.

  7. Relationships within the aldehyde dehydrogenase extended family.

    PubMed

    Perozich, J; Nicholas, H; Wang, B C; Lindahl, R; Hempel, J

    1999-01-01

    One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.

  8. An Electrophoretic Analysis of the Isozymes of Malate Dehydrogenase in Several Different Plants 1

    PubMed Central

    Weimberg, Ralph

    1968-01-01

    The particulate and soluble fractions of cell-free extracts from seeds, roots, and leaves of 10 different plants were examined electrophoretically for isozymes of malate dehydrogenase. Distinct isozyme patterns were observed for each plant and even for the individual tissues of each species. There were some isozymes in several different plant extracts with equal electrophoretic mobilities, but there was no isozyme band that was common to all tissues or to all plants. Images PMID:16656816

  9. Aldehyde dehydrogenase 1A1 in stem cells and cancer

    PubMed Central

    Tomita, Hiroyuki; Tanaka, Kaori; Tanaka, Takuji; Hara, Akira

    2016-01-01

    The human genome contains 19 putatively functional aldehyde dehydrogenase (ALDH) genes, which encode enzymes critical for detoxification of endogenous and exogenous aldehyde substrates through NAD(P)+-dependent oxidation. ALDH1 has three main isotypes, ALDH1A1, ALDH1A2, and ALDH1A3, and is a marker of normal tissue stem cells (SC) and cancer stem cells (CSC), where it is involved in self-renewal, differentiation and self-protection. Experiments with murine and human cells indicate that ALDH1 activity, predominantly attributed to isotype ALDH1A1, is tissue- and cancer-specific. High ALDH1 activity and ALDH1A1 overexpression are associated with poor cancer prognosis, though high ALDH1 and ALDH1A1 levels do not always correlate with highly malignant phenotypes and poor clinical outcome. In cancer therapy, ALDH1A1 provides a useful therapeutic CSC target in tissue types that normally do not express high levels of ALDH1A1, including breast, lung, esophagus, colon and stomach. Here we review the functions and mechanisms of ALDH1A1, the key ALDH isozyme linked to SC populations and an important contributor to CSC function in cancers, and we outline its potential in future anticancer strategies. PMID:26783961

  10. Demonstration of glutamate dehydrogenase isozymes in beef heart mitochondria.

    PubMed

    McDaniel, H; Bosing-Schneider, R; Jenkins, R; Rasched, I; Sund, H

    1986-01-15

    Glutamate dehydrogenase (GDH) has been purified from beef heart mitochondria and compared with crystalline beef liver GDH. The specific activity of heart GDH was 127 units and of liver GDH 80 units. Heart GDH subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis had a protein corresponding to liver GDH and a smaller molecular weight protein. On agarose gel electrophoresis heart GDH activity was resolved into two fractions (with or without protease inhibitors) while liver had only one fraction. One of the heart fractions moved with liver GDH on electrophoresis. Thermal stability studies showed heart and liver GDH activity differed. Mouse antibodies to liver GDH precipitated both liver and heart GDH on double immunodiffusion. Mouse antibodies to liver GDH identified on nitrocellulose paper the polypeptide band of liver and heart GDH that were the same molecular weight but did not cross-react with the smaller molecular weight polypeptide present in heart GDH. Trypsin digestion of the two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GDH from beef heart mitochondria did not show any overlapping peptides. We conclude beef heart GDH activity is composed of two isozymes. One is the same as beef liver GDH, and the other is a smaller molecular weight protein. We propose the terms GDH-LM for the liver GDH isozyme and GDH-HM for the smaller molecular weight isozyme present in heart mitochondria but not liver.

  11. Alcohol and aldehyde dehydrogenases: structures of the human liver enzymes, functional properties and evolutionary aspects.

    PubMed

    Jörnvall, H; Hempel, J; von Bahr-Lindström, H; Höög, J O; Vallee, B L

    1987-01-01

    polyol dehydrogenases are encountered. The two isozymes of human aldehyde dehydrogenase also exhibit considerable differences, with only 68% structural identity. The results show an early divergence into isozymes before the man/horse species radiation. Cys-302 is a functionally important residue and is located in one of the regions with conserved hydrophobic properties. Other regions with large differences in hydropathic properties may explain the absence of cross-hybridizing isozyme forms of human liver aldehyde dehydrogenase.

  12. SAXS fingerprints of aldehyde dehydrogenase oligomers

    PubMed Central

    Tanner, John J.

    2015-01-01

    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. PMID:26693506

  13. 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. PMID:26693506

  14. The Aldehyde Dehydrogenase Gene Superfamily Resource Center

    PubMed Central

    2009-01-01

    The website http://www.aldh.org is a publicly available database for nomenclature and functional and molecular sequence information for members of the aldehyde dehydrogenase (ALDH) gene superfamily for animals, plants, fungi and bacteria. The site has organised gene-specific records. It provides synopses of ALDH gene records, marries trivial terms to correct nomenclature and links global accession identifiers with source data. Server-side alignment software characterises the integrity of each sequence relative to the latest genomic assembly and provides identifier-specific detail reports, including a graphical presentation of the transcript's exon - intron structure, its size, coding sequence, genomic strand and locus. Also included are a summary of substrates, inhibitors and enzyme kinetics. The site provides reference lists and is designed to facilitate data mining by interested investigators. PMID:20038501

  15. Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

    PubMed Central

    Chen, Che-Hong; Ferreira, Julio Cesar Batista; Gross, Eric R.; Mochly-Rosen, Daria

    2014-01-01

    A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme. PMID:24382882

  16. HEPATOCYTE EXPRESION OF TUMOR ASSOCIATED ALDEHYDE DEHYDROGENASE (ALDH-3) AND P21 RAS FOLLOWING DIETHYLNITROSAMINE (DEN) INITIATION AND CHRONIC EXPOSURE TO DI(2-ETHYLHEXYL) PHTHALATE (DHEP)

    EPA Science Inventory

    Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP)either promote or inhibit rat liver tumorigenesis depending on the carcinogenesis protocol. In this study, we examined the expression of two histochemical markers, the tumor associated isozyme of aldehyde dehydrogenase (ALD...

  17. Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.

    PubMed

    Zalabák, David; Johnová, Patricie; Plíhal, Ondřej; Šenková, Karolina; Šamajová, Olga; Jiskrová, Eva; Novák, Ondřej; Jackson, David; Mohanty, Amitabh; Galuszka, Petr

    2016-07-01

    The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants. PMID:27031423

  18. DEVELOPMENTAL EXPRESSION OF ALDEHYDE DEHYDROGENASE IN RAT: A COMPARISON OF LIVER AND LUNG DEVELOPMENT

    EPA Science Inventory

    Metabolism is one of the major determinants for age-related susceptibility changes to chemicals. Aldehydes are highly reactive molecules present in the environment and can be produced during biotransformation of xenobiotics. Aldehyde dehydrogenases (ALDH) are important in aldehyd...

  19. Inactivation of cytosolic aldehyde dehydrogenase via-nitrosylation in ethanol-exposed rat liver

    PubMed Central

    Moon, Kwan-Hoon; Abdelmegeed, Mohamed A.; Song, Byoung-Joon

    2009-01-01

    Aldehyde dehydrogenase (ALDH) isozymes are critically important in the metabolism of acetaldehyde, thus preventing its accumulation after ethanol exposure. We previously reported that mitochondrial ALDH2 could be inactivated via -nitrosylation in ethanol-exposed rats. This study was aimed at investigating whether cytosolic ALDH1, with a relatively low- value (11–18 μM) for acetaldehyde, could be also inhibited in ethanol-exposed rats. Chronic or binge ethanol exposure significantly decreased ALDH1 activity, which was restored by addition of dithiothreitol. Immunoblot analysis with the anti--nitroso-Cys antibody showed one immunoreactive band in the immunoprecipiated ALDH1 only from ethanol-exposed rats, but not from pair-fed controls, suggesting -nitrosylation of ALDH1. Therefore inactivation of ALDH1 via -nitrosylation can result in accumulation of acetaldehyde upon ethanol exposure. PMID:17673211

  20. Early diagnosis of radiodermatitis using lactate dehydrogenase isozymes in hairless mice (SKH1-hr)

    PubMed Central

    Kim, Won-Dong

    2012-01-01

    In this study, we evaluate a method for the early diagnosis of radiodermatitis for use in the prevention and therapy of this condition. Hairless mice (SKH1-hr) were used to study the early diagnosis of radiodermatitis. Lactate dehydrogenase (LDH, EC 1.1.1.27) isozymes were analyzed using native-polyacrylamide gel electrophoresis and western blotting of blood serum and tissues collected from SKH1-hr mice. Radiodermatitis developed 24 days after the first X-irradiation. Reduced spleen weight was observed after the last X-irradiation (P<0.05). Thereafter the weight increased until 24 days after the first irradiation, finally reaching levels comparable to those in the sham-irradiated control group. LDH activity was the highest in skeletal muscle and lowest in blood serum. LDH C4, A4, A3B, A2B2, AB3, and B4 isozymes were detected, in the mentioned order, from the cathode. This result was similar in other mouse strains. In the irradiated group, LDH A4 isozyme levels were reduced in the serum until inflammation occurred, whereas those of B4 isozyme were elevated. The subunits A and B followed a similar trend to that of LDH A4 and B4 isozyme, respectively. Importantly, antibodies against LDH B4 isozyme could prove useful in the early diagnosis of radiodermatitis. PMID:23326284

  1. Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

    PubMed Central

    Hart, G J; Dickinson, F M

    1978-01-01

    The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed. PMID:217355

  2. Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid

    PubMed Central

    Moretti, Andrea; Li, Jianfeng; Donini, Stefano; Sobol, Robert W.; Rizzi, Menico; Garavaglia, Silvia

    2016-01-01

    The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest. PMID:27759097

  3. Effects of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity.

    PubMed

    Li, Sha; Gan, Li-Qin; Li, Shu-Ke; Zheng, Jie-Cong; Xu, Dong-Ping; Li, Hua-Bin

    2014-01-01

    Various alcoholic beverages containing different concentrations of ethanol are widely consumed, and excessive alcohol consumption may result in serious health problems. The consumption of alcoholic beverages is often accompanied by non-alcoholic beverages, such as herbal infusions, tea and carbonated beverages to relieve drunk symptoms. The aim of this study was to supply new information on the effects of these beverages on alcohol metabolism for nutritionists and the general public, in order to reduce problems associated with excessive alcohol consumption. The effects of 57 kinds of herbal infusions, tea and carbonated beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity were evaluated. Generally, the effects of these beverages on alcohol dehydrogenase and aldehyde dehydrogenase activity are very different. The results suggested that some beverages should not be drank after excessive alcohol consumption, and several beverages may be potential dietary supplements for the prevention and treatment of problems related to excessive alcohol consumption.

  4. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed

    Winberg, J O; McKinley-McKee, J S

    1998-02-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  5. Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

    PubMed Central

    Winberg, J O; McKinley-McKee, J S

    1998-01-01

    Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain

  6. Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

    PubMed Central

    Hart, G J; Dickinson, F M

    1977-01-01

    Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents. PMID:194582

  7. Functional Specialization of Maize Mitochondrial Aldehyde Dehydrogenases1

    PubMed Central

    Liu, Feng; Schnable, Patrick S.

    2002-01-01

    The maize (Zea mays) rf2a and rf2b genes both encode homotetrameric aldehyde dehydrogenases (ALDHs). The RF2A protein was shown previously to accumulate in the mitochondria. In vitro import experiments and ALDH assays on mitochondrial extracts from rf2a mutant plants established that the RF2B protein also accumulates in the mitochondria. RNA gel-blot analyses and immunohistolocation experiments revealed that these two proteins have only partially redundant expression patterns in organs and cell types. For example, RF2A, but not RF2B, accumulates to high levels in the tapetal cells of anthers. Kinetic analyses established that RF2A and RF2B have quite different substrate specificities; although RF2A can oxidize a broad range of aldehydes, including aliphatic aldehydes and aromatic aldehydes, RF2B can oxidize only short-chain aliphatic aldehydes. These two enzymes also have different pH optima and responses to changes in substrate concentration. In addition, RF2A, but not RF2B or any other natural ALDHs, exhibits positive cooperativity. These functional specializations may explain why many species have two mitochondrial ALDHs. This study provides data that serve as a basis for identifying the physiological pathway by which the rf2a gene participates in normal anther development and the restoration of Texas cytoplasm-based male sterility. For example, the observations that Texas cytoplasm anthers do not accumulate elevated levels of reactive oxygen species or lipid peroxidation and the kinetic features of RF2A make it unlikely that rf2a restores fertility by preventing premature programmed cell death. PMID:12481049

  8. A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

    PubMed

    Adeboye, Peter Temitope; Olsson, Lisbeth; Bettiga, Maurizio

    2016-07-01

    The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae. PMID:27070284

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

    PubMed

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

    2016-04-01

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

  10. Aldehyde Dehydrogenases in Cellular Responses to Oxidative/electrophilic Stress

    PubMed Central

    Singh, Surendra; Brocker, Chad; Koppaka, Vindhya; Ying, Chen; Jackson, Brian; Matsumoto, Akiko; Thompson, David C.; Vasiliou, Vasilis

    2013-01-01

    Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors like dehydration and ultraviolet radiation. The ability to act as an ‘aldehyde scavenger’ during lipid peroxidation is another ostensibly universal ALDH function found across species. Up-regulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation) and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that significantly contributes to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, underscoring the fundamental importance of these enzymes in physiological and pathological processes. PMID:23195683

  11. Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress.

    PubMed

    Singh, Surendra; Brocker, Chad; Koppaka, Vindhya; Chen, Ying; Jackson, Brian C; Matsumoto, Akiko; Thompson, David C; Vasiliou, Vasilis

    2013-03-01

    Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single-celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors such as dehydration and ultraviolet radiation. The ability to act as an "aldehyde scavenger" during lipid peroxidation is another ostensibly universal ALDH function found across species. Upregulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity, and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation), and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane, and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that contributes significantly to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, highlighting the fundamental importance of these enzymes in physiological and pathological processes. PMID:23195683

  12. Aldehyde dehydrogenases in cellular responses to oxidative/electrophilic stress.

    PubMed

    Singh, Surendra; Brocker, Chad; Koppaka, Vindhya; Chen, Ying; Jackson, Brian C; Matsumoto, Akiko; Thompson, David C; Vasiliou, Vasilis

    2013-03-01

    Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single-celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors such as dehydration and ultraviolet radiation. The ability to act as an "aldehyde scavenger" during lipid peroxidation is another ostensibly universal ALDH function found across species. Upregulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity, and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation), and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane, and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that contributes significantly to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, highlighting the fundamental importance of these enzymes in physiological and pathological processes.

  13. [Characterization of aldehyde dehydrogenase gene fragment from mung bean Vigna radiata using the polymerase chain reaction].

    PubMed

    Ponomarev, A G; Bubiakina, V V; Tatarinova, T D; Zelenin, S M

    1998-01-01

    Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean. The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach. Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical. PMID:9778740

  14. Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1).

    PubMed

    Yang, Shyh-Ming; Yasgar, Adam; Miller, Bettina; Lal-Nag, Madhu; Brimacombe, Kyle; Hu, Xin; Sun, Hongmao; Wang, Amy; Xu, Xin; Nguyen, Kimloan; Oppermann, Udo; Ferrer, Marc; Vasiliou, Vasilis; Simeonov, Anton; Jadhav, Ajit; Maloney, David J

    2015-08-13

    Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted. PMID:26207746

  15. Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1).

    PubMed

    Yang, Shyh-Ming; Yasgar, Adam; Miller, Bettina; Lal-Nag, Madhu; Brimacombe, Kyle; Hu, Xin; Sun, Hongmao; Wang, Amy; Xu, Xin; Nguyen, Kimloan; Oppermann, Udo; Ferrer, Marc; Vasiliou, Vasilis; Simeonov, Anton; Jadhav, Ajit; Maloney, David J

    2015-08-13

    Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted.

  16. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  17. Characteristics of aldehyde dehydrogenase 2 (Aldh2) knockout mice.

    PubMed

    Yu, Hsu-Sheng; Oyama, Tsunehiro; Isse, Toyohi; Kitakawa, Kyoko; Ogawa, Masanori; Pham, Thi-Thu-Phuong; Kawamoto, Toshihiro

    2009-11-01

    Acetaldehyde is an intermediate of ethanol oxidation. It covalently binds to DNA, and is known as a carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/*2 and ALDH2*1/*2), and Aldh2 knockout mice appear to be a valid animal model for humans with inactive ALDH2. This review gives an overview of published studies on Aldh2 knockout mice, which were treated with ethanol or acetaldehyde. According to these studies, it was found that Aldh2 -/- mice (Aldh2 knockout mice) are more susceptible to ethanol and acetaldehyde-induced toxicity than Aldh2 +/+ mice (wild type mice). When mice were fed with ethanol, the mortality was increased. When they were exposed to atmospheres containing acetaldehyde, the Aldh2 -/- mice showed more severe toxic symptoms, like weight loss and higher blood acetaldehyde levels, as compared with the Aldh2 +/+ mice. Thus, ethanol and acetaldehyde treatment affects Aldh2 knockout mice more than wild type mice. Based on these findings, it is suggested that ethanol consumption and acetaldehyde inhalation are inferred to pose a higher risk to ALDH2-inactive humans. These results also support that ALDH2-deficient humans who habitually consume alcohol have a higher rate of cancer than humans with functional ALDH2. PMID:19874182

  18. Aldehyde dehydrogenase induction in arsenic-exposed rat bladder epithelium.

    PubMed

    Huang, Ya-Chun; Yu, Hsin-Su; Chai, Chee-Yin

    2016-01-01

    Arsenic is widely distributed in the environment. Many human cancers, including urothelial carcinoma (UC), show a dose-dependent relationship with arsenic exposure in the south-west coast of Taiwan (also known as the blackfoot disease (BFD) areas). However, the molecular mechanisms of arsenic-mediated UC carcinogenesis has not yet been defined. In vivo study, the rat bladder epithelium were exposed with arsenic for 48 h. The proteins were extracted from untreated and arsenic-treated rat bladder cells and utilized two-dimensional gel electrophoresis and mass spectrometry. Selected peptides were extracted from the gel and identified by quadrupole-time of flight (Q-TOF) Ultima-Micromass spectra. The significantly difference expression of proteins in arsenic-treated groups as compared with untreated groups was confirmed by immunohistochemistry (IHC) and western blotting. We found that thirteen proteins were down-regulated and nine proteins were up-regulated in arsenic-treated rat bladder cells when compared with untreated groups. The IHC and western blotting results confirmed that aldehyde dehydrogenase (ALDH) protein was up-regulated in arsenic-treated rat bladder epithelium. Expression of ALDH protein was significantly higher in UC patients from BFD areas than those from non-BFD areas using IHC (p=0.018). In conclusion, the ALDH protein expression could be used as molecular markers for arsenic-induced transformation.

  19. An animal model of human aldehyde dehydrogenase deficiency

    SciTech Connect

    Chang, C.; Mann, J.; Yoshida, A.

    1994-09-01

    The genetic deficiency of ALDH2, a major mitochondrial aldehyde dehydrogenase, is intimately related to alcohol sensitivity and the degree of predisposition to alcoholic diseases in humans. The ultimate biological role of ALDH2 can be exposed by knocking out the ALDH2 gene in an animal model. As the first step for this line of studies, we cloned and characterized the ALDH2 gene from mouse C57/6J strain which is associated with a high alcohol preference. The gene spans 26 kbp and is composed of 13 exons. Embryonic stem cells were transfected with a replacement vector which contains a partially deleted exon3, a positive selection cassette (pPgk Neo), exon 4 with an artificial stop codon, exons 5, 6, 7, and a negative selection cassette (pMCI-Tk). Genomic DNAs prepared from drug resistant clones were analyzed by polymerase chain reaction and by Southern blot analysis to distinguish random integration from homologous recombination. Out of 132 clones examined, 8 had undergone homologous recombination at one of the ALDH2 alleles. The cloned transformed embryonic stem cells with a disrupted ALDH2 allele were injected into blastocysts. Transplantation of the blastocysts into surrogate mother mice yielded chimeric mice. The role of ALDH2 in alcohol preference, alcohol sensitivity and other biological and behavioral characteristics can be elucidated by examining the heterozygous and homozygous mutant strains produced by breeding of chimeric mice.

  20. Aldehyde dehydrogenase induction in arsenic-exposed rat bladder epithelium.

    PubMed

    Huang, Ya-Chun; Yu, Hsin-Su; Chai, Chee-Yin

    2016-01-01

    Arsenic is widely distributed in the environment. Many human cancers, including urothelial carcinoma (UC), show a dose-dependent relationship with arsenic exposure in the south-west coast of Taiwan (also known as the blackfoot disease (BFD) areas). However, the molecular mechanisms of arsenic-mediated UC carcinogenesis has not yet been defined. In vivo study, the rat bladder epithelium were exposed with arsenic for 48 h. The proteins were extracted from untreated and arsenic-treated rat bladder cells and utilized two-dimensional gel electrophoresis and mass spectrometry. Selected peptides were extracted from the gel and identified by quadrupole-time of flight (Q-TOF) Ultima-Micromass spectra. The significantly difference expression of proteins in arsenic-treated groups as compared with untreated groups was confirmed by immunohistochemistry (IHC) and western blotting. We found that thirteen proteins were down-regulated and nine proteins were up-regulated in arsenic-treated rat bladder cells when compared with untreated groups. The IHC and western blotting results confirmed that aldehyde dehydrogenase (ALDH) protein was up-regulated in arsenic-treated rat bladder epithelium. Expression of ALDH protein was significantly higher in UC patients from BFD areas than those from non-BFD areas using IHC (p=0.018). In conclusion, the ALDH protein expression could be used as molecular markers for arsenic-induced transformation. PMID:26482281

  1. Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase.

    PubMed

    Boulatnikov, Igor; Popov, Kirill M

    2003-02-21

    Pyruvate dehydrogenase kinase (PDK) is a mitochondrial enzyme responsible for regulation of the pyruvate dehydrogenase complex and, consequently, aerobic oxidation of carbohydrate fuels in general. In mammals, there are four genetically and biochemically distinct forms of PDK that are expressed in a tissue-specific manner (PDK1, PDK2, PDK3, and PDK4). These protein kinases have been shown to function as dimers, but the possibility of heterodimerization between various isozyme subunits has not yet been investigated. Here, we demonstrate that two members of the PDK family, PDK1 and PDK2, form heterodimeric species when coexpressed in the same Escherichia coli cell. The heterodimeric kinase produced in vivo was purified to near homogeneity by affinity chromatography. The purified kinase was stable and was not subjected to reassortment of the subunits. The heterodimeric kinase was catalytically active and was clearly distinct from homodimeric PDK1 or PDK2 with respect to kinetic parameters, site specificity and regulation. These data strongly suggest that heterodimerization between PDK1 and PDK2 adds another level of diversity to this protein family in addition to that which arises from gene multiplicity. PMID:12573248

  2. Vascular Bioactivation of Nitroglycerin by Aldehyde Dehydrogenase-2

    PubMed Central

    Lang, Barbara S.; Gorren, Antonius C. F.; Oberdorfer, Gustav; Wenzl, M. Verena; Furdui, Cristina M.; Poole, Leslie B.; Mayer, Bernd; Gruber, Karl

    2012-01-01

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes the bioactivation of nitroglycerin (glyceryl trinitrate, GTN) in blood vessels, resulting in vasodilation by nitric oxide (NO) or a related species. Because the mechanism of this reaction is still unclear we determined the three-dimensional structures of wild-type (WT) ALDH2 and of a triple mutant of the protein that exhibits low denitration activity (E268Q/C301S/C303S) in complex with GTN. The structure of the triple mutant showed that GTN binds to the active site via polar contacts to the oxyanion hole and to residues 268 and 301 as well as by van der Waals interactions to hydrophobic residues of the catalytic pocket. The structure of the GTN-soaked wild-type protein revealed a thionitrate adduct to Cys-302 as the first reaction intermediate, which was also found by mass spectrometry (MS) experiments. In addition, the MS data identified sulfinic acid as the irreversibly inactivated enzyme species. Assuming that the structures of the triple mutant and wild-type ALDH2 reflect binding of GTN to the catalytic site and the first reaction step, respectively, superposition of the two structures indicates that denitration of GTN is initiated by nucleophilic attack of Cys-302 at one of the terminal nitrate groups, resulting in formation of the observed thionitrate intermediate and release of 1,2-glyceryl dinitrate. Our results shed light on the molecular mechanism of the GTN denitration reaction and provide useful information on the structural requirements for high affinity binding of organic nitrates to the catalytic site of ALDH2. PMID:22988236

  3. Residues that influence coenzyme preference in the aldehyde dehydrogenases.

    PubMed

    González-Segura, Lilian; Riveros-Rosas, Héctor; Julián-Sánchez, Adriana; Muñoz-Clares, Rosario A

    2015-06-01

    To find out the residues that influence the coenzyme preference of aldehyde dehydrogenases (ALDHs), we reviewed, analyzed and correlated data from their known crystal structures and amino-acid sequences with their published kinetic parameters for NAD(P)(+). We found that the conformation of the Rossmann-fold loops participating in binding the adenosine ribose is very conserved among ALDHs, so that coenzyme specificity is mainly determined by the nature of the residue at position 195 (human ALDH2 numbering). Enzymes with glutamate or proline at 195 prefer NAD(+) because the side-chains of these residues electrostatically and/or sterically repel the 2'-phosphate group of NADP(+). But contrary to the conformational rigidity of proline, the conformational flexibility of glutamate may allow NADP(+)-binding in some enzymes by moving the carboxyl group away from the 2'-phosphate group, which is possible if a small neutral residue is located at position 224, and favored if the residue at position 53 interacts with Glu195 in a NADP(+)-compatible conformation. Of the residues found at position 195, only glutamate interacts with the NAD(+)-adenosine ribose; glutamine and histidine cannot since their side-chain points are opposite to the ribose, probably because the absence of the electrostatic attraction by the conserved nearby Lys192, or its electrostatic repulsion, respectively. The shorter side-chains of other residues-aspartate, serine, threonine, alanine, valine, leucine, or isoleucine-are distant from the ribose but leave room for binding the 2'-phosphate group. Generally, enzymes having a residue different from Glu bind NAD(+) with less affinity, but they can also bind NADP(+) even sometimes with higher affinity than NAD(+), as do enzymes containing Thr/Ser/Gln195. Coenzyme preference is a variable feature within many ALDH families, consistent with being mainly dependent on a single residue that apparently has no other structural or functional roles, and therefore can

  4. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle.

  5. Bifunctional aldehyde/alcohol dehydrogenase (ADHE) in chlorophyte algal mitochondria.

    PubMed

    Atteia, Ariane; van Lis, Robert; Mendoza-Hernández, Guillermo; Henze, Katrin; Martin, William; Riveros-Rosas, Hector; González-Halphen, Diego

    2003-09-01

    Protein profiles of mitochondria isolated from the heterotrophic chlorophyte Polytomella sp. grown on ethanol at pH 6.0 and pH 3.7 were analyzed by Blue Native and denaturing polyacrylamide gel electrophoresis. Steady-state levels of oxidative phosphorylation complexes were influenced by external pH. Levels of an abundant, soluble, mitochondrial protein of 85 kDa and its corresponding mRNA increased at pH 6.0 relative to pH 3.7. N-terminal and internal sequencing of the 85 kDa mitochondrial protein together with the corresponding cDNA identified it as a bifunctional aldehyde/alcohol dehydrogenase (ADHE) with strong similarity to homologues from eubacteria and amitochondriate protists. A mitochondrial targeting sequence of 27 amino acids precedes the N-terminus of the mature mitochondrial protein. A gene encoding an ADHE homologue was also identified in the genome of Chlamydomonas reinhardtii, a photosynthetic relative of Polytomella. ADHE reveals a complex picture of sequence similarity among homologues. The lack of ADHE from archaebacteria indicates a eubacterial origin for the eukaryotic enzyme. Among eukaryotes, ADHE has hitherto been characteristic of anaerobes since it is essential to cytosolic energy metabolism of amitochondriate protists such as Giardia intestinalis and Entamoeba histolytica. Its abundance and expression pattern suggest an important role for ADHE in mitochondrial metabolism of Polytomella under the conditions studied. The current data are compatible with the view that Polytomella ADHE could be involved either in ethanol production or assimilation, or both, depending upon environmental conditions. Presence of ADHE in an oxygen-respiring algal mitochondrion and co-expression at ambient oxygen levels with respiratory chain components is unexpected with respect to the view that eukaryotes acquired ADHE genes specifically as an adaptation to an anaerobic lifestyle. PMID:14756315

  6. Aldehyde dehydrogenases: From eye crystallins to metabolic disease and cancer stem cells

    PubMed Central

    Vasiliou, Vasilis; Thompson, David C.; Smith, Clay; Fujita, Mayumi; Chen, Ying

    2014-01-01

    The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)+)-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date, 24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity, ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development, neurotransmission, oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. PMID:23159885

  7. Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

    NASA Technical Reports Server (NTRS)

    White, H. B., III; Kaplan, N. O.

    1972-01-01

    The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

  8. Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: benefits of Alda-1

    PubMed Central

    Gomes, Katia M.S.; Bechara, Luiz R.G.; Lima, Vanessa M.; Ribeiro, Márcio A.C.; Campos, Juliane C.; Dourado, Paulo M.; Kowaltowski, Alicia J.; Mochly-Rosen, Daria; Ferreira, Julio C.B.

    2015-01-01

    Background/Objectives We previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy is unknown and should be established to determine the optimal time window for drug treatment. Methods Here we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4 weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. Results We observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24hrs after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. Conclusion Taken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction

  9. Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

    PubMed

    Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

    2016-09-27

    The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes. PMID:27580341

  10. Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

    PubMed

    Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

    2016-09-27

    The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

  11. Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity

    PubMed Central

    Brocker, Chad; Cantore, Miriam; Failli, Paola; Vasiliou, Vasilis

    2012-01-01

    Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H2O2) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent β-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation. PMID:21338592

  12. The use of tomato aminoaldehyde dehydrogenase 1 for the detection of aldehydes in fruit distillates.

    PubMed

    Frömmel, Jan; Tarkowski, Petr; Kopečný, David; Šebela, Marek

    2016-09-25

    Plant NAD(+)-dependent aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases. They participate in the metabolism of polyamines or osmoprotectants. The enzymes are characterized by their broad substrate specificity covering ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The isoenzyme 1 from tomato (Solanum lycopersicum; SlAMADH1) oxidizes aliphatic aldehydes very efficiently and converts also furfural, its derivatives or benzaldehyde, which are present at low concentrations in alcoholic distillates such as fruit brandy. In this work, SlAMADH1 was examined as a bioanalytical tool for their detection. These aldehydes arise from fermentation processes or thermal degradation of sugars and their presence is related to health complications after consumption including nausea, emesis, sweating, decrease in blood pressure, hangover headache, among others. Sixteen samples of slivovitz (plum brandy) from local producers in Moravia, Czech Republic, were analyzed for their aldehyde content using a spectrophotometric activity assay with SlAMADH1. In all cases, there were oxidative responses observed when monitoring NADH production in the enzymatic reaction. Aldehydes in the distillate samples were also subjected to a standard determination using reversed-phase HPLC with spectrophotometric and tandem mass spectrometric detection after a derivatization with 2,4-dinitrophenylhydrazine. Results obtained by both methods were found to correlate well for a majority of the analyzed samples. The possible applicability of SlAMADH1 for the evaluation of aldehyde content in food and beverages has now been demonstrated. PMID:26703808

  13. Cardiac Mitochondrial Respiratory Dysfunction and Tissue Damage in Chronic Hyperglycemia Correlate with Reduced Aldehyde Dehydrogenase-2 Activity

    PubMed Central

    Deshpande, Mandar; Thandavarayan, Rajarajan A.; Xu, Jiang; Yang, Xiao-Ping; Palaniyandi, Suresh S.

    2016-01-01

    Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial isozyme of the heart involved in the metabolism of toxic aldehydes produced from oxidative stress. We hypothesized that hyperglycemia-mediated decrease in ALDH2 activity may impair mitochondrial respiration and ultimately result in cardiac damage. A single dose (65 mg/kg; i.p.) streptozotocin injection to rats resulted in hyperglycemia with blood glucose levels of 443 ± 9 mg/dl versus 121 ± 7 mg/dl in control animals, p<0.0001, N = 7–11. After 6 months of diabetes mellitus (DM) induction, the rats were sacrificed after recording the functionality of their hearts. Increase in the cardiomyocyte cross sectional area (446 ± 32 μm2 Vs 221 ± 10 μm2; p<0.0001) indicated cardiac hypertrophy in DM rats. Both diastolic and systolic dysfunctions were observed with DM rats compared to controls. Most importantly, myocardial ALDH2 activity and levels were reduced, and immunostaining for 4HNE protein adducts was increased in DM hearts compared to controls. The mitochondrial oxygen consumption rate (OCR), an index of mitochondrial respiration, was decreased in mitochondria isolated from DM hearts compared to controls (p<0.0001). Furthermore, the rate of mitochondrial respiration and the increase in carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)-induced maximal respiration were also decreased with chronic hyperglycemia. Chronic hyperglycemia reduced mitochondrial OXPHOS proteins. Reduced ALDH2 activity was correlated with mitochondrial dysfunction, pathological remodeling and cardiac dysfunction, respectively. Our results suggest that chronic hyperglycemia reduces ALDH2 activity, leading to mitochondrial respiratory dysfunction and consequently cardiac damage and dysfunction. PMID:27736868

  14. Mesenchymal Stem/Stromal Cells Derived From a Reproductive Tissue Niche Under Oxidative Stress Have High Aldehyde Dehydrogenase Activity.

    PubMed

    Kusuma, Gina D; Abumaree, Mohamed H; Pertile, Mark D; Perkins, Anthony V; Brennecke, Shaun P; Kalionis, Bill

    2016-06-01

    The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother's tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDH(br)) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDH(br) cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDH(br) cells than CMSC. Finally, flow sorted ALDH(br) cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments.

  15. Mesenchymal Stem/Stromal Cells Derived From a Reproductive Tissue Niche Under Oxidative Stress Have High Aldehyde Dehydrogenase Activity.

    PubMed

    Kusuma, Gina D; Abumaree, Mohamed H; Pertile, Mark D; Perkins, Anthony V; Brennecke, Shaun P; Kalionis, Bill

    2016-06-01

    The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother's tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDH(br)) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDH(br) cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDH(br) cells than CMSC. Finally, flow sorted ALDH(br) cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments. PMID:26880140

  16. Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecane and hexadecanol metabolism

    SciTech Connect

    Singer, M.E.; Finnerty, W.R.

    1985-12-01

    The role of fatty aldehyde dehydrogenases (FALDHs) in hexadecane and hexadecanol metabolism was studied in Acinetobacter sp. strain HO1-N. Two distinct FALDHs were demonstrated in Acinetobacter sp. strain HO1-N: (i) a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9 fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and (ii) a constitutive, NAD-dependent, membrane-localized FALDH. Dodecyl aldehyde-negative mutants were isolated and grouped into two phenotypic classes based on growth: class 1 mutants were hexadecane and hexadecanol negative and class 2 mutants were hexadecane and hexadecanol positive. Specific activity of NADP-dependent FALDH in Ald21 (class 1 mutant) was 85% lower than that of wild-type FALDH, while the specific activity of Ald24 (class 2 mutant) was 55% greater than that of wild-type FALDH. Ald21R, a dodecyl aldehyde-positive revertant able to grow on hexadecane, hexadecanol, and dodecyl aldehyde, exhibited a 100% increase in the specific activity of the NADP-dependent FALDH. This study provides genetic and physiological evidence for the role of fatty aldehyde as an essential metabolic intermediate and NADP-dependent FALDH as a key enzyme in the dissimilation of hexadecane, hexadecanol, and dodecyl aldehyde in Acinetobacter sp. strain HO1-N.

  17. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

    PubMed

    Halavaty, Andrei S; Rich, Rebecca L; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R; Myszka, David G; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F; Anderson, Wayne F

    2015-05-01

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

  18. Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus

    PubMed Central

    Halavaty, Andrei S.; Rich, Rebecca L.; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R.; Myszka, David G.; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F.; Anderson, Wayne F.

    2015-01-01

    When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD+) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD+, NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme. PMID:25945581

  19. 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

  20. Lactic dehydrogenase isozyme patterns and alpha-hydroxybutyrate dehydrogenase activities in serum from newborns, patients with ovarian cancer or myocardial infarction.

    PubMed

    Kikuchi, Y; Kita, T; Furuya, K; Kato, K

    1988-11-01

    Lactic dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (HBD) and LDH isozyme patterns were studied in serum from newborns and patients with ovarian cancer or myocardial infarction. LDH and HBD activities from newborns and patients with ovarian cancer or myocardial infarction were significantly increased, compared with those from patients with benign ovarian tumor. These increases were accompanied with a decrease of LDH-H and an increase of LDH-M in serum from newborns and patients with ovarian cancer, while an increase of LDH-H in serum from patients with myocardial infarction was dominant. However, the raised HBD activities in serum from patients with benign ovarian tumor did not affect the LDH isozyme patterns. From analysis of linear regression, a negative correlation between LDH-1 or -2 and HBD activity in serum from patients with ovarian cancer was observed while there was a positive correlation between LDH-4 and HBD activity. Similar patterns in serum from newborns were observed. On the other hand, a positive correlation between LDH-1 and HBD activity and a negative correlation between LDH-4 and HBD activity were found in serum from patients with myocardial infarction.

  1. Purification and characterization of aldehyde dehydrogenase from rat liver mitochondria.

    PubMed

    Senior, D J; Tsai, C S

    1988-04-01

    Nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent dehydrogenase activities from rat liver mitochondria have been copurified to homogeneity using combined DEAE, Sepharose, and affinity chromatographic procedures. The enzyme has a native molecular weight of 240,000 and subunit molecular weight of 60,000. The enzyme is tetrameric consisting of four identical subunits as revealed by electrophoresis and terminal analyses. A partial summary of physical properties is provided. The amino acid composition by acid hydrolysis is reported. Specific activities for various NAD(P)+ analogs and alkanal substrates were compared. The action of the effectors chloral hydrate, disulfiram, diethylstilbestrol, and Mg2+ and K+ ions were also investigated. PMID:3355167

  2. Correlation of loss of activity of human aldehyde dehydrogenase with reaction of bromoacetophenone with glutamic acid-268 and cysteine-302 residues. Partial-sites reactivity of aldehyde dehydrogenase.

    PubMed Central

    Abriola, D P; MacKerell, A D; Pietruszko, R

    1990-01-01

    Bromoacetophenone (2-bromo-1-phenylethanone) has been characterized as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) [MacKerell, MacWright & Pietruszko (1986) Biochemistry 25, 5182-5189], and has been shown to react specifically with the Glu-268 residue [Abriola, Fields, Stein, MacKerell & Pietruszko (1987) Biochemistry 26, 5679-5684] with an apparent inactivation stoichiometry of two molecules of bromoacetophenone per molecule of enzyme. The specificity of bromoacetophenone for reaction with Glu-268, however, is not absolute, owing to the extreme reactivity of this reagent. When bromo[14C]acetophenone was used to label the human cytoplasmic E1 isoenzyme radioactively and tryptic fragmentation was carried out, peptides besides that containing Glu-268 were found to have reacted with reagent. These peptides were purified by h.p.l.c. and analysed by sequencing and scintillation counting to quantify radioactive label in the material from each cycle of sequencing. Reaction of bromoacetophenone with the aldehyde dehydrogenase molecule during enzyme activity loss occurs with two residues, Glu-268 and Cys-302. The activity loss, however, appears to be proportional to incorporation of label at Glu-268. The large part of incorporation of label at Cys-302 occurs after the activity loss is essentially complete. With both Glu-268 and Cys-302, however, the incorporation of label stops after one molecule of bromoacetophenone has reacted with each residue. Reaction with other residues continues after activity loss is complete. PMID:1968743

  3. The activity of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in the sera of patients with brain cancer.

    PubMed

    Jelski, Wojciech; Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2014-12-01

    Human brain tissue contains various alcohol dehydrogenase (ADH) isoenzymes and possess also aldehyde dehydrogenase (ALDH) activity. In our last experiments we have shown that ADH and ALDH are present also in the brain tumour cells. Moreover the activities of total ADH and class I isoenzymes were significantly higher in cancer tissue than healthy cells. It can suggests that these changes may be reflected by enzyme activity in the serum of patients with brain cancer. Serum samples were taken for routine biochemical investigation from 62 patients suffering from brain cancer (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. A statistically significant increase of class I alcohol dehydrogenase isoenzymes was found in the sera of patients with brain cancer. The median activity of this class isoenzyme in the patients group increased about 24 % in the comparison to the control level. The total alcohol dehydrogenase activity was also significantly higher (26 %) among patients with brain tumour than healthy ones. The activities of other tested ADH isoenzymes and total ALDH were unchanged. The increase of the activity of total ADH and class I alcohol dehydrogenase isoenzyme in the sera of patients with brain cancer seems to be caused by the release of this isoenzyme from tumour's cells.

  4. Mitochondrial aldehyde dehydrogenase 2 protects gastric mucosa cells against DNA damage caused by oxidative stress.

    PubMed

    Duan, Yantao; Gao, Yaohui; Zhang, Jun; Chen, Yinan; Jiang, Yannan; Ji, Jun; Zhang, Jianian; Chen, Xuehua; Yang, Qiumeng; Su, Liping; Zhang, Jun; Liu, Bingya; Zhu, Zhenggang; Wang, Lishun; Yu, Yingyan

    2016-04-01

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a member of the aldehyde dehydrogenase superfamily and is involved with the metabolic processing of aldehydes. ALDH2 plays a cytoprotective role by removing aldehydes produced during normal metabolism. We examined the cytoprotective role of ALDH2 specifically in gastric mucosa cells. Overexpression of ALDH2 increased the viability of gastric mucosa cells treated with H2O2, while knockdown of ALDH2 had an opposite effect. Moreover, overexpression of ALDH2 protected gastric mucosa cells against oxidative stress-induced apoptosis as determined by flow cytometry, Hoechst 33342, and TUNEL assays. Consistently, ALDH2 knockdown had an opposite effect. Additionally, DNA damage was ameliorated in ALDH2-overexpressing gastric mucosa cells treated with H2O2. We further identified that this cytoprotective role of ALDH2 was mediated by metabolism of 4-hydroxynonenal (4-HNE). Consistently, 4-HNE mimicked the oxidative stress induced by H2O2 in gastric mucosa cells. Treatment with 4-HNE increased levels of DNA damage in ALDH2-knockdown GES-1 cells, while overexpression of ALDH2 decreased 4-HNE-induced DNA damage. These findings suggest that ALDH2 can protect gastric mucosa cells against DNA damage caused by oxidative stress by reducing levels of 4-HNE.

  5. Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

  6. Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde

    PubMed Central

    Ma, Heng; Guo, Rui; Yu, Lu; Zhang, Yingmei; Ren, Jun

    2011-01-01

    Aims The present study was designed to examine the mechanism involved in mitochondrial aldehyde dehydrogenase (ALDH2)-induced cardioprotection against ischaemia/reperfusion (I/R) injury with a focus on autophagy. Methods Wild-type (WT), ALDH2 overexpression, and knockout (KO) mice (n = 4–6 for each index measured) were subjected to I/R, and myocardial function was assessed using echocardiographic, Langendroff, and edge-detection systems. Western blotting was used to evaluate AMP-dependent protein kinase (AMPK), Akt, autophagy, and the AMPK/Akt upstream signalling LKB1 and PTEN. Results ALDH2 overexpression and KO significantly attenuated and accentuated, respectively, infarct size, factional shortening, and recovery of post-ischaemic left ventricular function following I/R as well as hypoxia/reoxygenation-induced cardiomyocyte contractile dysfunction. Autophagy was induced during ischaemia and remained elevated during reperfusion. ALDH2 significantly promoted autophagy during ischaemia, which was accompanied by AMPK activation and mammalian target of rapamycin (mTOR) inhibition. On the contrary, ALDH2 overtly inhibited autophagy during reperfusion accompanied by the activation of Akt and mTOR. Inhibition and induction of autophagy mitigated ALDH2-induced protection against cell death in hypoxia and reoxygenation, respectively. In addition, levels of the endogenous toxic aldehyde 4-hydroxy-2-nonenal (4-HNE) were elevated by ischaemia and reperfusion, which was abrogated by ALDH2. Furthermore, ALDH2 ablated 4-HNE-induced cardiomyocyte dysfunction and protein damage, whereas 4-HNE directly decreased pan and phosphorylated LKB1 and PTEN expression. Conclusion Our data suggest a myocardial protective effect of ALDH2 against I/R injury possibly through detoxification of toxic aldehyde and a differential regulation of autophagy through AMPK- and Akt-mTOR signalling during ischaemia and reperfusion, respectively. PMID:20705694

  7. A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. Enzymes catalyzing aldehyde oxidation in an adult moth

    SciTech Connect

    Tasayco, M.L.; Prestwich, G.D. )

    1990-02-25

    Aldehyde dehydrogenase (ALDH) and oxidase (AO) enzymes from the tissue extracts of male and female tobacco budworm moth (Heliothis virescens) were identified after electrophoretic protein separation. AO activity was visualized using formazan- or horseradish peroxidase-mediated staining coupled to the AO-catalyzed oxidation of benzaldehyde. A set of six soluble AO enzymes with isoelectric points from pI 4.6 to 5.3 were detected primarily in the antennal extracts. Partially purified antennal AO enzymes also oxidized both (Z)-9-tetradecenal and (Z)-11-hexadecenal, the two major pheromone components of this moth. ALDH activity was detected using a tritium-labeled affinity reagent based on a known irreversible inhibitor of this enzyme. This labeled vinyl ketone, (3H)(Z)-1,11-hexadecadien-3-one, was synthesized and used to covalently modify the soluble ALDH enzymes from tissue extracts. Molecular subunits of potential ALDH enzymes were visualized in the fluorescence autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the antenna, head, and leg tissues. Covalent modification of these protein subunits decreased specifically in the presence of excess pheromone aldehyde or benzaldehyde. Labeled vinyl ketones are thus novel tools for the identification of molecular subunits of ALDH enzymes.

  8. Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592.

    PubMed Central

    Yan, R T; Chen, J S

    1990-01-01

    Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay. Images PMID:2275527

  9. 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.

  10. A gatekeeper helix determines the substrate specificity of Sjögren–Larsson Syndrome enzyme fatty aldehyde dehydrogenase

    PubMed Central

    Keller, Markus A.; Zander, Ulrich; Fuchs, Julian E.; Kreutz, Christoph; Watschinger, Katrin; Mueller, Thomas; Golderer, Georg; Liedl, Klaus R.; Ralser, Markus; Kräutler, Bernhard; Werner, Ernst R.; Marquez, Jose A.

    2014-01-01

    Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren–Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a ‘gatekeeper’ helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations. PMID:25047030

  11. Corneal aldehyde dehydrogenase and glutathione S-transferase activity after excimer laser keratectomy in guinea pigs

    PubMed Central

    Bilgihan, K.; Bilgihan, A.; Turkozkan, N.

    1998-01-01

    BACKGROUND—The free radical balance of the eye may be changed by excimer laser keratectomy. Previous studies have demonstrated that excimer laser keratectomy increases the corneal temperature, decreases the superoxide dismutase activity of the aqueous, and induces lipid peroxidation in the superficial corneal stroma. Aldehyde dehydrogenase (ALDH) and glutathione S-transferase (GST) are known to play an important role in corneal metabolism, particularly in detoxification of aldehydes, which are generated from free radical reactions.
METHODS—In three groups of guinea pigs mechanical corneal de-epithelialisation was performed in group I, superficial corneal photoablation in group II, and deep corneal photoablation in group III, and the corneal ALDH and GST activities measured after 48 hours.
RESULTS—The mean ALDH and GST activities of group I and II showed no differences compared with the controls (p>0.05). The corneal ALDH activities were found to be significantly decreased (p<0.05) and GST activities increased (p<0.05) in group III.
CONCLUSION—These results suggest that excimer laser treatment of high myopia may change the ALDH and GST activities, metabolism, and free radical balance of the cornea.

 Keywords: excimer laser keratectomy; aldehyde dehydrogenase; glutathione S-transferase PMID:9602629

  12. Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain

    PubMed Central

    Zambelli, Vanessa O.; Gross, Eric R.; Chen, Che-Hong; Gutierrez, Vanessa P.; Cury, Yara; Mochly-Rosen, Daria

    2014-01-01

    Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase 2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R2=0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than wild-type mice. Finally, Alda-1 treatment was also beneficial when given even after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians’ apparent lower pain tolerance. PMID:25163478

  13. Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases

    SciTech Connect

    Khanna, Mary; Chen, Che-Hong; Kimble-Hill, Ann; Parajuli, Bibek; Perez-Miller, Samantha; Baskaran, Sulochanadevi; Kim, Jeewon; Dria, Karl; Vasiliou, Vasilis; Mochly-Rosen, Daria; Hurley, Thomas D.

    2012-10-23

    Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated {beta}-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

  14. Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver.

    PubMed Central

    Hart, G J; Dickinson, F M

    1978-01-01

    In the presence of acetic anhydride or butyric anhydride, liver aldehyde dehydrogenases catalyse the oxidation of NADH at pH 7.0 and 25 degrees C. The maximum velocities and Michaelis constants for NADH at saturating anhydride concentrations are independent of which anhydride is used, the values being V'max. = 12 min-1 and Km for NADH = 9 micrometer for the mitochondrial enzyme and V'max = 25 min-1 and Km for NADH = 20 micrometer for the cytoplasmic enzyme. Substitution of [4A-2H]NADH for NADH resulted in 2-fold and 4-fold decreases in rate for the mitochondrial and cytoplasmic enzymes respectively. PMID:217349

  15. Role and structural characterization of plant aldehyde dehydrogenases from family 2 and family 7.

    PubMed

    Končitíková, Radka; Vigouroux, Armelle; Kopečná, Martina; Andree, Tomáš; Bartoš, Jan; Šebela, Marek; Moréra, Solange; Kopečný, David

    2015-05-15

    Aldehyde dehydrogenases (ALDHs) are responsible for oxidation of biogenic aldehyde intermediates as well as for cell detoxification of aldehydes generated during lipid peroxidation. So far, 13 ALDH families have been described in plants. In the present study, we provide a detailed biochemical characterization of plant ALDH2 and ALDH7 families by analysing maize and pea ALDH7 (ZmALDH7 and PsALDH7) and four maize cytosolic ALDH(cALDH)2 isoforms RF2C, RF2D, RF2E and RF2F [the first maize ALDH2 was discovered as a fertility restorer (RF2A)]. We report the crystal structures of ZmALDH7, RF2C and RF2F at high resolution. The ZmALDH7 structure shows that the three conserved residues Glu(120), Arg(300) and Thr(302) in the ALDH7 family are located in the substrate-binding site and are specific to this family. Our kinetic analysis demonstrates that α-aminoadipic semialdehyde, a lysine catabolism intermediate, is the preferred substrate for plant ALDH7. In contrast, aromatic aldehydes including benzaldehyde, anisaldehyde, cinnamaldehyde, coniferaldehyde and sinapaldehyde are the best substrates for cALDH2. In line with these results, the crystal structures of RF2C and RF2F reveal that their substrate-binding sites are similar and are formed by an aromatic cluster mainly composed of phenylalanine residues and several nonpolar residues. Gene expression studies indicate that the RF2C gene, which is strongly expressed in all organs, appears essential, suggesting that the crucial role of the enzyme would certainly be linked to the cell wall formation using aldehydes from phenylpropanoid pathway as substrates. Finally, plant ALDH7 may significantly contribute to osmoprotection because it oxidizes several aminoaldehydes leading to products known as osmolytes. PMID:25734422

  16. Structural Basis of Substrate Recognition by Aldehyde Dehydrogenase 7A1

    PubMed Central

    2016-01-01

    Aldehyde dehydrogenase 7A1 (ALDH7A1) is part of lysine catabolism and catalyzes the NAD+-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Herein, we describe a structural study of human ALDH7A1 focused on substrate recognition. Five crystal structures and small-angle X-ray scattering data are reported, including the first crystal structure of any ALDH7 family member complexed with α-aminoadipate. The product binds with the ε-carboxylate in the oxyanion hole, the aliphatic chain packed into an aromatic box, and the distal end of the product anchored by electrostatic interactions with five conserved residues. This binding mode resembles that of glutamate bound to the proline catabolic enzyme ALDH4A1. Analysis of ALDH7A1 and ALDH4A1 structures suggests key interactions that underlie substrate discrimination. Structures of apo ALDH7A1 reveal dramatic conformational differences from the product complex. Product binding is associated with a 16 Å movement of the C-terminus into the active site, which stabilizes the active conformation of the aldehyde substrate anchor loop. The fact that the C-terminus is part of the active site was hitherto unknown. Interestingly, the C-terminus and aldehyde anchor loop are disordered in a new tetragonal crystal form of the apoenzyme, implying that these parts of the enzyme are highly flexible. Our results suggest that the active site of ALDH7A1 is disassembled when the aldehyde site is vacant, and the C-terminus is a mobile element that forms quaternary structural interactions that aid aldehyde binding. These results are relevant to the c.1512delG genetic deletion associated with pyridoxine-dependent epilepsy, which alters the C-terminus of ALDH7A1. PMID:26260980

  17. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.

    PubMed

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O; Wood, Andrew J; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W; Vasiliou, Vasilis

    2013-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD(+)- or NADP(+)-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as 'aldehyde scavengers' by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species-including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  18. The effects of alcohol and aldehyde dehydrogenases on disorders of hematopoiesis.

    PubMed

    Smith, Clay; Gasparetto, Maura; Jordan, Craig; Pollyea, Daniel A; Vasiliou, Vasilis

    2015-01-01

    Hematopoiesis involves the orderly production of millions of blood cells per second from a small number of essential bone marrow cells termed hematopoietic stem cells (HSCs). Ethanol suppresses normal hematopoiesis resulting in leukopenia, anemia, and thrombocytopenia and may also predispose to the development of diseases such as myelodysplasia (MDS) and acute leukemia. Currently the exact mechanisms by which ethanol perturbs hematopoiesis are unclear. The aldehyde dehydrogenase (ALDH) gene family plays a major role in the metabolism of reactive aldehydes derived from ethanol in the liver and other organs. At least one of the ALDH isoforms, ALDH1A1, is expressed at high levels in HSCs in humans, mice, and other organisms. Recent data indicate that ALDH1A1 and possibly other ALDH isoforms may metabolize reactive aldehydes in HSCs and other hematopoietic cells as they do in the liver and elsewhere. In addition, loss of these ALDHs leads to perturbation of a variety of cell processes that may predispose HSCs to disorders in growth and leukemic transformation. From these findings, we suggest a hypothesis that the cytopenias and possible increased risk of MDS and acute leukemia in heavy alcohol users is due to polymorphisms in genes responsible for metabolism of alcohol derived reactive aldehydes and repair of their DNA adducts in HSCs and other hematopoietic cells. In the article, we will summarize the biological properties of hematopoietic cells and diseases related to ethanol consumption, discuss molecular characteristics of ethanol metabolism, and describe a model to explain how ethanol derived reactive aldehydes may promote HSC damage. PMID:25427917

  19. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics

    PubMed Central

    Brocker, Chad; Vasiliou, Melpomene; Carpenter, Sarah; Carpenter, Christopher; Zhang, Yucheng; Wang, Xiping; Kotchoni, Simeon O.; Wood, Andrew J.; Kirch, Hans-Hubert; Kopečný, David; Nebert, Daniel W.

    2012-01-01

    In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD+- or NADP+-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as ‘aldehyde scavengers’ by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried outgenome-wide identification of ALDH genes in a number of plant species—including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies. PMID:23007552

  20. Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes.

    PubMed

    Choi, Myung-Min; Kim, Eun-A; Yang, Seung-Ju; Choi, Soo Young; Cho, Sung-Woo; Huh, Jae-Wan

    2007-07-01

    Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

  1. The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

    PubMed Central

    Tottmar, S. O. C.; Pettersson, H.; Kiessling, K.-H.

    1973-01-01

    1. Kinetic experiments suggested the possible existence of at least two different NAD+-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP+-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The Km for acetaldehyde was apparently less than 10μm for enzyme I and 0.9–1.7mm for enzyme II. The Km for NAD+ was similar for both enzymes (20–30μm). The Km for NADP+ was 2–3mm and for acetaldehyde 0.5–0.7mm for the NADP+-dependent activity. 4. The NAD+-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources. PMID:4149764

  2. Genomic organization and expression of the human fatty aldehyde dehydrogenase gene (FALDH)

    SciTech Connect

    Rogers, G.R.; Markova, N.G.; Compton, J.G.

    1997-01-15

    Mutations in the fatty aldehyde dehydrogenase (FALDH) gene cause Sjoegren-Larsson syndrome (SLS) - a disease characterized by mental retardation, spasticity, and congenital ichthyosis. To facilitate mutation analysis in SLS and to study the pathogenesis of FALDH deficiency, we have determined the structural organization and characterized expression of the FALDH (proposed designation ALDH10) gene. The gene consists of 10 exons spanning about 30.5 kb. A TATA-less promoter is associated with the major transcription initiation site found to be 258 hp upstream of the ATG codon. The G4C-rich sequences surrounding the transcription initiation site encompassed regulatory elements that interacted with proteins in HeLa nuclear extracts and were able to promote transcription in vitro. FALDH is widely expressed as three transcripts of 2, 3.8, and 4.0 kb, which originate from multiple polyadenylation signals in the 3{prime} UTR. An alternatively spliced mRNA was detected that contains an extra exon and encodes an enzyme that is likely to have altered membrane-binding properties. The FALDH gene lies only 50-85 kb from ALDH3, an aldehyde dehydrogenase gene that has homologous sequence and intron/exon structure. 25 refs., 4 figs., 1 tab.

  3. Ocular Aldehyde Dehydrogenases: Protection against Ultraviolet Damage and Maintenance of Transparency for Vision

    PubMed Central

    Chen, Ying; Thompson, David C.; Koppaka, Vindhya; Jester, James V.; Vasiliou, Vasilis

    2012-01-01

    Aldehyde dehydrogenase (ALDH) enzymes catalyze the NAD(P)+-dependent oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some members of the ALDH superfamily of enzymes are abundantly expressed in the mammalian cornea and lens in a taxon-specific manner. Considered to be corneal and lens crystallins, they confer protective and transparent properties upon these ocular tissues. ALDH3A1 is highly expressed in the cornea of most mammals, with the exception of rabbit that expresses exclusively ALDH1A1 in the cornea. ALDH1A1 is present in both the cornea and lens of several animal species. As a result of their catalytic and non-catalytic functions, ALDH3A1 and ALDH1A1 proteins protect inner ocular tissues from ultraviolet radiation and reactive oxygen-induced damage. In addition, these corneal crystallins contribute to cellular transparency in corneal stromal keratocytes, supporting a structural role of these ALDH proteins. A putative regulatory function of ALDH3A1 on corneal cell proliferation has also been proposed. Finally, the three retinaldehye dehydrogenases cooperatively mediate retinoic acid signaling during the eye development. PMID:23098688

  4. Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells.

    PubMed

    Van Wassenhove, Lauren D; Mochly-Rosen, Daria; Weinberg, Kenneth I

    2016-09-01

    Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35-45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia. PMID:27650066

  5. Characterization of Two Distinct Structural Classes of Selective Aldehyde Dehydrogenase 1A1 Inhibitors

    PubMed Central

    Morgan, Cynthia A.; Hurley, Thomas D.

    2015-01-01

    Aldehyde dehydrogenases (ALDH) catalyze the irreversible oxidation of aldehydes to their corresponding carboxylic acid. Alterations in ALDH1A1 activity are associated with such diverse diseases as cancer, Parkinson’s disease, obesity, and cataracts. Inhibitors of ALDH1A1 could aid in illuminating the role of this enzyme in disease processes. However, there are no commercially available selective inhibitors for ALDH1A1. Here we characterize two distinct chemical classes of inhibitors that are selective for human ALDH1A1 compared to eight other ALDH isoenzymes. The prototypical members of each structural class, CM026 and CM037, exhibit sub-micromolar inhibition constants, but have different mechanisms of inhibition. The crystal structures of these compounds bound to ALDH1A1 demonstrate that they bind within the aldehyde binding pocket of ALDH1A1 and exploit the presence of a unique Glycine residue to achieve their selectivity. These two novel and selective ALDH1A1 inhibitors may serve as chemical tools to better understand the contributions of ALDH1A1 to normal biology and to disease states. PMID:25634381

  6. 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

    2015-01-01

    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.

  7. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of gastric cancer patients.

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Laniewska, Magdalena; Szmitkowski, Maciej

    2010-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in gastric cancer cells (GC). Moreover, the activity of total ADH and class IV isoenzymes is significantly higher in cancer tissue than in healthy mucosa. The activity of these enzymes in cancer cells is probably reflected in the sera and could thus be helpful for diagnostics of gastric cancer. The aim of this study was to investigate a potential role of ADH and ALDH as tumor markers for gastric cancer. We defined diagnostic sensitivity, specificity, predictive value for positive and negative results, and receiver-operating characteristics (ROC) curve for tested enzymes. Serum samples were taken from 168 patients with gastric cancer before treatment and from 168 control subjects. Total ADH activity and class III and IV isoenzymes were measured by photometric but ALDH activity and ADH I and II by the fluorometric method, with class-specific fluorogenic substrates. There was significant increase in the activity of ADH IV isoenzyme and ADH total in the sera of gastric cancer patients compared to the control. The diagnostic sensitivity for ADH IV was 73%, specificity 79%, positive and negative predictive values were 81 and 72% respectively. Area under ROC curve for ADH IV was 0.67. The results suggest a potential role for ADH IV as marker of gastric cancer.

  8. NAD(P)-Dependent Aldehyde Dehydrogenases Induced during Growth of Ralstonia eutropha Strain Bo on Tetrahydrofurfuryl Alcohol

    PubMed Central

    Schräder, Thomas; Zarnt, Grit; Andreesen, Jan R.

    2001-01-01

    Different aldehyde dehydrogenases (AlDHs) were formed during growth of Ralstonia eutropha Bo on tetrahydrofurfuryl alcohol (THFA). One of these enzymes, AlDH 4, was purified and characterized as a homodimer containing no prosthetic groups, showing a strong substrate inhibition, and having an N-terminal sequence similar to those of various NAD(P)-dependent AlDHs. The conversion rate of THFA by the quinohemoprotein THFA dehydrogenase was increased by AlDH 4. PMID:11717302

  9. Cloning and molecular evolution of the aldehyde dehydrogenase 2 gene (Aldh2) in bats (Chiroptera).

    PubMed

    Chen, Yao; Shen, Bin; Zhang, Junpeng; Jones, Gareth; He, Guimei

    2013-02-01

    Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.

  10. Benomyl, Aldehyde Dehydrogenase, DOPAL, and the Catecholaldehyde Hypothesis for the Pathogenesis of Parkinson’s Disease

    PubMed Central

    2015-01-01

    The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is detoxified mainly by aldehyde dehydrogenase (ALDH). We find that the fungicide benomyl potently and rapidly inhibits ALDH and builds up DOPAL in vivo in mouse striatum and in vitro in PC12 cells and human cultured fibroblasts and glial cells. The in vivo results resemble those noted previously with knockouts of the genes encoding ALDH1A1 and 2, a mouse model of aging-related Parkinson’s disease (PD). Exposure to pesticides that inhibit ALDH may therefore increase PD risk via DOPAL buildup. This study lends support to the “catecholaldehyde hypothesis” that the autotoxic dopamine metabolite DOPAL plays a pathogenic role in PD. PMID:25045800

  11. A Personalized Medicine Approach for Asian Americans with the Aldehyde Dehydrogenase 2*2 Variant

    PubMed Central

    Gross, Eric R.; Zambelli, Vanessa O.; Small, Bryce A.; Ferreira, Julio C.B.; Chen, Che-Hong; Mochly-Rosen, Daria

    2015-01-01

    Asian Americans are one of the fastest-growing populations in the United States. A relatively large subset of this population carries a unique loss-of-function point mutation in aldehyde dehydrogenase 2 (ALDH2), ALDH2*2. Found in approximately 560 million people of East Asian descent, ALDH2*2 reduces enzymatic activity by approximately 60% to 80% in heterozygotes. Furthermore, this variant is associated with a higher risk for several diseases affecting many organ systems, including a particularly high incidence relative to the general population of esophageal cancer, myocardial infarction, and osteoporosis. In this review, we discuss the pathophysiology associated with the ALDH2*2 variant, describe why this variant needs to be considered when selecting drug treatments, and suggest a personalized medicine approach for Asian American carriers of this variant. We also discuss future clinical and translational perspectives regarding ALDH2*2 research. PMID:25292432

  12. Biological Evaluation and 3D-QSAR Studies of Curcumin Analogues as Aldehyde Dehydrogenase 1 Inhibitors

    PubMed Central

    Wang, Hui; Du, Zhiyun; Zhang, Changyuan; Tang, Zhikai; He, Yan; Zhang, Qiuyan; Zhao, Jun; Zheng, Xi

    2014-01-01

    Aldehyde dehydrogenase 1 (ALDH1) is reported as a biomarker for identifying some cancer stem cells, and down-regulation or inhibition of the enzyme can be effective in anti-drug resistance and a potent therapeutic for some tumours. In this paper, the inhibitory activity, mechanism mode, molecular docking and 3D-QSAR (three-dimensional quantitative structure activity relationship) of curcumin analogues (CAs) against ALDH1 were studied. Results demonstrated that curcumin and CAs possessed potent inhibitory activity against ALDH1, and the CAs compound with ortho di-hydroxyl groups showed the most potent inhibitory activity. This study indicates that CAs may represent a new class of ALDH1 inhibitor. PMID:24840575

  13. Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring

    PubMed Central

    Ahadome, Sarah D.; Abraham, David J.; Rayapureddi, Suryanarayana; Saw, Valerie P.; Saban, Daniel R.; Calder, Virginia L.; Norman, Jill T.; Ponticos, Markella; Daniels, Julie T.; Dart, John K.

    2016-01-01

    Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.

  14. Aldehyde Dehydrogenase 2 Has Cardioprotective Effects on Myocardial Ischaemia/Reperfusion Injury via Suppressing Mitophagy

    PubMed Central

    Ji, Wenqing; Wei, Shujian; Hao, Panpan; Xing, Junhui; Yuan, Qiuhuan; Wang, Jiali; Xu, Feng; Chen, Yuguo

    2016-01-01

    Mitophagy, a selective form of autophagy, is excessively activated in myocardial ischemia/reperfusion (I/R). The study investigated whether aldehyde dehydrogenase 2 (ALDH2) exerted its cardioprotective effect by regulating mitophagy. Myocardial infarct size and apoptosis after I/R in rats were ameliorated by Alda-1, an ALDH2 activator, and aggravated by ALDH2 inhibition. Both in I/R rats and hypoxia/reoxygenation H9C2 cells, ALDH2 activation suppressed phosphatase and tensin homolog-induced putative kinase 1 (PINK1)/Parkin expression, regulating mitophagy, by preventing 4-hydroxynonenal, reactive oxygen species and mitochondrial superoxide accumulation. Furthermore, the effect was enhanced by ALDH2 inhibition. Thus, ALDH2 may protect hearts against I/R injury by suppressing PINK1/Parkin–dependent mitophagy. PMID:27148058

  15. Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring

    PubMed Central

    Ahadome, Sarah D.; Abraham, David J.; Rayapureddi, Suryanarayana; Saw, Valerie P.; Saban, Daniel R.; Calder, Virginia L.; Norman, Jill T.; Ponticos, Markella; Daniels, Julie T.; Dart, John K.

    2016-01-01

    Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy. PMID:27699226

  16. 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

    2015-10-01

    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.

  17. IN VITRO ORGANIC NITRATE BIOACTIVATION TO NITRIC OXIDE BY RECOMBINANT ALDEHYDE DEHYDROGENASE 3A1

    PubMed Central

    Lin, Shunxin; Page, Nathaniel A.; Fung, Sun Mi; Fung, Ho-Leung

    2013-01-01

    Organic nitrates (ORNs) are commonly used anti-ischemic and anti-anginal agents, which serve as an exogenous source of the potent vasodilator nitric oxide (NO). Recently, both mitochondrial aldehyde dehydrogenase-2 (ALDH2) and cytosolic aldehyde dehydrogenase-1a1 (ALDH1A1) have been shown to exhibit the ability to selectively bioactivate various ORNs in vitro. The objective of the present research was to examine the potential role of ALDH3A1, another major cytosolic isoform of ALDH, in the in vitro bioactivation of various ORNs, and to estimate the enzyme kinetic parameters toward ORNs through mechanistic modeling. The extent of bioactivation was assayed by exposing recombinant ALDH3A1 to various concentrations of ORNs, and measuring the concentration-time profiles of released NO via a NO-specific electrode. Metabolite formation kinetics was monitored for nitroglycerin (NTG) using LC/MS/MS. Our results showed that ALDH3A1 mRNA and protein were highly expressed in C57BL/6 mouse aortic, cardiac, and hepatic tissues, and it was able to release NO from several ORNs, including NTG, isosorbide dinitrate (ISDN), isosorbide-2-mononitrate (IS-2-MN), and nicorandil with similar Vmax (0.175 – 0.503 nmol/min/mg of ALDH3A1), and Km values of 4.01, 46.5, 818 and 5.75 × 103 μM respectively. However, activation of isosorbide-5-mononitrate (IS-5-MN) by ALDH3A1 was undetectable in vitro. ALDH3A1 was also shown to denitrate NTG, producing primarily glyceryl 1, 2-dinitrate (1, 2-GDN) in preference to glyceryl 1, 3-dinitrate (1, 3-GDN). Therefore, ALDH3A1 may contribute to the bioactivation of ORNs in vivo. PMID:24126018

  18. Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

    PubMed

    Wenzl, M Verena; Beretta, Matteo; Griesberger, Martina; Russwurm, Michael; Koesling, Doris; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2011-08-01

    To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

  19. Changes in the inducibility of a hepatic aldehyde dehydrogenase by various effectors.

    PubMed

    Vasiliou, V; Marselos, M

    1989-01-01

    A hepatic soluble aldehyde dehydrogenase (ALDH), inducible by polycyclic aromatic hydrocarbons, was studied in Wistar rats in connection with substances known to affect drug metabolism or aldehyde dehydrogenase activity, such as phenobarbital (PB), disulfiram (DS), beta-diethylaminoethyl diphenylpropylacetate (SKF 525A) and calcium cyanamide (CC). 3-Methylcholanthrene (MC) was given as a model inducer of ALDH (100 mg/kg, i.p., as a single dose) and the animals were killed after 3 days. Pretreatment with PB (1 g/l drinking water, for 2 weeks) enhanced the inducing effect of MC. On the contrary, pretreatment with DS (100 mg/kg, i.p., daily x 4) reduced by 70% the expected increase in ALDH activity. Neither SKF 525A (25 mg/kg, i.p., daily x 4), nor CC (5 mg/kg, i.p., daily x 4) could affect the action of the inducer. At the above doses, basal ALDH activity was inhibited by DS (30%) and CC (70%), but was not affected at all by PB or SKF 525A. The results were somewhat different when the various effectors tested were administered to animals already treated with MC (20 mg/kg, i.p., daily x 6). In this case, DS did not affect the already induced ALDH activity. On the contrary, CC was still an effective inhibitor. Unexpectedly, post-treatment with SKF 525A further enhanced the initial induction brought about by MC. Our findings show that substances affecting microsomal drug metabolism can interfere with the process of ALDH induction by MC. The additive result of PB pretreatment is probably due to the enhanced accumulation of an active metabolite of MC.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Betaine Accumulation and Betaine-Aldehyde Dehydrogenase in Spinach Leaves 1

    PubMed Central

    Pan, Shu-Mei; Moreau, Robert A.; Yu, Charles; Huang, Anthony H. C.

    1981-01-01

    Spinach leaf discs accumulated betaine when exposed to a mannitol solution of −20 bars. The accumulation was 12 micromoles per gram original fresh weight in a 24-hour period. Betaine-aldehyde dehydrogenase (EC 1.2.1.8) was assayed in various subcellular fractions prepared from spinach leaves, and it was found only in the soluble fraction. This cytosolic enzyme was purified 175-fold, and its properties were studied. The enzyme was relatively specific for betaine aldehyde as the substrate with an apparent Km value of 2.08 × 10−4 molar. It also exerted activity on other aldehyde analogs tested, but with lower Vmax and higher Km values. The enzyme was relatively specific for nicotinamide adenine dinucleotide as the coenzyme, having an apparent Km value of 9.46 × 10−6 molar; lower activities were observed when nicotinamide adenine dinucleotide phosphate or 3-acetyl pyridine adenine dinucleotide were tested as electron acceptors. The activity was enhanced by dithiothreitol and inhibited by p-chloromercuribenzoate, and the inhibition by p-chloromercuribenzoate was partially reversed by the subsequent addition of dithiothreitol. The activity was inhibited by high concentrations of NaCl and, to a lesser extent, proline. The equilibrium of the enzymic reaction was strongly in favor of betaine formation. The in vitro activity of the enzyme under optimal assay conditions was high enough to account for the amount of betaine accumulated under water stress conditions. The enzyme activity was the same in unstressed leaves and in leaves that had been water stressed for 24 hours. PMID:16661818

  1. Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant

    PubMed Central

    Perez-Miller, Samantha; Younus, Hina; Vanam, Ram; Chen, Che-Hong; Mochly-Rosen, Daria; Hurley, Thomas D.

    2010-01-01

    In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant, acrolein. ALDH2 also bioactivates nitroglycerin, but it is best known for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian Alcohol-induced Flushing Syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semi-dominant and heterozygotic individuals exhibit a similar, but not as severe phenotype. We recently identified a small molecule, Alda-1, which activates wild-type ALDH2 and restores near wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone. PMID:20062057

  2. Cloning and characterization of a novel betaine aldehyde dehydrogenase gene from Suaeda corniculata.

    PubMed

    Wang, F W; Wang, M L; Guo, C; Wang, N; Li, X W; Chen, H; Dong, Y Y; Chen, X F; Wang, Z M; Li, H Y

    2016-01-01

    Glycine betaine is an important quaternary ammonium compound that is produced in response to several abiotic stresses in many organisms. The synthesis of glycine betaine requires the catalysis of betaine aldehyde dehydrogenase (BADH), which can convert betaine aldehyde into glycine betaine in plants, especially in halotolerant plants. In this study, we isolated the full-length cDNA of BADH from Suaeda corniculata (ScBADH) using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends. Next, we analyzed the expression profile of ScBADH using real-time PCR. The results showed that ScBADH expression was induced in the roots, stems, and leaves of S. corniculata seedlings under salt and drought stress. Next, ScBADH was overexpressed in Arabidopsis, resulting in the transgenic plants exhibiting enhanced tolerance over wild-type plants under salt and drought stress. We then analyzed the levels of glycine betaine and proline, as well as superoxide dismutase (SOD) activity, during salt stress in WT and transgenic Arabidopsis. The results indicated that overexpression of ScBADH produced more glycine betaine and proline, and increased SOD activity under NaCl treatment. Our results suggest that ScBADH might be a positive regulator in plants during the response to NaCl. PMID:27420939

  3. Alda-1 is an agonist and chemical chaperone for the common human aldehyde dehydrogenase 2 variant

    SciTech Connect

    Perez-Miller, Samantha; Younus, Hina; Vanam, Ram; Chen, Che-Hong; Mochly-Rosen, Daria; Hurley, Thomas D.

    2010-04-19

    In approximately one billion people, a point mutation inactivates a key detoxifying enzyme, aldehyde dehydrogenase (ALDH2). This mitochondrial enzyme metabolizes toxic biogenic and environmental aldehydes, including the endogenously produced 4-hydroxynonenal (4HNE) and the environmental pollutant acrolein, and also bioactivates nitroglycerin. ALDH2 is best known, however, for its role in ethanol metabolism. The accumulation of acetaldehyde following the consumption of even a single alcoholic beverage leads to the Asian alcohol-induced flushing syndrome in ALDH2*2 homozygotes. The ALDH2*2 allele is semidominant, and heterozygotic individuals show a similar but less severe phenotype. We recently identified a small molecule, Alda-1, that activates wild-type ALDH2 and restores near-wild-type activity to ALDH2*2. The structures of Alda-1 bound to ALDH2 and ALDH2*2 reveal how Alda-1 activates the wild-type enzyme and how it restores the activity of ALDH2*2 by acting as a structural chaperone.

  4. Genome-Wide Identification and Functional Classification of Tomato (Solanum lycopersicum) Aldehyde Dehydrogenase (ALDH) Gene Superfamily

    PubMed Central

    Lopez-Valverde, Francisco J.; Robles-Bolivar, Paula; Lima-Cabello, Elena; Gachomo, Emma W.; Kotchoni, Simeon O.

    2016-01-01

    Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of S. lycopersicum ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving Cys-Glu couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of S. lycopersicum ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling. PMID:27755582

  5. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11{beta}-hydroxysteroid dehydrogenase

    SciTech Connect

    Agarwal, A.K.; Rogerson, F.M.; Mune, T.; White, P.C.

    1995-09-01

    11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11{beta}HSD. In vitro, the NAD{sup +}-dependent kidney (type 2) isozyme catalyzes 11{beta}-dehydrogenase but not reductase reactions, whereas the NADP{sup +}-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11{beta}HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11{beta}HSD. 25 refs., 5 figs.

  6. Effects of Alda-1, an Aldehyde Dehydrogenase-2 Agonist, on Hypoglycemic Neuronal Death.

    PubMed

    Ikeda, Tetsuhiko; Takahashi, Tetsuya; Tsujita, Mika; Kanazawa, Masato; Toriyabe, Masafumi; Koyama, Misaki; Itoh, Kosuke; Nakada, Tsutomu; Nishizawa, Masatoyo; Shimohata, Takayoshi

    2015-01-01

    Hypoglycemic encephalopathy (HE) is caused by a lack of glucose availability to neuronal cells, and no neuroprotective drugs have been developed as yet. Studies on the pathogenesis of HE and the development of new neuroprotective drugs have been conducted using animal models such as the hypoglycemic coma model and non-coma hypoglycemia model. However, both models have inherent problems, and establishment of animal models that mimic clinical situations is desirable. In this study, we first developed a short-term hypoglycemic coma model in which rats could be maintained in an isoelectric electroencephalogram (EEG) state for 2 min and subsequent hyperglycemia without requiring anti-seizure drugs and an artificial ventilation. This condition caused the production of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic aldehyde, in neurons of the hippocampus and cerebral cortex, and a marked increase in neuronal death as evaluated by Fluoro-Jade B (FJB) staining. We also investigated whether N-(1,3-benzodioxole-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1), a small-molecule agonist of aldehyde dehydrogenase-2, could attenuate 4-HNE levels and reduce hypoglycemic neuronal death. After confirming that EEG recordings remained isoelectric for 2 min, Alda-1 (8.5 mg/kg) or vehicle (dimethyl sulfoxide; DMSO) was administered intravenously with glucose to maintain a blood glucose level of 250 to 270 mg/dL. Fewer 4-HNE and FJB-positive cells were observed in the cerebral cortex of Alda-1-treated rats than in DMSO-treated rats 24 h after glucose administration (P = 0.002 and P = 0.020). Thus, activation of the ALDH2 pathway could be a molecular target for HE treatment, and Alda-1 is a potentially neuroprotective agent that exerts a beneficial effect on neurons when intravenously administered simultaneously with glucose. PMID:26083658

  7. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs

    PubMed Central

    Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

    2015-01-01

    Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants. PMID:25909656

  8. Aldehyde dehydrogenase 1 defines and protects a nigrostriatal dopaminergic neuron subpopulation

    PubMed Central

    Liu, Guoxiang; Yu, Jia; Ding, Jinhui; Xie, Chengsong; Sun, Lixin; Rudenko, Iakov; Zheng, Wang; Sastry, Namratha; Luo, Jing; Rudow, Gay; Troncoso, Juan C.; Cai, Huaibin

    2014-01-01

    Subpopulations of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) display a differential vulnerability to loss in Parkinson’s disease (PD); however, it is not clear why these subsets are preferentially selected in PD-associated neurodegeneration. In rodent SNpc, DA neurons can be divided into two subpopulations based on the expression of aldehyde dehydrogenase 1 (ALDH1A1). Here, we have shown that, in α-synuclein transgenic mice, a murine model of PD-related disease, DA neurodegeneration occurs mainly in a dorsomedial ALDH1A1-negative subpopulation that is also prone to cytotoxic aggregation of α-synuclein. Notably, the topographic ALDH1A1 pattern observed in α-synuclein transgenic mice was conserved in human SNpc. Postmortem evaluation of brains of patients with PD revealed a severe reduction of ALDH1A1 expression and neurodegeneration in the ventral ALDH1A1-positive DA subpopulations. ALDH1A1 expression was also suppressed in α-synuclein transgenic mice. Deletion of Aldh1a1 exacerbated α-synuclein–mediated DA neurodegeneration and α-synuclein aggregation, whereas Aldh1a1-null and control DA neurons were comparably susceptible to 1-methyl-4-phenylpyridinium–, glutamate-, or camptothecin-induced cell death. ALDH1A1 overexpression appeared to preferentially protect against α-synuclein–mediated DA neurodegeneration but did not rescue α-synuclein–induced loss of cortical neurons. Together, our findings suggest that ALDH1A1 protects subpopulations of SNpc DA neurons by preventing the accumulation of dopamine aldehyde intermediates and formation of cytotoxic α-synuclein oligomers. PMID:24865427

  9. Parallel Functional Changes in Independent Testis-Specific Duplicates of Aldehyde dehydrogenase in Drosophila

    PubMed Central

    Chakraborty, Mahul; Fry, James D.

    2015-01-01

    A large proportion of duplicates, originating from ubiquitously expressed genes, acquire testis-biased expression. Identifying the underlying cause of this observation requires determining whether the duplicates have altered functions relative to the parental genes. Typically, statistical methods are used to test for positive selection, signature of which in protein sequence of duplicates implies functional divergence. When assumptions are violated, however, such tests can lead to false inference of positive selection. More convincing evidence for naturally selected functional changes would be the occurrence of structural changes with similar functional consequences in independent duplicates of the same gene. We investigated two testis-specific duplicates of the broadly expressed enzyme gene Aldehyde dehydrogenase (Aldh) that arose in different Drosophila lineages. The duplicates show a typical pattern of accelerated amino acid substitutions relative to their broadly expressed paralogs, with statistical evidence for positive selection in both cases. Importantly, in both duplicates, width of the entrance to the substrate binding site, known a priori to influence substrate specificity, and otherwise conserved throughout the genus Drosophila, has been reduced, resulting in narrowing of the entrance. Protein structure modeling suggests that the reduction of the size of the enzyme’s substrate entry channel, which is likely to shift substrate specificity toward smaller aldehydes, is accounted for by the positively selected parallel substitutions in one duplicate but not the other. Evolution of the testis-specific duplicates was accompanied by reduction in expression of the ancestral Aldh in males, supporting the hypothesis that the duplicates may have helped resolve intralocus sexual conflict over Aldh function. PMID:25564519

  10. Aldehyde Dehydrogenase Gene Superfamily in Populus: Organization and Expression Divergence between Paralogous Gene Pairs.

    PubMed

    Tian, Feng-Xia; Zang, Jian-Lei; Wang, Tan; Xie, Yu-Li; Zhang, Jin; Hu, Jian-Jun

    2015-01-01

    Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of reactive aldehydes to their corresponding nontoxic carboxylic acids. ALDHs have been studied in many organisms from bacteria to mammals; however, no systematic analyses incorporating genome organization, gene structure, expression profiles, and cis-acting elements have been conducted in the model tree species Populus trichocarpa thus far. In this study, a comprehensive analysis of the Populus ALDH gene superfamily was performed. A total of 26 Populus ALDH genes were found to be distributed across 12 chromosomes. Genomic organization analysis indicated that purifying selection may have played a pivotal role in the retention and maintenance of PtALDH gene families. The exon-intron organizations of PtALDHs were highly conserved within the same family, suggesting that the members of the same family also may have conserved functionalities. Microarray data and qRT-PCR analysis indicated that most PtALDHs had distinct tissue-specific expression patterns. The specificity of cis-acting elements in the promoter regions of the PtALDHs and the divergence of expression patterns between nine paralogous PtALDH gene pairs suggested that gene duplications may have freed the duplicate genes from the functional constraints. The expression levels of some ALDHs were up- or down-regulated by various abiotic stresses, implying that the products of these genes may be involved in the adaptation of Populus to abiotic stresses. Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus ALDH gene superfamily and provide insights into the function and evolution of ALDH gene families in vascular plants.

  11. Parallel functional changes in independent testis-specific duplicates of Aldehyde dehydrogenase in Drosophila.

    PubMed

    Chakraborty, Mahul; Fry, James D

    2015-04-01

    A large proportion of duplicates, originating from ubiquitously expressed genes, acquire testis-biased expression. Identifying the underlying cause of this observation requires determining whether the duplicates have altered functions relative to the parental genes. Typically, statistical methods are used to test for positive selection, signature of which in protein sequence of duplicates implies functional divergence. When assumptions are violated, however, such tests can lead to false inference of positive selection. More convincing evidence for naturally selected functional changes would be the occurrence of structural changes with similar functional consequences in independent duplicates of the same gene. We investigated two testis-specific duplicates of the broadly expressed enzyme gene Aldehyde dehydrogenase (Aldh) that arose in different Drosophila lineages. The duplicates show a typical pattern of accelerated amino acid substitutions relative to their broadly expressed paralogs, with statistical evidence for positive selection in both cases. Importantly, in both duplicates, width of the entrance to the substrate binding site, known a priori to influence substrate specificity, and otherwise conserved throughout the genus Drosophila, has been reduced, resulting in narrowing of the entrance. Protein structure modeling suggests that the reduction of the size of the enzyme's substrate entry channel, which is likely to shift substrate specificity toward smaller aldehydes, is accounted for by the positively selected parallel substitutions in one duplicate but not the other. Evolution of the testis-specific duplicates was accompanied by reduction in expression of the ancestral Aldh in males, supporting the hypothesis that the duplicates may have helped resolve intralocus sexual conflict over Aldh function.

  12. Effects of Alda-1, an Aldehyde Dehydrogenase-2 Agonist, on Hypoglycemic Neuronal Death

    PubMed Central

    Ikeda, Tetsuhiko; Takahashi, Tetsuya; Tsujita, Mika; Kanazawa, Masato; Toriyabe, Masafumi; Koyama, Misaki; Itoh, Kosuke; Nakada, Tsutomu; Nishizawa, Masatoyo; Shimohata, Takayoshi

    2015-01-01

    Hypoglycemic encephalopathy (HE) is caused by a lack of glucose availability to neuronal cells, and no neuroprotective drugs have been developed as yet. Studies on the pathogenesis of HE and the development of new neuroprotective drugs have been conducted using animal models such as the hypoglycemic coma model and non-coma hypoglycemia model. However, both models have inherent problems, and establishment of animal models that mimic clinical situations is desirable. In this study, we first developed a short-term hypoglycemic coma model in which rats could be maintained in an isoelectric electroencephalogram (EEG) state for 2 min and subsequent hyperglycemia without requiring anti-seizure drugs and an artificial ventilation. This condition caused the production of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic aldehyde, in neurons of the hippocampus and cerebral cortex, and a marked increase in neuronal death as evaluated by Fluoro-Jade B (FJB) staining. We also investigated whether N-(1,3-benzodioxole-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1), a small-molecule agonist of aldehyde dehydrogenase-2, could attenuate 4-HNE levels and reduce hypoglycemic neuronal death. After confirming that EEG recordings remained isoelectric for 2 min, Alda-1 (8.5 mg/kg) or vehicle (dimethyl sulfoxide; DMSO) was administered intravenously with glucose to maintain a blood glucose level of 250 to 270 mg/dL. Fewer 4-HNE and FJB-positive cells were observed in the cerebral cortex of Alda-1-treated rats than in DMSO-treated rats 24 h after glucose administration (P = 0.002 and P = 0.020). Thus, activation of the ALDH2 pathway could be a molecular target for HE treatment, and Alda-1 is a potentially neuroprotective agent that exerts a beneficial effect on neurons when intravenously administered simultaneously with glucose. PMID:26083658

  13. Potent inhibition of aldehyde dehydrogenase-2 by diphenyleneiodonium: focus on nitroglycerin bioactivation.

    PubMed

    Neubauer, Regina; Neubauer, Andrea; Wölkart, Gerald; Schwarzenegger, Christine; Lang, Barbara; Schmidt, Kurt; Russwurm, Michael; Koesling, Doris; Gorren, Antonius C F; Schrammel, Astrid; Mayer, Bernd

    2013-09-01

    Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.

  14. Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes.

    PubMed Central

    Klibanov, A M; Giannousis, P P

    1982-01-01

    The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated. All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart. Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well. All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde. The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography. PMID:7048306

  15. Aldehyde Dehydrogenase 1B1: Molecular Cloning and Characterization of a Novel Mitochondrial Acetaldehyde-Metabolizing Enzyme

    PubMed Central

    Stagos, Dimitrios; Chen, Ying; Brocker, Chad; Donald, Elizabeth; Jackson, Brian C.; Orlicky, David J.; Thompson, David C.

    2010-01-01

    Ethanol-induced damage is largely attributed to its toxic metabolite, acetaldehyde. Clearance of acetaldehyde is achieved by its oxidation, primarily catalyzed by the mitochondrial class II aldehyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial aldehyde dehydrogenase (ALDH) that shares 75% peptide sequence homology with ALDH2. Recent population studies in whites suggest a role for ALDH1B1 in ethanol metabolism. However, to date, no formal documentation of the biochemical properties of ALDH1B1 has been forthcoming. In this current study, we cloned and expressed human recombinant ALDH1B1 in Sf9 insect cells. The resultant enzyme was purified by affinity chromatography to homogeneity. The kinetic properties of purified human ALDH1B1 were assessed using a wide range of aldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD+ as the cofactor and was catalytically active toward short- and medium-chain aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Most importantly, human ALDH1B1 exhibited an apparent Km of 55 μM for acetaldehyde, making it the second low Km ALDH for metabolism of this substrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature also shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and humans was examined by quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis. The highest expression occurred in the liver, followed by the intestinal tract, implying a potential physiological role for ALDH1B1 in these tissues. The current study is the first report on the expression, purification, and biochemical characterization of human ALDH1B1 protein. PMID:20616185

  16. Cloning and sequence analysis of the safflower betaine aldehyde dehydrogenase gene.

    PubMed

    Wang, Y B; Guan, L L; Xu, Y W; Shen, H; Wu, W

    2014-01-01

    In response to salinity or drought stress, many plants accumulate glycine betaine, which is a regulator of osmosis. In plants, the last step in betaine synthesis is catalyzed by betaine aldehyde dehydrogenase (BADH), a nuclear-encoded chloroplastic enzyme. Based on the conserved oligo amino acid residues of the published BADH genes from other higher plant species, a cDNA sequence, designated CtBADH, was isolated from safflower (Carthamus tinctorius L.) using a polymerase chain reaction approach. The clones were 1.7 kb on average, and contained an open reading frame predicting a polypeptide of 503 amino acids with 84.5% identity to that of Helianthus annuus. The deduced amino acid sequence showed a decapeptide, Val-Thr-Leu-Geu-Leu-Gly-Gly-Lys-Ser-Pro and Cys, which is essential for proper functioning of BADH. Phylogenetic analysis indicated that CtBADH grouped with other dicotyledonous plant BADH genes, and subgrouped in the composite family. Prediction of secondary structure and subcellular localization suggested that the protein encoded by CtBADH contains 33 coils, 15 alpha helixes, and 21 beta strands, and most likely targets the chloroplast or mitochondria.

  17. Aldehyde dehydrogenase 1a3 defines a subset of failing pancreatic β cells in diabetic mice.

    PubMed

    Kim-Muller, Ja Young; Fan, Jason; Kim, Young Jung R; Lee, Seung-Ah; Ishida, Emi; Blaner, William S; Accili, Domenico

    2016-01-01

    Insulin-producing β cells become dedifferentiated during diabetes progression. An impaired ability to select substrates for oxidative phosphorylation, or metabolic inflexibility, initiates progression from β-cell dysfunction to β-cell dedifferentiation. The identification of pathways involved in dedifferentiation may provide clues to its reversal. Here we isolate and functionally characterize failing β cells from various experimental models of diabetes and report a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (ALDH(+)) as β cells become dedifferentiated. Flow-sorted ALDH(+) islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrates that ALDH(+) cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of β-cell failure. PMID:27572106

  18. High current density PQQ-dependent alcohol and aldehyde dehydrogenase bioanodes.

    PubMed

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

    2015-10-15

    In this paper, we explore the bioelectrooxidation of ethanol using pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenase (ADH and AldDH) enzymes for biofuel cell applications. The bioanode architectures were designed with both direct electron transfer (DET) and mediated electron transfer (MET) mechanisms employing high surface area materials such as multi-walled carbon nanotubes (MWCNTs) and MWCNT-decorated gold nanoparticles, along with different immobilization techniques. Three different polymeric matrices were tested (tetrabutyl ammonium bromide (TBAB)-modified Nafion; octyl-modified linear polyethyleneimine (C8-LPEI); and cellulose) in the DET studies. The modified Nafion membrane provided the best electrical communication between enzymes and the electrode surface, with catalytic currents as high as 16.8 ± 2.1 µA cm(-2). Then, a series of ferrocene redox polymers were evaluated for MET. The redox polymer 1,1'-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI) provided the best electrochemical response. Using this polymer, the electrochemical assays conducted in the presence of MWCNTs and MWCNTs-Au indicated a Jmax of 781 ± 59 µA cm(-2) and 925 ± 68 µA cm(-2), respectively. Overall, from the results obtained here, DET using the PQQ-dependent ADH and AldDH still lacks high current density, while the bioanodes that operate via MET employing ferrocene-modified LPEI redox polymers show efficient energy conversion capability in ethanol/air biofuel cells.

  19. Aldehyde dehydrogenase variation enhances effect of pesticides associated with Parkinson disease

    PubMed Central

    Fitzmaurice, Arthur G.; Rhodes, Shannon L.; Cockburn, Myles; Ritz, Beate

    2014-01-01

    Objective: The objective of this study was to determine whether environmental and genetic alterations of neuronal aldehyde dehydrogenase (ALDH) enzymes were associated with increased Parkinson disease (PD) risk in an epidemiologic study. Methods: A novel ex vivo assay was developed to identify pesticides that can inhibit neuronal ALDH activity. These were investigated for PD associations in a population-based case-control study, the Parkinson's Environment & Genes (PEG) Study. Common variants in the mitochondrial ALDH2 gene were genotyped to assess effect measure modification (statistical interaction) of the pesticide effects by genetic variation. Results: All of the metal-coordinating dithiocarbamates tested (e.g., maneb, ziram), 2 imidazoles (benomyl, triflumizole), 2 dicarboxymides (captan, folpet), and 1 organochlorine (dieldrin) inhibited ALDH activity, potentially via metabolic byproducts (e.g., carbon disulfide, thiophosgene). Fifteen screened pesticides did not inhibit ALDH. Exposures to ALDH-inhibiting pesticides were associated with 2- to 6-fold increases in PD risk; genetic variation in ALDH2 exacerbated PD risk in subjects exposed to ALDH-inhibiting pesticides. Conclusion: ALDH inhibition appears to be an important mechanism through which environmental toxicants contribute to PD pathogenesis, especially in genetically vulnerable individuals, suggesting several potential interventions to reduce PD occurrence or slow or reverse its progression. PMID:24491970

  20. Aldehyde dehydrogenase 2 is associated with cognitive functions in patients with Parkinson’s disease

    PubMed Central

    Yu, Rwei-Ling; Tan, Chun-Hsiang; Lu, Ying-Che; Wu, Ruey-Meei

    2016-01-01

    Neurotransmitter degradation has been proposed to cause the accumulation of neurotoxic metabolites. The metabolism of these metabolites involves aldehyde dehydrogenase 2 (ALDH2). The Asian-specific single nucleotide polymorphism rs671 causes reduced enzyme activity. This study aims to explore whether Parkinson’s disease (PD) patients with reduced ALDH2 activity owing to the rs671 polymorphism are at risk for neuropsychological impairments. A total of 139 PD patients were recruited. Each participant was assessed for medical characteristics and their ALDH2 genotype. The Mini-Mental State Examination (MMSE), the Clinical Dementia Rating Scale and the Frontal Behavioral Inventory were used to measure neuropsychological functions. We found that the MMSE scores were significantly lower in patients with inactive ALDH2 (U = 1873.5, p = 0.02). The presence of cognitive impairments was significantly more frequent in the inactive ALDH2 group (46.0%) than in the active ALDH2 group (26.3%) (χ2 = 5.886, p = 0.01). The inactive group showed significant deterioration in hobbies and exhibited more severe “disorganization” and “hyper-sexuality” behaviours. The additive effects of the allele on the development of cognitive impairments in PD patients may be an important finding that provides further insight into the pathogenic mechanism of cognitive dysfunction in PD. PMID:27453488

  1. Aldehyde Dehydrogenase Activity Identifies a Population of Human Skeletal Muscle Cells With High Myogenic Capacities

    PubMed Central

    Vauchez, Karine; Marolleau, Jean-Pierre; Schmid, Michel; Khattar, Patricia; Chapel, Alain; Catelain, Cyril; Lecourt, Séverine; Larghéro, Jérôme; Fiszman, Marc; Vilquin, Jean-Thomas

    2009-01-01

    Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM), umbilical cord blood (UCB), and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study, we have identified ALDH+ cells within human skeletal muscles, and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH, Aldefluor, identified brightly stained (ALDHbr) cells with low side scatter (SSClo), in enzymatically dissociated muscle biopsies, thereafter abbreviated as SMALD+ (for skeletal muscle ALDH+) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD+/CD34− and SMALD+/CD34+ cells. These sub-populations did not initially express endothelial (CD31), hematopoietic (CD45), and myogenic (CD56) markers. Upon sorting, however, whereas SMALD+/CD34+ cells developed in vitro as a heterogeneous population of CD56− cells able to differentiate in adipoblasts, the SMALD+/CD34− fraction developed in vitro as a highly enriched population of CD56+ myoblasts able to form myotubes. Moreover, only the SMALD+/CD34− population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. PMID:19738599

  2. NOTCH-induced aldehyde dehydrogenase 1A1 deacetylation promotes breast cancer stem cells

    PubMed Central

    Zhao, Di; Mo, Yan; Li, Meng-Tian; Zou, Shao-Wu; Cheng, Zhou-Li; Sun, Yi-Ping; Xiong, Yue; Guan, Kun-Liang; Lei, Qun-Ying

    2014-01-01

    High aldehyde dehydrogenase (ALDH) activity is a marker commonly used to isolate stem cells, particularly breast cancer stem cells (CSCs). Here, we determined that ALDH1A1 activity is inhibited by acetylation of lysine 353 (K353) and that acetyltransferase P300/CBP–associated factor (PCAF) and deacetylase sirtuin 2 (SIRT2) are responsible for regulating the acetylation state of ALDH1A1 K353. Evaluation of breast carcinoma tissues from patients revealed that cells with high ALDH1 activity have low ALDH1A1 acetylation and are capable of self-renewal. Acetylation of ALDH1A1 inhibited both the stem cell population and self-renewal properties in breast cancer. Moreover, NOTCH signaling activated ALDH1A1 through the induction of SIRT2, leading to ALDH1A1 deacetylation and enzymatic activation to promote breast CSCs. In breast cancer xenograft models, replacement of endogenous ALDH1A1 with an acetylation mimetic mutant inhibited tumorigenesis and tumor growth. Together, the results from our study reveal a function and mechanism of ALDH1A1 acetylation in regulating breast CSCs. PMID:25384215

  3. Aldehyde dehydrogenase 1a3 defines a subset of failing pancreatic β cells in diabetic mice

    PubMed Central

    Kim-Muller, Ja Young; Fan, Jason; Kim, Young Jung R.; Lee, Seung-Ah; Ishida, Emi; Blaner, William S.; Accili, Domenico

    2016-01-01

    Insulin-producing β cells become dedifferentiated during diabetes progression. An impaired ability to select substrates for oxidative phosphorylation, or metabolic inflexibility, initiates progression from β-cell dysfunction to β-cell dedifferentiation. The identification of pathways involved in dedifferentiation may provide clues to its reversal. Here we isolate and functionally characterize failing β cells from various experimental models of diabetes and report a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (ALDH+) as β cells become dedifferentiated. Flow-sorted ALDH+ islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrates that ALDH+ cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of β-cell failure. PMID:27572106

  4. Roles of histamine on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma cell line.

    PubMed

    Wang, Yi; Jiang, Yang; Ikeda, Jun-Ichiro; Tian, Tian; Sato, Atsushi; Ohtsu, Hiroshi; Morii, Eiichi

    2014-10-01

    Cancer-initiating cells (CICs) are a limited number of cells that are essential for maintenance, recurrence, and metastasis of tumors. Aldehyde dehydrogenase 1 (ALDH1) has been recognized as a marker of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and that ALDH1 high population was more tumorigenic, invasive, and resistant to apoptosis than ALDH1 low population. Histamine plays a critical role in cancer cell proliferation, migration, and invasion. Here, we examined the effect of histamine on ALDH1 expression in endometrioid adenocarcinoma cell line. The addition of histamine increased ALDH1 high population, which was consistent with the result that histamine enhanced the invasive ability and the resistance to anticancer drug. Among 4 types of histamine receptors, histamine H1 and H2 receptor (H1R and H2R) were expressed in endometrioid adenocarcinoma cell line. The addition of H1R agonist but not H2R agonist increased ALDH1. The antagonist H1R but not H2R inhibited the effect of histamine on ALDH1 expression. These results indicated that histamine increased the expression of ALDH1 via H1R but not H2R. These findings may provide the evidence for exploring a new strategy to suppress CICs by inhibiting ALDH1 expression with histamine.

  5. Cloning and heterologous expression of two aryl-aldehyde dehydrogenases from the white-rot basidiomycete Phanerochaete chrysosporium

    SciTech Connect

    Nakamura, Tomofumi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-04-09

    We identified two aryl-aldehyde dehydrogenase proteins (PcALDH1 and PcALDH2) from the white-rot basidiomycete Phanerochaete chrysosporium. Both PcALDHs were translationally up-regulated in response to exogenous addition of vanillin, one of the key aromatic compounds in the pathway of lignin degradation by basidiomycetes. To clarify the catalytic functions of PcALDHs, we isolated full-length cDNAs encoding these proteins and heterologously expressed the recombinant enzymes using a pET/Escherichia coli system. The open reading frames of both PcALDH1 and PcALDH2 consisted of 1503 nucleotides. The deduced amino acid sequences of both proteins showed high homologies with aryl-aldehyde dehydrogenases from other organisms and contained ten conserved domains of ALDHs. Moreover, a novel glycine-rich motif 'GxGxxxG' was located at the NAD{sup +}-binding site. The recombinant PcALDHs catalyzed dehydrogenation reactions of several aryl-aldehyde compounds, including vanillin, to their corresponding aromatic acids. These results strongly suggested that PcALDHs metabolize aryl-aldehyde compounds generated during fungal degradation of lignin and various aromatic xenobiotics.

  6. Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans.

    PubMed

    Sobreira, Tiago J P; Marlétaz, Ferdinand; Simões-Costa, Marcos; Schechtman, Deborah; Pereira, Alexandre C; Brunet, Frédéric; Sweeney, Sarah; Pani, Ariel; Aronowicz, Jochanan; Lowe, Christopher J; Davidson, Bradley; Laudet, Vincent; Bronner, Marianne; de Oliveira, Paulo S L; Schubert, Michael; Xavier-Neto, José

    2011-01-01

    Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

  7. Activator Protein-1 Regulation of Murine Aldehyde Dehydrogenase 1a1

    PubMed Central

    Makia, N. L.; Amunom, I.; Falkner, K. C.; Conklin, D. J.; Surapureddi, S.; Goldstein, J. A.

    2012-01-01

    Previously we demonstrated that aldehyde dehydrogenase (ALDH) 1a1 is the major ALDH expressed in mouse liver and is an effective catalyst in metabolism of lipid aldehydes. Quantitative real-time polymerase chain reaction analysis revealed a ≈2.5- to 3-fold induction of the hepatic ALDH1A1 mRNA in mice administered either acrolein (5 mg/kg acrolein p.o.) or butylated hydroxylanisole (BHA) (0.45% in the diet) and of cytosolic NAD+-dependent ALDH activity. We observed ≈2-fold increases in ALDH1A1 mRNA levels in both Nrf2(+/+) and Nrf2(−/−) mice treated with BHA compared with controls, suggesting that BHA-induced expression is independent of nuclear factor E2-related factor 2 (Nrf2). The levels of activator protein-1 (AP-1) mRNA and protein, as well as the amount of phosphorylated c-Jun were significantly increased in mouse liver or Hepa1c1c7 cells treated with either BHA or acrolein. With use of luciferase reporters containing the 5′-flanking sequence of Aldh1a1 (−1963/+27), overexpression of c-Jun resulted in an ≈4-fold induction in luciferase activity, suggesting that c-Jun transactivates the Aldh1a1 promoter as a homodimer and not as a c-Jun/c-Fos heterodimer. Promoter deletion and mutagenesis analyses demonstrated that the AP-1 site at position −758 and possibly −1069 relative to the transcription start site was responsible for c-Jun-mediated transactivation. Electrophoretic mobility shift assay analysis with antibodies against c-Jun and c-Fos showed that c-Jun binds to the proximal AP-1 site at position −758 but not at −1069. Recruitment of c-Jun to this proximal AP-1 site by BHA was confirmed by chromatin immunoprecipitation analysis, indicating that recruitment of c-Jun to the mouse Aldh1a1 gene promoter results in increased transcription. This mode of regulation of an ALDH has not been described before. PMID:22740640

  8. Ethanol oxidation and the inhibition by drugs in human liver, stomach and small intestine: Quantitative assessment with numerical organ modeling of alcohol dehydrogenase isozymes.

    PubMed

    Chi, Yu-Chou; Lee, Shou-Lun; Lai, Ching-Long; Lee, Yung-Pin; Lee, Shiao-Pieng; Chiang, Chien-Ping; Yin, Shih-Jiun

    2016-10-25

    Alcohol dehydrogenase (ADH) is the principal enzyme responsible for metabolism of ethanol. Human ADH constitutes a complex isozyme family with striking variations in kinetic function and tissue distribution. Liver and gastrointestinal tract are the major sites for first-pass metabolism (FPM). Their relative contributions to alcohol FPM and degrees of the inhibitions by aspirin and its metabolite salicylate, acetaminophen and cimetidine remain controversial. To address this issue, mathematical organ modeling of ethanol-oxidizing activities in target tissues and that of the ethanol-drug interactions were constructed by linear combination of the corresponding numerical rate equations of tissue constituent ADH isozymes with the documented isozyme protein contents, kinetic parameters for ethanol oxidation and the drug inhibitions of ADH isozymes/allozymes that were determined in 0.1 M sodium phosphate at pH 7.5 and 25 °C containing 0.5 mM NAD(+). The organ simulations reveal that the ADH activities in mucosae of the stomach, duodenum and jejunum with ADH1C*1/*1 genotype are less than 1%, respectively, that of the ADH1B*1/*1-ADH1C*1/*1 liver at 1-200 mM ethanol, indicating that liver is major site of the FPM. The apparent hepatic KM and Vmax for ethanol oxidation are simulated to be 0.093 ± 0.019 mM and 4.0 ± 0.1 mmol/min, respectively. At 95% clearance in liver, the logarithmic average sinusoidal ethanol concentration is determined to be 0.80 mM in accordance with the flow-limited gradient perfusion model. The organ simulations indicate that higher therapeutic acetaminophen (0.5 mM) inhibits 16% of ADH1B*1/*1 hepatic ADH activity at 2-20 mM ethanol and that therapeutic salicylate (1.5 mM) inhibits 30-31% of the ADH1B*2/*2 activity, suggesting potential significant inhibitions of ethanol FPM in these allelotypes. The result provides systematic evaluations and predictions by computer simulation on potential ethanol FPM in target tissues and hepatic

  9. Ethanol oxidation and the inhibition by drugs in human liver, stomach and small intestine: Quantitative assessment with numerical organ modeling of alcohol dehydrogenase isozymes.

    PubMed

    Chi, Yu-Chou; Lee, Shou-Lun; Lai, Ching-Long; Lee, Yung-Pin; Lee, Shiao-Pieng; Chiang, Chien-Ping; Yin, Shih-Jiun

    2016-10-25

    Alcohol dehydrogenase (ADH) is the principal enzyme responsible for metabolism of ethanol. Human ADH constitutes a complex isozyme family with striking variations in kinetic function and tissue distribution. Liver and gastrointestinal tract are the major sites for first-pass metabolism (FPM). Their relative contributions to alcohol FPM and degrees of the inhibitions by aspirin and its metabolite salicylate, acetaminophen and cimetidine remain controversial. To address this issue, mathematical organ modeling of ethanol-oxidizing activities in target tissues and that of the ethanol-drug interactions were constructed by linear combination of the corresponding numerical rate equations of tissue constituent ADH isozymes with the documented isozyme protein contents, kinetic parameters for ethanol oxidation and the drug inhibitions of ADH isozymes/allozymes that were determined in 0.1 M sodium phosphate at pH 7.5 and 25 °C containing 0.5 mM NAD(+). The organ simulations reveal that the ADH activities in mucosae of the stomach, duodenum and jejunum with ADH1C*1/*1 genotype are less than 1%, respectively, that of the ADH1B*1/*1-ADH1C*1/*1 liver at 1-200 mM ethanol, indicating that liver is major site of the FPM. The apparent hepatic KM and Vmax for ethanol oxidation are simulated to be 0.093 ± 0.019 mM and 4.0 ± 0.1 mmol/min, respectively. At 95% clearance in liver, the logarithmic average sinusoidal ethanol concentration is determined to be 0.80 mM in accordance with the flow-limited gradient perfusion model. The organ simulations indicate that higher therapeutic acetaminophen (0.5 mM) inhibits 16% of ADH1B*1/*1 hepatic ADH activity at 2-20 mM ethanol and that therapeutic salicylate (1.5 mM) inhibits 30-31% of the ADH1B*2/*2 activity, suggesting potential significant inhibitions of ethanol FPM in these allelotypes. The result provides systematic evaluations and predictions by computer simulation on potential ethanol FPM in target tissues and hepatic

  10. Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors

    PubMed Central

    Venton, G; Pérez-Alea, M; Baier, C; Fournet, G; Quash, G; Labiad, Y; Martin, G; Sanderson, F; Poullin, P; Suchon, P; Farnault, L; Nguyen, C; Brunet, C; Ceylan, I; Costello, R T

    2016-01-01

    The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38− leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38− subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells. PMID:27611922

  11. Aldehyde dehydrogenase-derived omega-crystallins of squid and octopus. Specialization for lens expression.

    PubMed

    Zinovieva, R D; Tomarev, S I; Piatigorsky, J

    1993-05-25

    omega-Crystallin of the octopus lens is related to aldehyde dehydrogenases (ALDH) of vertebrates (Tomarev, S. I., Zinovieva, R. D., and Piatigorsky, J. (1991) J. Biol. Chem. 266, 24226-24231) and ALDH1/eta-crystallin of elephant shrews (Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269). Only very low amounts of omega-crystallin are present in the squid lens. Here, we have cloned omega-crystallin cDNAs of the octopus (Octopus dofleini) and squid (Ommastrephes sloani pacificus) lenses. The deduced amino acid sequences of omega-crystallin from these species are 78% identical to each other, 56-58% identical to cytoplasmic ALDH1 and mitochondrial ALDH2 of vertebrates (which are 66-68% identical to each other), and 40% identical to Escherichia coli and spinach ALDHs. These data are consistent with the idea that the ALDH1/ALDH2 gene duplication in vertebrates occurred after divergence of cephalopods from the line giving rise to vertebrates, but before the separation of squid and octopus. Southern blot hybridization indicated that omega-crystallin is encoded by few genes (possibly just one) in octopus and squid. Northern blot hybridization revealed two bands (2.7 and 9.0 kilobases) of omega-crystallin RNA in the octopus lens and one band (4.2 kilobases) in the squid lens; omega-crystallin RNAs were undetectable in numerous non-lens tissues of octopus and squid, suggesting lens-specific expression of this gene(s). Finally, extracts of the octopus lens had no detectable ALDH activity using different substrates, consistent with omega-crystallin having no enzymatic activity. Taken together, our results suggest that omega-crystallin evolved by duplication of an ancestral gene encoding ALDH and subsequently specialized for refraction in the transparent lens while losing ALDH activity and expression in other tissues. PMID:7684383

  12. Aldehyde dehydrogenase 1A1 circumscribes high invasive glioma cells and predicts poor prognosis

    PubMed Central

    Xu, Sen-Lin; Liu, Sha; Cui, Wei; Shi, Yu; Liu, Qin; Duan, Jiang-Jie; Yu, Shi-Cang; Zhang, Xia; Cui, You-Hong; Kung, Hsiang-Fu; Bian, Xiu-Wu

    2015-01-01

    Glioma is the most aggressive brain tumor with high invasiveness and poor prognosis. More reliable, sensitive and practical biomarkers to reveal glioma high invasiveness remain to be explored for the guidance of therapy. We herein evaluated the diagnostic and prognostic value of aldehyde dehydrogenase 1A1 (ALDH1A1) in the glioma specimens from 237 patients, and found that ADLH1A1 was frequently overexpressed in the high-grade glioma (WHO grade III-IV) as compared to the low-grade glioma (WHO grade I-II) patients. The tumor cells with ALDH1A1 expression were more abundant in the region between tumor and the borderline of adjacent tissue as compared to the central part of the tumor. ALDH1A1 overexpression was associated with poor differentiation and dismal prognosis. Notably, the overall and disease-free survivals of the patients who had ALDH1A1+ tumor cells sparsely located in the adjacent tissue were much worse. Furthermore, ALDH1A1 expression was correlated with the “classical-like” (CL) subtype as we examined GBM specimens from 72 patients. Multivariate Cox regression analysis revealed that ALDH1A1 was an independent marker for glioma patients’ outcome. Mechanistically, both in vitro and in vivo studies revealed that ALDH1A1+ cells isolated from either a glioblastoma cell line U251 or primary glioblastoma cells displayed significant invasiveness, clonogenicity, and proliferation as compared to ALDH1A1- cells, due to increased levels of mRNA and protein for matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9). These results indicate that ALDH1A1+ cells contribute to the progression of glioma including invasion, proliferation and poor prognosis, and suggest that targeting ALDH1A1 may have important implications for the treatment of highly invasive glioma. PMID:26101711

  13. Aldehyde Dehydrogenase 2 Knockout Accentuates Ethanol-Induced Cardiac Depression: Role of Protein Phosphatases

    PubMed Central

    Ma, Heng; Byra, Emily A.; Yu, Lu; Hu, Nan; Kitagawa, Kyoko; Nakayama, Keiichi I.; Kawamoto, Toshihiro; Ren, Jun

    2010-01-01

    Alcohol consumption leads to myocardial contractile dysfunction possibly due to the toxicity of ethanol and its major metabolite acetaldehyde. This study was designed to examine the influence of mitochondrial aldehyde dehydrogenase-2 (ALDH2) knockout (KO) on acute ethanol exposure-induced cardiomyocyte dysfunction. Wild-type (WT) and ALDH2 KO mice were subjected to acute ethanol (3 g/kg, i.p.) challenge and cardiomyocyte contractile function was assessed 24 hrs later using an IonOptix® edge-detection system. Western blot analysis was performed to evaluate ALDH2, protein phosphatase 2A (PP2A), phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β). ALDH2 KO accentuated ethanol-induced elevation in cardiac acetaldehyde levels. Ethanol exposure depressed cardiomyocyte contractile function including decreased cell shortening amplitude and maximal velocity of shortening/relengthening as well as prolonged relengthening duration and a greater decline in peak shortening in response to increasing stimulus frequency, the effect of which was significantly exaggerated by ALDH2 KO. ALDH2 KO also unmasked an ethanol-induced prolongation of shortening duration. In addition, short-term in vitro incubation of ethanol-induced cardiomyocyte mechanical defects were exacerbated by the ALDH inhibitor cyanamide. Ethanol treatment dampened phosphorylation of Akt and GSK-3β associated with up-regulated PP2A, which was accentuated by ALDH2 KO. ALDH2 KO aggravated ethanol-induced decrease in mitochondrial membrane potential. These results suggested that ALDH2 deficiency led to worsened ethanol-induced cardiomyocyte function, possibly due to upregulated expression of protein phosphatase, depressed Akt activation and subsequently impaired mitochondrial function. These findings depict a critical role of ALDH2 in the pathogenesis of alcoholic cardiomyopathy. PMID:20362583

  14. Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels

    PubMed Central

    Neubauer, Regina; Wölkart, Gerald; Opelt, Marissa; Schwarzenegger, Christine; Hofinger, Marielies; Neubauer, Andrea; Kollau, Alexander; Schmidt, Kurt; Schrammel, Astrid; Mayer, Bernd

    2015-01-01

    The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2. PMID:25576686

  15. Aldehyde Dehydrogenase-2 Deficiency Aggravates Cardiac Dysfunction Elicited by Endoplasmic Reticulum Stress Induction

    PubMed Central

    Liao, Jianquan; Sun, Aijun; Xie, Yeqing; Isse, Toyoshi; Kawamoto, Toshihiro; Zou, Yunzeng; Ge, Junbo

    2012-01-01

    Mitochondrial aldehyde dehydrogenase-2 (ALDH2) has been characterized as an important mediator of endogenous cytoprotection in the heart. This study was designed to examine the role of ALDH2 knockout (KO) in the regulation of cardiac function after endoplasmic reticulum (ER) stress. Wild-type (WT) and ALDH2 KO mice were subjected to a tunicamycin challenge, and the echocardiographic property was examined. Protein levels of six items—78 kDa glucose-regulated protein (GRP78), phosphorylation of eukaryotic initiation factor 2 subunit α (p-eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP), phosphorylation of Akt, p47phox nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 4-hydroxynonenal—were determined by using Western blot analysis. Cytotoxicity and apoptosis were estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay and caspase-3 activity, respectively. ALDH2 deficiency exacerbated cardiac contractile dysfunction and promoted ER stress after ER stress induction, manifested by the changes of ejection fraction and fractional shortening. In vitro study revealed that tunicamycin significantly upregulated the levels of GRP78, p-eIF2α, CHOP, p47phox NADPH oxidase and 4-hydroxynonenal, which was exacerbated by ALDH2 knockdown and abolished by ALDH2 overexpression, respectively. Overexpression of ALDH2 abrogated tunicamycin-induced dephosphorylation Akt. Inhibition of phosphatidylinositol 3-kinase using LY294002 did not affect ALDH2-conferred protection against ER stress, although LY294002 reversed the antiapoptotic action of ALDH2 associated with p47phox NADPH oxidase. These results suggest a pivotal role of ALDH2 in the regulation of ER stress and ER stress–induced apoptosis. The protective role of ALDH2 against ER stress–induced cell death was probably mediated by Akt via a p47phox NADPH oxidase-dependent manner. These findings indicate the critical role of ALDH2 in the pathogenesis of ER stress

  16. Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus.

    PubMed

    Lee, Jin Ju; Kim, Jae Hong; Kim, Dae Geun; Kim, Dong Hyeok; Simborio, Hannah Leah; Min, Won Gi; Rhee, Man Hee; Lim, Jong Hwan; Chang, Hong Hee; Kim, Suk

    2014-01-10

    The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.

  17. Mining distinct aldehyde dehydrogenase 1 (ALDH1) isoenzymes in gastric cancer

    PubMed Central

    Li, Guan-Wu; Huang, Yi-Teng; Wu, Hua-Tao

    2016-01-01

    Aldehyde dehydrogenase 1 (ALDH1) consists of a family of intracellular enzymes, highly expressed in stem cells populations of leukemia and some solid tumors. Up to now, 6 isoforms of ALDH1 have been reported. However, the expression patterns and the identity of ALDH1 isoenzymes contributing to ALDH1 activity, as well as the prognostic values of ALDH1 isoenzymes in cancers all remain to be elucidated. Here, we studied the expressions of ALDH1 transcripts in gastric cancer (GC) compared with the normal controls using the ONCOMINE database. Through the Kaplan-Meier plotter database, which contains updated gene expression data and survival information of 876 GC patients, we also investigated the prognostic values of ALDH1 isoenzymes in GC patients. It was found that when compared with normal tissues, ALDH1A1 mRNA expression was downregulated, whereas ALDH1A3 and ALDH1B1 were upregulated in GC patients. In survival analyses, high ALDH1A1 and ALDH1B1 expressions were associated with better overall survival (OS) in all GC patients. In addition, high transcription activity of ALDH1A1 predicted better OS in gastric intestinal type adenocarcinoma, but not in diffuse gastric adenocarcinoma. GC patients with high mRNA level of ALDH1B1 showed better OS in gastric intestinal type, and worse OS in diffuse type. Oppositely, high transcription activities of ALDH1A2, ALDH1A3 and ALDH1L1 predicted worsen overall survival in GC patients, suggesting that these isoenzymes might be responsible mainly for the ALDH1 activities in GC. These data provides ALDH1A2, ALDH1A3 and ALDH1L1 as excellent potential targets for individualized treatment of GC patients. PMID:27015121

  18. Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels.

    PubMed

    Neubauer, Regina; Wölkart, Gerald; Opelt, Marissa; Schwarzenegger, Christine; Hofinger, Marielies; Neubauer, Andrea; Kollau, Alexander; Schmidt, Kurt; Schrammel, Astrid; Mayer, Bernd

    2015-02-15

    The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.

  19. Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors.

    PubMed

    Venton, G; Pérez-Alea, M; Baier, C; Fournet, G; Quash, G; Labiad, Y; Martin, G; Sanderson, F; Poullin, P; Suchon, P; Farnault, L; Nguyen, C; Brunet, C; Ceylan, I; Costello, R T

    2016-01-01

    The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38- leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38- subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells. PMID:27611922

  20. Xanthine dehydrogenase and aldehyde oxidase impact plant hormone homeostasis and affect fruit size in 'Hass' avocado.

    PubMed

    Taylor, Nicky J; Cowan, A Keith

    2004-04-01

    The contribution of xanthine dehydrogenase (XDH, EC 1.1.1.204) to fruit size was investigated using the normal and small-fruit variants of Persea americana Mill. cv. 'Hass'. Inhibition of XDH by treatment of normal fruit, in the linear phase of growth (phase II), with allopurinol (Allo) arrested fruit growth. Adenine (Ade), a less effective inhibitor of this enzyme, also arrested fruit growth when applied in phase II and slowed fruit growth when applied in phase III. A time-course study on the activity of XDH in mesocarp tissue from normal and small fruit showed that maximum activity occurred late in phase II and that the peak in activity was absent in mesocarp of the small fruit. Feeding Ade to growing fruit in phase III caused a transient decline in fruit growth (measured as change in fruit length). Thereafter, growth resumed although fruit size was irreversibly affected. Treatment of fruit with Ade and Ade-containing cytokinins altered activity of another molybdenum enzyme, aldehyde oxidase (EC 1.2.3.1). Cytokinin oxidase was induced by cytokinin and auxin. Purine catabolism via hypoxanthine/xanthine was operative in normal fruit and in mesocarp from the small-fruit variant and as expected, Allo treatment caused accumulation of xanthine and adenine. In the absence of an increase in XDH during growth of the small-fruit phenotype, low levels of Ade were interpreted as resulting from respiration-enhanced adenylate depletion. Stress and/or pathogen induction of the alternative oxidase pathway is proposed as a possible cause.

  1. Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

    PubMed Central

    Tuck, Laura R.; Altenbach, Kirsten; Ang, Thiau Fu; Crawshaw, Adam D.; Campopiano, Dominic J.; Clarke, David J.; Marles-Wright, Jon

    2016-01-01

    The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD+. This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes. PMID:26899032

  2. A comparison of the primary structures of lactate dehydrogenase isozymes M4 from giant panda, red panda, black bear and dog.

    PubMed

    Liang, S P; Zhang, L X

    1987-03-01

    Lactate dehydrogenase isozymes M4 have been isolated and purified from red panda (Ailurus fulgens), black bear (Selenarctos thibetanus) and dog (Canis familiars) by affinity chromatography and compared with that from giant panda (Ailuropoda melanoleuca). Experimental results have shown that the N-termini, C-termini and the molecular weights of LDH-M subunits of red panda, black bear and dog are the same as those of the LDH-M subunit of giant panda. Analysis and comparison of HPLC peptide maps from the tryptic digests of the isozymes of red panda, black bear and dog have shown that most of their peptide fragments had the same retention time and amino acid composition as the corresponding peptide fragments from giant panda. Fragments with different retention times and/or amino acid compositions were sequenced. Careful examination of those variant amino acid residues demonstrated clearly that the primary structure of giant panda LDH-M subunit is unique and it appears that the giant panda might be classified as an independent family.

  3. Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but worsens liver inflammation and fibrosis in mice

    PubMed Central

    Kwon, Hyo-Jung; Won, Young-Suk; Park, Ogyi; Chang, Binxia; Duryee, Michael J.; Thiele, Geoffrey E.; Matsumoto, Akiko; Singh, Surendra; Abdelmegeed, Mohamed A.; Song, Byoung-Joon; Kawamoto, Toshihiro; Vasiliou, Vasilis; Thiele, Geoffrey M.; Gao, Bin

    2014-01-01

    Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40~50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. In the present study, wild-type and ALDH2−/− mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4) treatment, and liver injury was assessed. Compared with wild-type mice, ethanol-fed ALDH2−/− mice had higher levels of malondialdehyde-acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic IL-6 expression but surprisingly lower levels of steatosis and serum ALT. Higher IL-6 levels were also detected in ethanol-treated precision-cut-liver-slices from ALDH2−/− mice and in Kupffer cells isolated from ethanol-fed ALDH2−/− mice than those levels in wild-type mice. In vitro incubation with MAA enhanced the LPS-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2−/− mice than in wild-type mice. An additional deletion of hepatic STAT3 increased steatosis and hepatocellular damage in ALDH2−/− mice. Finally, ethanol-fed ALDH2−/− mice were more prone to CCl4-induced liver inflammation and fibrosis than ethanol-fed wild-type mice. Conclusions: ALDH2−/− mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis via MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that alcohol, via acetaldehyde and its associated adducts, stimulates hepatic inflammation and fibrosis independent from causing hepatocyte death, and that ALDH2-deficient individuals may be resistant to steatosis and blood ALT elevation, but are

  4. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  5. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  6. Alcohol dehydrogenases from Scheffersomyces stipitis involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

    PubMed

    Ma, Menggen; Wang, Xu; Zhang, Xiaoping; Zhao, Xianxian

    2013-09-01

    Aldehyde inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) are generated from biomass pretreatment. Scheffersomyces stipitis is able to reduce furfural and HMF to less toxic furanmethanol and furan-2,5-dimethanol; however, the enzymes involved in the reductive reaction still remain unknown. In this study, transcription responses of two known and five putative alcohol dehydrogenase genes from S. stipitis were analyzed under furfural and HMF stress conditions. All the seven alcohol dehydrogenase genes were also cloned and overexpressed for their activity analyses. Our results indicate that transcriptions of SsADH4 and SsADH6 were highly induced under furfural and HMF stress conditions, and the proteins encoded by them exhibited NADH- and/or NADPH-dependent activities for furfural and HMF reduction, respectively. For furfural reduction, NADH-dependent activity was also observed in SsAdh1p and NAD(P)H-dependent activities were also observed in SsAdh5p and SsAdh7p. For HMF reduction, NADPH-dependent activities were also observed in SsAdh5p and SsAdh7p. SsAdh4p displayed the highest NADPH-dependent specific activity and catalytic efficiency for reduction of both furfural and HMF among the seven alcohol dehydrogenases. Enzyme activities of all SsADH proteins were more stable under acidic condition. For most SsADH proteins, the optimum temperature for enzyme activities was 30 °C and more than 50 % enzyme activities remained at 60 °C. Reduction activities of formaldehyde, acetaldehyde, isovaleraldehyde, benzaldehyde, and phenylacetaldehyde were also observed in some SsADH proteins. Our results indicate that multiple alcohol dehydrogenases in S. stipitis are involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion. PMID:23912116

  7. Molecular cloning of a plant betaine-aldehyde dehydrogenase, an enzyme implicated in adaptation to salinity and drought.

    PubMed Central

    Weretilnyk, E A; Hanson, A D

    1990-01-01

    Many plants, as well as other organisms, accumulate betaine (N,N,N-trimethylglycine) as a nontoxic or protective osmolyte under saline or dry conditions. In plants, the last step in betaine synthesis is catalyzed by betaine-aldehyde dehydrogenase (BADH, EC 1.2.1.8), a nuclear-encoded chloroplastic enzyme. A cDNA clone for BADH (1812 base pairs) was selected from a lambda gt10 cDNA library derived from leaves of salt-stressed spinach (Spinacia oleracea L.). The library was screened with oligonucleotide probes corresponding to amino acid sequences of two peptides prepared from purified BADH. The authenticity of the clone was confirmed by nucleotide sequence analysis; this analysis demonstrated the presence of a 1491-base-pair open reading frame that contained sequences encoding 12 peptide fragments of BADH. The clone hybridized to a 1.9-kilobase mRNA from spinach leaves; this mRNA was more abundant in salt-stressed plants, consistent with the known salt induction of BADH activity. The amino acid sequence deduced from the BADH cDNA sequence showed substantial similarities to those for nonspecific aldehyde dehydrogenases (EC 1.2.1.3 and EC 1.2.1.5) from several sources, including absolute conservation of a decapeptide in the probable active site. Comparison of deduced and determined amino acid sequences indicated that the transit peptide may comprise only 7 or 8 residues, which is atypically short for precursors to stromal proteins. Images PMID:2320587

  8. The activity of class I, II, III and IV of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) in brain cancer.

    PubMed

    Laniewska-Dunaj, Magdalena; Jelski, Wojciech; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2013-07-01

    The brain being highly sensitive to the action of alcohol is potentially susceptible to its carcinogenic effects. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the main enzymes involved in ethanol metabolism, which leads to the generation of carcinogenic acetaldehyde. Human brain tissue contains various ADH isoenzymes and possess also ALDH activity. The purpose of this study was to compare the capacity for ethanol metabolism measured by ADH isoenzymes and ALDH activity in cancer tissues and healthy brain cells. The samples were taken from 62 brain cancer patients (36 glioblastoma, 26 meningioma). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, the fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. The total activity of ADH, and activity of class I ADH were significantly higher in cancer cells than in healthy tissues. The other tested classes of ADH and ALDH did not show statistically significant differences of activity in cancer and in normal cells. Analysis of the enzymes activity did not show significant differences depending on the location of the tumor. The differences in the activity of total alcohol dehydrogenase, and class I isoenzyme between cancer tissues and healthy brain cells might be a factor for metabolic changes and disturbances in low mature cancer cells and additionally might be a reason for higher level of acetaldehyde which can intensify the carcinogenesis.

  9. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

    PubMed

    Chiang, Chien-Ping; Jao, Shu-Wen; Lee, Shiao-Pieng; Chen, Pei-Chi; Chung, Chia-Chi; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2012-02-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained

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

    PubMed Central

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

    2016-01-01

    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. PMID:27321949

  11. Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

    PubMed Central

    Jörnvall, H; Hempel, J; Vallee, B L; Bosron, W F; Li, T K

    1984-01-01

    The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. PMID:6374651

  12. The bifunctional aldehyde-alcohol dehydrogenase controls ethanol and acetate production in Entamoeba histolytica under aerobic conditions.

    PubMed

    Pineda, Erika; Encalada, Rusely; Olivos-García, Alfonso; Néquiz, Mario; Moreno-Sánchez, Rafael; Saavedra, Emma

    2013-01-16

    By applying metabolic control analysis and inhibitor titration we determined the degree of control (flux control coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde-alcohol dehydrogenase (ADHE) over the fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The flux-control coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and -0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant flux-control on the carbon end-product formation of amoebas subjected to aerobic conditions. PMID:23201265

  13. The mechanism of discrimination between oxidized and reduced coenzyme in the aldehyde dehydrogenase domain of Aldh1l1.

    PubMed

    Tsybovsky, Yaroslav; Malakhau, Yuryi; Strickland, Kyle C; Krupenko, Sergey A

    2013-02-25

    Aldh1l1, also known as 10-formyltetrahydrofolate dehydrogenase (FDH), contains the carboxy-terminal domain (Ct-FDH), which is a structural and functional homolog of aldehyde dehydrogenases (ALDHs). This domain is capable of catalyzing the NADP(+)-dependent oxidation of short chain aldehydes to their corresponding acids, and similar to most ALDHs it has two conserved catalytic residues, Cys707 and Glu673. Previously, we demonstrated that in the Ct-FDH mechanism these residues define the conformation of the bound coenzyme and the affinity of its interaction with the protein. Specifically, the replacement of Cys707 with an alanine resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme. We suggested that this was due to the loss of a covalent bond between the cysteine and the C4N atom of nicotinamide ring of NADP(+) formed during Ct-FDH catalysis. To obtain further insight into the functional significance of the covalent bond between Cys707 and the coenzyme, and the overall role of the two catalytic residues in the coenzyme binding and positioning, we have now solved crystal structures of Ct-FDH in the complex with thio-NADP(+) and the complexes of the C707S mutant with NADP(+) and NADPH. This study has allowed us to trap the coenzyme in the contracted conformation, which provided a snapshot of the conformational processing of the coenzyme during the transition from oxidized to reduced form. Overall, the results of this study further support the previously proposed mechanism by which Cys707 helps to differentiate between the oxidized and reduced coenzyme during ALDH catalysis.

  14. Functional and expression analyses of two kinds of betaine aldehyde dehydrogenases in a glycinebetaine-hyperaccumulating graminaceous halophyte, Leymus chinensis.

    PubMed

    Mitsuya, Shiro; Tsuchiya, Asumi; Kono-Ozaki, Keiko; Fujiwara, Takashi; Takabe, Teruhiro; Takabe, Tetsuko

    2015-01-01

    Glycinebetaine (GB) is an important compatible solute for salinity tolerance in many plants. In this study, we analyzed the enzymatic activity and the expression level of betaine aldehyde dehydrogenase (BADH), an important enzyme that catalyzes the last step in the GB synthesis in Leymus chinensis, a GB-hyperaccumulating graminaceous halophyte, and compared with those of barley, a graminaceous glycophyte. We have isolated cDNAs for two BADH genes, LcBADH1 and LcBADH2. LcBADH1 has a putative peroxisomal signal peptide (PTS1) at its C-terminus, while LcBADH2 does not have any typical signal peptide. Using immunofluorescent labeling, we showed that BADH proteins were localized to the cytosol and dot-shaped organelles in the mesophyll and bundle sheath cells of L.chinensis leaves. The affinity of recombinant LcBADH2 for betaine aldehyde was comparable to other plant BADHs, whereas recombinant LcBADH1 showed extremely low affinity for betaine aldehyde, indicating that LcBADH2 plays a major role in GB synthesis in L. chinensis. In addition, the recombinant LcBADH2 protein was tolerant to NaCl whereas LcBADH1 wasn't. The kinetics, subcellular and tissue localization of BADH proteins were comparable between L. chinensis and barley. The activity and expression level of BADH proteins were higher in L. chinensis compared with barley under both normal and salinized conditions, which may be related to the significant difference in the amount of GB accumulation between two plants.

  15. The longitudinal effect of the aldehyde dehydrogenase 2*2 allele on the risk for nonalcoholic fatty liver disease

    PubMed Central

    Oniki, K; Morita, K; Watanabe, T; Kajiwara, A; Otake, K; Nakagawa, K; Sasaki, Y; Ogata, Y; Saruwatari, J

    2016-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies toxic aldehydes and has a key role in protecting the liver. An elevated gamma-glutamyl transferase (GGT) level is related to oxidative stress and nonalcoholic fatty liver disease (NAFLD). We herein investigated the association between inactive ALDH2*2 allele (rs671) and the risk of NAFLD, including the relationship to the GGT level. A retrospective follow-up study (mean 5.4±1.1 years) was conducted among 341 Japanese health screening program participants. The receiver operating characteristic curve indicated that the GGT level predicted the development of NAFLD (area under the curve: 0.65, P<0.05) with a cutoff value of 25.5 IUl−1. The longitudinal risk of NAFLD was higher in the ALDH2*2 allele carriers than in the noncarriers (odds ratio (OR): 2.30, 95% confidence interval (CI): 1.21–4.40), and the risk was further increased among the *2 allele carriers with GGT values ⩾25.5 IUl−1 (OR: 4.28, 95% CI: 1.80–10.19). On the other hand, there were no significant changes in the subjects' body weight and body mass index during observation period. The ALDH2*2 allele, in relation to the GGT level, may potentially be a novel risk factor for NAFLD. PMID:27214654

  16. The longitudinal effect of the aldehyde dehydrogenase 2*2 allele on the risk for nonalcoholic fatty liver disease.

    PubMed

    Oniki, K; Morita, K; Watanabe, T; Kajiwara, A; Otake, K; Nakagawa, K; Sasaki, Y; Ogata, Y; Saruwatari, J

    2016-05-23

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies toxic aldehydes and has a key role in protecting the liver. An elevated gamma-glutamyl transferase (GGT) level is related to oxidative stress and nonalcoholic fatty liver disease (NAFLD). We herein investigated the association between inactive ALDH2*2 allele (rs671) and the risk of NAFLD, including the relationship to the GGT level. A retrospective follow-up study (mean 5.4±1.1 years) was conducted among 341 Japanese health screening program participants. The receiver operating characteristic curve indicated that the GGT level predicted the development of NAFLD (area under the curve: 0.65, P<0.05) with a cutoff value of 25.5 IUl(-1). The longitudinal risk of NAFLD was higher in the ALDH2*2 allele carriers than in the noncarriers (odds ratio (OR): 2.30, 95% confidence interval (CI): 1.21-4.40), and the risk was further increased among the *2 allele carriers with GGT values ⩾25.5 IUl(-1) (OR: 4.28, 95% CI: 1.80-10.19). On the other hand, there were no significant changes in the subjects' body weight and body mass index during observation period. The ALDH2*2 allele, in relation to the GGT level, may potentially be a novel risk factor for NAFLD.

  17. Glu504Lys Single Nucleotide Polymorphism of Aldehyde Dehydrogenase 2 Gene and the Risk of Human Diseases

    PubMed Central

    Zhao, Yan; Wang, Chuancai

    2015-01-01

    Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial enzyme that is known for its important role in oxidation and detoxification of ethanol metabolite acetaldehyde. ALDH2 also metabolizes other reactive aldehydes such as 4-hydroxy-2-nonenal and acrolein. The Glu504Lys single nucleotide polymorphism (SNP) of ALDH2 gene, which is found in approximately 40% of the East Asian populations, causes defect in the enzyme activity of ALDH2, leading to alterations in acetaldehyde metabolism and alcohol-induced “flushing” syndrome. Evidence suggests that ALDH2 Glu504Lys SNP is a potential candidate genetic risk factor for a variety of chronic diseases such as cardiovascular disease, cancer, and late-onset Alzheimer's disease. In addition, the association between ALDH2 Glu504Lys SNP and the development of these chronic diseases appears to be affected by the interaction between the SNP and lifestyle factors such as alcohol consumption as well as by the presence of other genetic variations. PMID:26491656

  18. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

    PubMed

    Baggetto, L G; Lehninger, A L

    1987-05-29

    Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

  19. Ginsenoside Rh1 eliminates the cytoprotective phenotype of human immunodeficiency virus type 1-transduced human macrophages by inhibiting the phosphorylation of pyruvate dehydrogenase lipoamide kinase isozyme 1.

    PubMed

    Jeong, Jin-Ju; Kim, Baek; Kim, Dong-Hyun

    2013-01-01

    Red ginseng (the steamed root of Panax ginseng C.A. MEYER, Araliaceae), which contains ginsenosides as its main constituents, is frequently used to treat tumor, inflammation, diabetes, stress and acquired immunodeficiency syndrome in Asian countries. Of these ginsenosides, only protopanaxadiol compound K has been reported to abolish the cytoprotective phenotype of human immunodeficiency virus type 1 (HIV-1)-transfected human macrophages. Here, we investigated the anti-cytoprotective effect of protopanaxatriol ginsenoside Rh1 on Tat-expressing cytoprotective CHME5 cells and D3-infected human primary macrophages. Treatment with ginsenoside Rh1 in the presence of lipopolysaccharide/cycloheximide (LPS/CHX) potently abolished the cytoprotective phenotype of Tat-transduced CHME5 cells as well as D3-infected human primary macrophages. Ginsenoside Rh1 significantly inhibited LPS/CHX-induced Akt phosphorylation, as well as mammalian target of rapamycin and Bcl-2-associated death promoter activation in both cell types. Furthermore, ginsenoside Rh1 inhibited pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK-1) phosphorylation. However, ginsenoside Rh1 did not inhibit phosphoinositide 3-kinase phosphorylation. Ginsenosides Rh1 in the presence of miltefosine (5 µM) additively increased the anti-cytoprotective activity against HIV-1 Tat-expressing macrophages. On the basis of these findings, we propose that ginsenoside Rh1 could possibly eliminate HIV-1 infected macrophages by inhibiting the PDK1/Akt pathway. PMID:23811558

  20. Acyclovir-induced nephrotoxicity: the role of the acyclovir aldehyde metabolite.

    PubMed

    Gunness, Patrina; Aleksa, Katarina; Bend, John; Koren, Gideon

    2011-11-01

    For decades, acyclovir-induced nephrotoxicity was believed to be secondary to crystalluria. Clinical evidence of nephrotoxicity in the absence of crystalluria suggests that acyclovir induces direct insult to renal tubular cells. We postulated that acyclovir is metabolized by the alcohol dehydrogenase (ADH) enzyme to acyclovir aldehyde, which is metabolized by the aldehyde dehydrognase 2 (ALDH2) enzyme to 9-carboxymethoxymethylguanine (CMMG). We hypothesized that acyclovir aldehyde plays a role in acyclovir-induced nephrotoxicity. Human renal proximal tubular (HK-2) cells were used as our in vitro model. Western blot and enzymes activities assays were performed to determine whether the HK-2 cells express ADH and ALDH2 isozymes, respectively. Cytotoxicity (measured as a function of cell viability) assays were conducted to determine (1) whether the acyclovir aldehyde plays a role in acyclovir-induced nephrotoxicity and (2) whether CMMG induces cell death. A colorimetric assay was performed to determine whether acyclovir was metabolized to an aldehyde in vitro. Our results illustrated that (1) HK-2 cells express ADH and ALDH2 isozymes, (2) 4-methylpyrazole rendered significant protection against cell death, (3) CMMG does not induce cell death, and (4) acyclovir was metabolized to an aldehyde in tubular cells. These data indicate that acyclovir aldehyde is produced in HK-2 cells and that inhibition of its production by 4-methylpyrazole offers significant protection from cell death in vitro, suggesting that acyclovir aldehyde may cause the direct renal tubular insult associated with acyclovir.

  1. Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.

    PubMed

    Danielsson, O; Shafqat, J; Estonius, M; el-Ahmad, M; Jörnvall, H

    1996-11-19

    The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a

  2. Isolation of an Aldehyde Dehydrogenase Involved in the Oxidation of Fluoroacetaldehyde to Fluoroacetate in Streptomyces cattleya

    PubMed Central

    Murphy, Cormac D.; Moss, Steven J.; O'Hagan, David

    2001-01-01

    Streptomyces cattleya is unusual in that it produces fluoroacetate and 4-fluorothreonine as secondary metabolites. We now report the isolation of an NAD+-dependent fluoroacetaldehyde dehydrogenase from S. cattleya that mediates the oxidation of fluoroacetaldehyde to fluoroacetate. This is the first enzyme to be identified that is directly involved in fluorometabolite biosynthesis. Production of the enzyme begins in late exponential growth and continues into the stationary phase. Measurement of kinetic parameters shows that the enzyme has a high affinity for fluoroacetaldehyde and glycoaldehyde, but not acetaldehyde. PMID:11571203

  3. Mitochondrial Aldehyde Dehydrogenase Activation by Alda‐1 Inhibits Atherosclerosis and Attenuates Hepatic Steatosis in Apolipoprotein E‐Knockout Mice

    PubMed Central

    Stachowicz, Aneta; Olszanecki, Rafał; Suski, Maciej; Wiśniewska, Anna; Totoń‐Żurańska, Justyna; Madej, Józef; Jawień, Jacek; Białas, Magdalena; Okoń, Krzysztof; Gajda, Mariusz; Głombik, Katarzyna; Basta‐Kaim, Agnieszka; Korbut, Ryszard

    2014-01-01

    Background Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice. Methods and Results Alda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids. Conclusions Collectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation. PMID:25392542

  4. Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.).

    PubMed

    Li, Xiaoqin; Guo, Rongrong; Li, Jun; Singer, Stacy D; Zhang, Yucheng; Yin, Xiangjing; Zheng, Yi; Fan, Chonghui; Wang, Xiping

    2013-10-01

    Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance.

  5. Simultaneous involvement of a tungsten-containing aldehyde:ferredoxin oxidoreductase and a phenylacetaldehyde dehydrogenase in anaerobic phenylalanine metabolism.

    PubMed

    Debnar-Daumler, Carlotta; Seubert, Andreas; Schmitt, Georg; Heider, Johann

    2014-01-01

    Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.

  6. Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzaldehyde.

    PubMed

    Scharschmidt, M; Fisher, M A; Cleland, W W

    1984-11-01

    Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.

  7. Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat.

    PubMed

    Ding, Yu; Ma, Qing-Hu

    2004-08-01

    Malate dehydrogenase (MDH), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Cytosolic NAD-dependent MDH (cyMDH) is one class of MDH that has not been extensively characterized in plants. Here we report the cloning of a cDNA from wheat by RT-PCR and cDNA library screening, which is designated as TaMDH. Sequence analysis indicated that TaMDH exhibits a highly similarity to other plant cyMDHs. Immunological analysis confirmed that TaMDH encoded a cytosolic NAD-dependent MDH. The secondary and three-dimensional structures of TaMDH were analyzed by molecular modeling. DNA gel-blot analyses demonstrated that TaMDH gene exists as two copies in the wheat genome. RNA and protein gel-blot hybridization indicated that both TaMDH mRNA and protein were constitutively expressed in vegetative tissues of wheat, with slightly lower levels in roots than in leaves and stems. In silico analysis indicated that TaMDH was also expressed in various reproductive tissues and tissues under many different stress conditions. Kinetic analysis of bacterially expressed and purified protein confirmed that TaMDH catalyzed a reaction driven towards malate synthesis, which is consistent with other cyMDHs. Evolutionary analysis showed that this class of genes evolved from a very ancestral gene. The cyMDH represents an ancestral form of MDH, which is highly conserved in plants, animals and bacteria. This implies that cyMDHs are housekeeping genes and may have very essential functions in plant metabolism.

  8. Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme.

    PubMed

    Tsybovsky, Yaroslav; Krupenko, Sergey A

    2011-07-01

    The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.

  9. Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production

    PubMed Central

    Zarei, Adel; Trobacher, Christopher P.; Shelp, Barry J.

    2016-01-01

    Polyamines represent a potential source of 4-aminobutyrate (GABA) in plants exposed to abiotic stress. Terminal catabolism of putrescine in Arabidopsis thaliana involves amine oxidase and the production of 4-aminobutanal, which is a substrate for NAD+-dependent aminoaldehyde dehydrogenase (AMADH). Here, two AMADH homologs were chosen (AtALDH10A8 and AtALDH10A9) as candidates for encoding 4-aminobutanal dehydrogenase activity for GABA synthesis. The two genes were cloned and soluble recombinant proteins were produced in Escherichia coli. The pH optima for activity and catalytic efficiency of recombinant AtALDH10A8 with 3-aminopropanal as substrate was 10.5 and 8.5, respectively, whereas the optima for AtALDH10A9 were approximately 9.5. Maximal activity and catalytic efficiency were obtained with NAD+ and 3-aminopropanal, followed by 4-aminobutanal; negligible activity was obtained with betaine aldehyde. NAD+ reduction was accompanied by the production of GABA and β-alanine, respectively, with 4-aminobutanal and 3-aminopropanal as substrates. Transient co-expression systems using Arabidopsis cell suspension protoplasts or onion epidermal cells and several organelle markers revealed that AtALDH10A9 was peroxisomal, but AtALDH10A8 was cytosolic, although the N-terminal 140 amino acid sequence of AtALDH10A8 localized to the plastid. Root growth of single loss-of-function mutants was more sensitive to salinity than wild-type plants, and this was accompanied by reduced GABA accumulation. PMID:27725774

  10. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

  11. Relative inhibitory potency of molinate and metabolites with aldehyde dehydrogenase2: implications for the mechanism of enzyme inhibition

    PubMed Central

    Allen, Erin M.G.; Anderson, David G.R.; Florang, Virginia R.; Khanna, May; Hurley, Thomas D.; Doorn, Jonathan A.

    2010-01-01

    Molinate is a thiocarbamate herbicide used as a pre-emergent in rice patty fields. It has two predominant sulfoxidation metabolites, molinate sulfoxide and molinate sulfone. Previous work demonstrated an in vivo decrease in liver aldehyde dehydrogenase (ALDH) activity in rats treated with molinate and motor function deficits in dogs dosed chronically with this compound. ALDH is an enzyme important in the catabolism of many neurotransmitters, such as dopamine. Inhibition of this enzyme may lead to the accumulation of endogenous neurotoxic metabolites such as 3,4-dihydroxyphenylacetaldehyde (DOPAL), a dopamine metabolite, which may account for the observed neurotoxicity. In this study, the relative reactivity of molinate and both of its sulfoxidation metabolites towards ALDH were investigated, as well as the mechanism of inhibition. ALDH activity was monitored in two different model systems, human recombinant ALDH (hALDH2) and mouse striatal synaptosomes. Molinate sulfone was found to be the most potent ALDH inhibitor, compared to molinate and molinate sulfoxide. The reactivity of these three compounds was also assessed, using N-acetyl Cys, model peptides, and hALDH2. It was determined that molinate sulfone is capable of covalently modifying Cys residues, including catalytic Cys302 of ALDH, accounting for the observed enzyme inhibition. PMID:20954713

  12. Relative inhibitory potency of molinate and metabolites with aldehyde dehydrogenase 2: implications for the mechanism of enzyme inhibition.

    PubMed

    Allen, Erin M G; Anderson, David G R; Florang, Virginia R; Khanna, May; Hurley, Thomas D; Doorn, Jonathan A

    2010-11-15

    Molinate is a thiocarbamate herbicide used as a pre-emergent in rice patty fields. It has two predominant sulfoxidation metabolites, molinate sulfoxide and molinate sulfone. Previous work demonstrated an in vivo decrease in liver aldehyde dehydrogenase (ALDH) activity in rats treated with molinate and motor function deficits in dogs dosed chronically with this compound. ALDH is an enzyme important in the catabolism of many neurotransmitters, such as dopamine. Inhibition of this enzyme may lead to the accumulation of endogenous neurotoxic metabolites such as 3,4-dihydroxyphenylacetaldehyde, a dopamine metabolite, which may account for the observed neurotoxicity. In this study, the relative reactivity of molinate and both of its sulfoxidation metabolites toward ALDH was investigated, as well as the mechanism of inhibition. The ALDH activity was monitored in two different model systems, human recombinant ALDH (hALDH2) and mouse striatal synaptosomes. Molinate sulfone was found to be the most potent ALDH inhibitor, as compared to molinate and molinate sulfoxide. The reactivity of these three compounds was also assessed, using N-acetyl Cys, model peptides, and hALDH2. It was determined that molinate sulfone is capable of covalently modifying Cys residues, including catalytic Cys302 of ALDH, accounting for the observed enzyme inhibition.

  13. Impairment of aldehyde dehydrogenase 2 increases accumulation of acetaldehyde-derived DNA damage in the esophagus after ethanol ingestion

    PubMed Central

    Yukawa, Yoshiyuki; Ohashi, Shinya; Amanuma, Yusuke; Nakai, Yukie; Tsurumaki, Mihoko; Kikuchi, Osamu; Miyamoto, Shin’ichi; Oyama, Tsunehiro; Kawamoto, Toshihiro; Chiba, Tsutomu; Matsuda, Tomonari; Muto, Manabu

    2014-01-01

    Ethanol and its metabolite, acetaldehyde, are the definite carcinogens for esophageal squamous cell carcinoma (ESCC), and reduced catalytic activity of aldehyde dehydrogenase 2 (ALDH2), which detoxifies acetaldehyde, increases the risk for ESCC. However, it remains unknown whether the ALDH2 genotype influences the level of acetaldehyde-derived DNA damage in the esophagus after ethanol ingestion. In the present study, we administered ethanol orally or intraperitoneally to Aldh2-knockout and control mice, and we quantified the level of acetaldehyde-derived DNA damage, especially N2-ethylidene-2’-deoxyguanosine (N2-ethylidene-dG), in the esophagus. In the model of oral ethanol administration, the esophageal N2-ethylidene-dG level was significantly higher in Aldh2-knockout mice compared with control mice. Similarly, in the model of intraperitoneal ethanol administration, in which the esophagus is not exposed directly to the alcohol solution, the esophageal N2-ethylidene-dG level was also elevated in Aldh2-knockout mice. This result indicates that circulating ethanol-derived acetaldehyde causes esophageal DNA damage, and that the extent of damage is influenced by knockout of Aldh2. Taken together, our findings strongly suggest the importance of acetaldehyde-derived DNA damage which is induced in the esophagus of individuals with ALDH2 gene impairment. This provides a physiological basis for understanding alcohol-related esophageal carcinogenesis. PMID:24959382

  14. Impairment of aldehyde dehydrogenase 2 increases accumulation of acetaldehyde-derived DNA damage in the esophagus after ethanol ingestion.

    PubMed

    Yukawa, Yoshiyuki; Ohashi, Shinya; Amanuma, Yusuke; Nakai, Yukie; Tsurumaki, Mihoko; Kikuchi, Osamu; Miyamoto, Shin'ichi; Oyama, Tsunehiro; Kawamoto, Toshihiro; Chiba, Tsutomu; Matsuda, Tomonari; Muto, Manabu

    2014-01-01

    Ethanol and its metabolite, acetaldehyde, are the definite carcinogens for esophageal squamous cell carcinoma (ESCC), and reduced catalytic activity of aldehyde dehydrogenase 2 (ALDH2), which detoxifies acetaldehyde, increases the risk for ESCC. However, it remains unknown whether the ALDH2 genotype influences the level of acetaldehyde-derived DNA damage in the esophagus after ethanol ingestion. In the present study, we administered ethanol orally or intraperitoneally to Aldh2-knockout and control mice, and we quantified the level of acetaldehyde-derived DNA damage, especially N(2) -ethylidene-2'-deoxyguanosine (N(2) -ethylidene-dG), in the esophagus. In the model of oral ethanol administration, the esophageal N(2) -ethylidene-dG level was significantly higher in Aldh2-knockout mice compared with control mice. Similarly, in the model of intraperitoneal ethanol administration, in which the esophagus is not exposed directly to the alcohol solution, the esophageal N(2) -ethylidene-dG level was also elevated in Aldh2-knockout mice. This result indicates that circulating ethanol-derived acetaldehyde causes esophageal DNA damage, and that the extent of damage is influenced by knockout of Aldh2. Taken together, our findings strongly suggest the importance of acetaldehyde-derived DNA damage which is induced in the esophagus of individuals with ALDH2 gene impairment. This provides a physiological basis for understanding alcohol-related esophageal carcinogenesis. PMID:24959382

  15. Lack of aldehyde dehydrogenase ameliorates oxidative stress induced by single-dose ethanol administration in mouse liver.

    PubMed

    Matsumoto, Akiko; Ichiba, Masayoshi; Horita, Mikako; Yamashita, Zenko; Takahashi, Tatsuya; Isse, Toyohi; Oyama, Tsunehiro; Kawamoto, Toshihiro; Tomokuni, Katsumaro

    2007-02-01

    Polymorphism of aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2, is far more common in East Asian countries. Acetaldehyde, an intermediate metabolite of ethanol, is metabolized very slowly in people who have ALDH2*2, as the mutated ALDH2 lacks acetaldehyde metabolizing activity. On the other hand, it is well established that metabolism of ethanol causes oxidative stress in liver tissue. To examine the consequences of this polymorphism on ethanol-induced oxidative stress in liver tissue, we conducted a study using Aldh2 knockout mice. Aldh2+/+ and Aldh2-/- mice were orally administered ethanol at a dose of 5g/kg body weight. Levels of malondialdehyde, an indicator of oxidative stress, and glutathione, a key antioxidant, in liver tissue were analyzed 0-24h after administration. Levels of malondialdehyde were significantly lower in Aldh2-/- mice than in Aldh2+/+ mice at 12h after injection, while levels of glutathione were higher in Aldh2-/- mice than in Aldh2+/+ mice at 6 and 12h after injection. Our results suggest that a lack of ALDH ameliorates ethanol-induced oxidative stress in liver tissue. PMID:17452299

  16. Salt-induction of betaine aldehyde dehydrogenase mRNA, protein, and enzymatic activity in sugar beet. [Beta vulgaris L

    SciTech Connect

    McCue, K.F.; Hanson, A.D. )

    1991-05-01

    In Chenopodiaceae such as sugar beet (Beta vulgaris L.), glycine betaine (betaine) accumulates in response to drought or salinity stress and functions in the cytoplasm as a compatible osmolyte. The last enzyme in the biosynthetic pathway, betaine aldehyde dehydrogenase (BADH), increases as much as 4-fold in response to rising salinity in the external medium. This increase is accompanied by an increase in both protein and mRNA levels. The steady state increases in BADH were examined at a series of NaCl concentrations from 100 to 500 mM NaCl. BADH protein levels were examined by native PAGE, and by western blot analysis using antibodies raised against BADH purified from spinach. mRNA levels were examined by northern plot analysis of total RNA isolated from the leaves and hybridized with a sugar beet BADH cDNA clone. The time course for BADH mRNA induction was determined in a salt shock experiment utilizing 400 mM NaCl added to the external growth medium. Disappearance of BADH was examined in a salt relief experiment using plants step-wise salinized to 500 mM NaCl and then returned to 0 mM NaCl.

  17. cDNA cloning and analysis of betaine aldehyde dehydrogenase, a salt inducible enzyme in sugar beet

    SciTech Connect

    McCue, K.F.; Hanson, A.D. )

    1990-05-01

    Betaine accumulates and serves as a compatible osmolyte in some plants subjected to drought or salinity stress. The last enzyme in the betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). The activity of BADH increases in response to increasing salinity levels. This increase in activity corresponds to an increase in protein detectable by immunoblotting, and to an increase in the translatable BADH mRNA. BADH was cloned from a cDNA library constructed in {lambda}gt10 using poly(A){sup +} RNA from sugar beets salinized to 500 mM NaCl. cDNAs were size selected (>1kb) before ligation into the vector, and the library was screened with a spinach BADH cDNA probe. Three nearly full length clones obtained were confirmed as BADH by their nucleotide and deduced amino acid homology to spinach BADH. Clones averaged 1.8 kb and contained open reading frames of 500 amino acids at 80% identity with spinach BADH. RNA gel blot analysis of poly(A){sup +} RNA indicated that salinization to 500 mM NaCl resulted in a 5-fold increase of BADH mRNA level.

  18. Aldehyde Dehydrogenase 2 (ALDH2) Polymorphism and the Risk of Alcoholic Liver Cirrhosis among East Asians: A Meta-Analysis

    PubMed Central

    He, Lei; Luo, Hesheng

    2016-01-01

    Purpose The aldehyde dehydrogenase 2 (ALDH2) gene has been implicated in the development of alcoholic liver cirrhosis (ALC) in East Asians. However, the results are inconsistent. In this study, a meta-analysis was performed to assess the associations between the ALDH2 polymorphism and the risk of ALC. Materials and Methods Relevant studies were retrieved by searching PubMed, Web of Science, CNKI, Wanfang and Veipu databases up to January 10, 2015. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using either the fixed- or random effects model. Results A total of twelve case-control studies included 1003 cases and 2011 controls were included. Overall, the ALDH2 polymorphism was associated with a decreased risk of ALC (*1/*2 vs. *1/*1: OR=0.78, 95% CI: 0.61–0.99). However, in stratification analysis by country, we failed to detect any association among Chinese, Korean or Japanese populations. Conclusion The pooled evidence suggests that ALDH2 polymorphism may be an important protective factor for ALC in East Asians. PMID:27189280

  19. Pigs fed camelina meal increase hepatic gene expression of cytochrome 8b1, aldehyde dehydrogenase, and thiosulfate transferase

    PubMed Central

    2014-01-01

    Camelina sativa is an oil seed crop which can be grown on marginal lands. Camelina seed oil is rich in omega-3 fatty acids (>35%) and γ-tocopherol but is also high in erucic acid and glucosinolates. Camelina meal, is the by-product after the oil has been extracted. Camelina meal was fed to 28 d old weaned pigs at 3.7% and 7.4% until age 56 d. The camelina meal supplements in the soy based diets, improved feed efficiency but also significantly increased the liver weights. Gene expression analyses of the livers, using intra-species microarrays, identified increased expression of phase 1 and phase 2 drug metabolism enzymes. The porcine versions of the enzymes were confirmed by real time PCR. Cytochrome 8b1 (CYP8B1), aldehyde dehydrogenase 2 (Aldh2), and thiosulfate transferase (TST) were all significantly stimulated. Collectively, these genes implicate the camelina glucosinolate metabolite, methyl-sulfinyldecyl isothiocyanate, as the main xeniobiotic, causing increased hepatic metabolism and increased liver weight. PMID:24383433

  20. Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

    SciTech Connect

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; Anderson, Lindsey N.; Sadler, Natalie C.; Piehowski, Paul D.; Gache, Yannick; Weber, Thomas J.

    2013-11-27

    Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profiles by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.

  1. Elucidating the reaction mechanism of the benzoate oxidation pathway encoded aldehyde dehydrogenase from Burkholderia xenovorans LB400

    PubMed Central

    Bains, Jasleen; Leon, Rafael; Temke, Kevin G; Boulanger, Martin J

    2011-01-01

    Oxidation of cis-3,4-dehydroadipyl-CoA semialdehyde to cis-3,4-dehydroadipyl-CoA by the aldehyde dehydrogenase, ALDHC (EC.1.2.1.77), is an essential step in the metabolism of benzoate in Burkholderia xenovorans LB400. In a previous study, we established a structural blueprint for this novel group of ALDH enzymes. Here, we build significantly on this initial work and propose a detailed reaction mechanism for ALDHC based on comprehensive structural and functional investigations of active site residues. Kinetic analyses reveal essential roles for C296 as the nucleophile and E257 as the associated general base. Structural analyses of E257Q and C296A variants suggest a dynamic charge repulsion relationship between E257 and C296 that contributes to the inherent flexibility of E257 in the native enzyme, which is further regulated by E496 and E167. A proton relay network anchored by E496 and supported by E167 and K168 serves to reset E257 for the second catalytic step. We also propose that E167, which is unique to ALDHC and its homologs, serves a critical role in presenting the catalytic water to the newly reset E257 such that the enzyme can proceed with deacylation and product release. Collectively, the reaction mechanism proposed for ALDHC promotes a greater understanding of these novel ALDH enzymes, the ALDH super-family in general, and benzoate degradation in B. xenovorans LB400. PMID:21495107

  2. Solanum Incanum Extract Downregulates Aldehyde Dehydrogenase 1-Mediated Stemness and Inhibits Tumor Formation in Ovarian Cancer Cells

    PubMed Central

    Wu, Yi-Hui; Chiu, Wen-Tai; Young, Ming-Jer; Chang, Tzu-Hao; Huang, Yu-Fang; Chou, Cheng-Yang

    2015-01-01

    Solanum incanum extract (SR-T100), containing the active ingredient solamargine, can induce apoptosis via upregulation of tumor necrosis factor receptor expression and activation of the mitochondrial apoptosis pathway, and has therapeutic effects in patients with actinic keratosis. Here, we evaluate the novel molecular mechanisms underlying SR-T100-regulated stemness and chemoresistance. The concentration of SR-T100 that inhibited 50% cell viability (IC50) was lower in ovarian cancer cells than in nonmalignant cells. Furthermore, the SR-T100 IC50 in chemoresistant cells was similar to the IC50 in chemosensitive cells. Additionally, SR-T100 increased cisplatin and paclitaxel sensitivity in chemoresistant cells. SR-T100 downregulated the expression of stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), Notch1, and FoxM1, and reduced sphere formation in ovarian cancer cells. Using microarray analyses, immunoblotting, luciferase activity, and chromatin immunoprecipitation (ChIP) assays, we showed that SR-T100 suppressed the expression of c/EBPβ and COL11A1, and its promoter activity, in resistant cells, but not sensitive cells. SR-T100, paclitaxel, and cisplatin inhibited the growth of A2780CP70 cells in mouse xenografts, as compared to the vehicle control, and the combination of cisplatin and SR-T100 was more effective than either treatment alone. SR-T100 may represent a potential therapeutic adjunct to chemotherapy for ovarian cancer treatment. PMID:26366215

  3. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  4. Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat.

    PubMed

    Badejo, Adeleke M; Hodnette, Chris; Dhaliwal, Jasdeep S; Casey, David B; Pankey, Edward; Murthy, Subramanyam N; Nossaman, Bobby D; Hyman, Albert L; Kadowitz, Philip J

    2010-09-01

    It has been reported that mitochondrial aldehyde dehydrogenase (ALDH2) catalyzes the formation of glyceryl dinitrate and inorganic nitrite from glyceryl trinitrate (GTN), leading to an increase in cGMP and vasodilation in the coronary and systemic vascular beds. However, the role of nitric oxide (NO) formed from nitrite in mediating the response to GTN in the pulmonary vascular bed is uncertain. The purpose of the present study was to determine if nitrite plays a role in mediating vasodilator responses to GTN. In this study, intravenous injections of GTN and sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure under baseline and elevated tone conditions and decreases in systemic arterial pressure in response to GTN and sodium nitrite were attenuated by cyanamide, an ALDH2 inhibitor, whereas responses to the NO donor, sodium nitroprusside (SNP), were not altered. The decreases in pulmonary and systemic arterial pressure in response to GTN and SNP were not altered by allopurinol, an inhibitor of xanthine oxidoreductase, whereas responses to sodium nitrite were attenuated. GTN was approximately 1,000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures. These results suggest that ALDH2 plays an important role in the bioactivation of GTN and nitrite in the pulmonary and systemic vascular beds and that the reduction of nitrite to vasoactive NO does not play an important role in mediating vasodilator responses to GTN in the intact chest rat.

  5. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.

    PubMed

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  6. Aldehyde dehydrogenase 1A1 stabilizes transcription factor Gli2 and enhances the activity of Hedgehog signaling in hepatocellular cancer.

    PubMed

    Yan, Zhengwei; Xu, Liyao; Zhang, Junyan; Lu, Quqin; Luo, Shiwen; Xu, Linlin

    2016-03-18

    The Gli transcription factors are primary transcriptional regulators that mediate the activation of Hedgehog (Hh) signaling. Recent studies have revealed that Gli proteins are also regulated transcriptionally and post-translationally through noncanonical mechanisms, independent of Hh signaling. However, the precise mechanisms involved in the regulation of Gli proteins remain unclear. Using a differential mass-spectrometry approach, we found that aldehyde dehydrogenase 1A1 (ALDH1A1) is associated with transcription factor Gli2. Overexpression of ALDH1A1 increased Gli2 protein levels; in contrast, ALDH1A1 depletion facilitated Gli2 degradation. In addition, Gli2 mRNA expression was not affected by ectopic expression of ALDH1A1, indicating the role of ALDH1A1 in the stabilization of Gli2. Further investigation showed that ALDH1A1 prolonged the stability of Gli2 protein in a catalytic-independent manner. Finally, we showed that overexpression of ALDH1A1 activated the Hh signaling pathway and promoted cell growth, migration and invasion in hepatocellular cancer cells. Together, these results illustrate regulatory roles of ALDH1A1 in the activation of the Hh signaling pathway and highlight a novel mechanism for the aberrant activation of the Hh signaling pathway in hepatocellular cancer cells. PMID:26896768

  7. Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

    DOE PAGES

    Wright, Aaron T.; Magnaldo, Thierry; Sontag, Ryan L.; Anderson, Lindsey N.; Sadler, Natalie C.; Piehowski, Paul D.; Gache, Yannick; Weber, Thomas J.

    2013-11-27

    Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of relevant pathways/networks. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol-reactive probes with a flexible click chemistry functional group to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. We observe qualitative differences in protein thiol profilesmore » by SDS-PAGE analysis when detection by iodoacetamide vs maleimide probe chemistries are compared, and pretreatment of cells with hydrogen peroxide eliminates detection of the majority of SDS-PAGE bands. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent donors, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and this deficiency was confirmed by Western blot. Redox probes revealed additional protein thiol differences between GDFs and NHDFs, including radiation responsive annexin family members. Our results indicate a multifactorial basis for the unusual sensitivity of Gorlin syndrome to radiation carcinogenesis, and the pathways identified have plausible implications for radiation health effects.« less

  8. Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids

    PubMed Central

    1994-01-01

    Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauchi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536- 19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from cDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli beta-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in ER targeting. PMID:8089174

  9. Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping.

    PubMed

    Vasiliou, V; Bairoch, A; Tipton, K F; Nebert, D W

    1999-08-01

    As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out

  10. Alcohol and Aldehyde Dehydrogenases Contribute to Sex-Related Differences in Clearance of Zolpidem in Rats

    PubMed Central

    Peer, Cody J.; Strope, Jonathan D.; Beedie, Shaunna; Ley, Ariel M.; Holly, Alesia; Calis, Karim; Farkas, Ronald; Parepally, Jagan; Men, Angela; Fadiran, Emmanuel O.; Scott, Pamela; Jenkins, Marjorie; Theodore, William H.; Sissung, Tristan M.

    2016-01-01

    Objectives: The recommended zolpidem starting dose was lowered in females (5 mg vs. 10 mg) since side effects were more frequent and severe than those of males; the mechanism underlying sex differences in pharmacokinetics (PK) is unknown. We hypothesized that such differences were caused by known sex-related variability in alcohol dehydrogenase (ADH) expression. Methods: Male, female, and castrated male rats were administered 2.6 mg/kg zolpidem, ± disulfiram (ADH/ALDH pathway inhibitor) to compare PK changes induced by sex and gonadal hormones. PK analyses were conducted in rat plasma and rat brain. Key findings: Sex differences in PK were evident: females had a higher CMAX (112.4 vs. 68.1 ug/L) and AUC (537.8 vs. 231.8 h∗ug/L) than uncastrated males. Castration induced an earlier TMAX (0.25 vs. 1 h), greater CMAX (109.1 vs. 68.1 ug/L), and a corresponding AUC increase (339.7 vs. 231.8 h∗ug/L). Administration of disulfiram caused more drastic CMAX and TMAX changes in male vs. female rats that mirrored the effects of castration on first-pass metabolism, suggesting that the observed PK differences may be caused by ADH/ALDH expression. Brain concentrations paralleled plasma concentrations. Conclusion: These findings indicate that sex differences in zolpidem PK are influenced by variation in the expression of ADH/ALDH due to gonadal androgens. PMID:27574509

  11. The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE, Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Lo, Jonathan; Zheng, Tianyong; Hon, Shuen; Olson, Daniel G.

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  12. 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 Central

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

    2015-01-01

    Background 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. Methods 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. Results 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). Conclusions 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

  13. Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity.

    PubMed

    Stiti, Naim; Podgórska, Karolina; Bartels, Dorothea

    2014-03-01

    The cofactor-binding site of the NAD(+)-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2'- and 3'-hydroxyl groups of the adenosyl ribose ring of NAD(+) and repels the 2'-phosphate moiety of NADP(+). If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD(+) is altered and rendered the enzyme capable of using NADP(+). This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2'-phosphate of NADP(+). Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP(+). The double mutant ALDH3H1Glu149Thr/Ile200Val showed a good catalysis with NADP(+). Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a "closed" cofactor binding site. The cofactor specificity was shifted even further in favor of NADP(+), as the mutant ALDH3H1E149T/V178R/I200V uses NADP(+) with almost 7-fold higher catalytic efficiency compared to NAD(+). Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.

  14. Improved tolerance to various abiotic stresses in transgenic sweet potato (Ipomoea batatas) expressing spinach betaine aldehyde dehydrogenase.

    PubMed

    Fan, Weijuan; Zhang, Min; Zhang, Hongxia; Zhang, Peng

    2012-01-01

    Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea batatas), a root crop with worldwide importance. The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavorable conditions. The gene encoding betaine aldehyde dehydrogenase (BADH) is involved in the biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic stress tolerance in plants. This study is to improve sweet potato, a GB accumulator, resistant to multiple abiotic stresses by promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet potato cultivar Sushu-2 via Agrobacterium-mediated transformation. The overexpression of SoBADH in the transgenic sweet potato improved tolerance to various abiotic stresses, including salt, oxidative stress, and low temperature. The increased BADH activity and GB accumulation in the transgenic plant lines under normal and multiple environmental stresses resulted in increased protection against cell damage through the maintenance of cell membrane integrity, stronger photosynthetic activity, reduced reactive oxygen species (ROS) production, and induction or activation of ROS scavenging by the increased activity of free radical-scavenging enzymes. The increased proline accumulation and systemic upregulation of many ROS-scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-scavenging system and proline biosynthesis via an integrative mechanism. This study demonstrates that the enhancement of GB biosynthesis in sweet potato is an effective and feasible approach to improve its tolerance to multiple abiotic stresses without causing phenotypic defects. This strategy for trait improvement in

  15. Improved Tolerance to Various Abiotic Stresses in Transgenic Sweet Potato (Ipomoea batatas) Expressing Spinach Betaine Aldehyde Dehydrogenase

    PubMed Central

    Fan, Weijuan; Zhang, Min; Zhang, Hongxia; Zhang, Peng

    2012-01-01

    Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea batatas), a root crop with worldwide importance. The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavorable conditions. The gene encoding betaine aldehyde dehydrogenase (BADH) is involved in the biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic stress tolerance in plants. This study is to improve sweet potato, a GB accumulator, resistant to multiple abiotic stresses by promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet potato cultivar Sushu-2 via Agrobacterium-mediated transformation. The overexpression of SoBADH in the transgenic sweet potato improved tolerance to various abiotic stresses, including salt, oxidative stress, and low temperature. The increased BADH activity and GB accumulation in the transgenic plant lines under normal and multiple environmental stresses resulted in increased protection against cell damage through the maintenance of cell membrane integrity, stronger photosynthetic activity, reduced reactive oxygen species (ROS) production, and induction or activation of ROS scavenging by the increased activity of free radical-scavenging enzymes. The increased proline accumulation and systemic upregulation of many ROS-scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-scavenging system and proline biosynthesis via an integrative mechanism. This study demonstrates that the enhancement of GB biosynthesis in sweet potato is an effective and feasible approach to improve its tolerance to multiple abiotic stresses without causing phenotypic defects. This strategy for trait improvement in

  16. Different effects of ascorbate deprivation and classical vascular nitrate tolerance on aldehyde dehydrogenase-catalysed bioactivation of nitroglycerin

    PubMed Central

    Wenzl, MV; Wölkart, G; Stessel, H; Beretta, M; Schmidt, K; Mayer, B

    2009-01-01

    Background and purpose: Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance. Experimental approach: Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg·kg−1 GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2-and 1,3-glyceryl dinitrate (GDN). Key results: Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN ∼10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the ∼10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance. Conclusions and implications: These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression. PMID:19254277

  17. Aldehyde dehydrogenase 2 ameliorates doxorubicin-induced myocardial dysfunction through detoxification of 4-HNE and suppression of autophagy.

    PubMed

    Sun, Aijun; Cheng, Yong; Zhang, Yingmei; Zhang, Qian; Wang, Shijun; Tian, Shan; Zou, Yunzeng; Hu, Kai; Ren, Jun; Ge, Junbo

    2014-06-01

    Mitochondrial aldehyde dehydrogenase (ALDH2) protects against cardiac injury via reducing production of 4-hydroxynonenal (4-HNE) and ROS. This study was designed to examine the impact of ALDH2 on doxorubicin (DOX)-induced cardiomyopathy and mechanisms involved with a focus on autophagy. 4-HNE and autophagic markers were detected by Western blotting in ventricular tissues from normal donors and patients with idiopathic dilated cardiomyopathy. Cardiac function, 4-HNE and levels of autophagic markers were detected in WT, ALDH2 knockout or ALDH2 transfected mice treated with or without DOX. Autophagy regulatory signaling including PI-3K, AMPK and Akt was examined in DOX-treated cardiomyocytes incubated with or without ALDH2 activator Alda-1. DOX-induced myocardial dysfunction, upregulation of 4-HNE and autophagic proteins were further aggravated in ALDH2 knockout mice while they were ameliorated in ALDH2 transfected mice. DOX downregulated Class I and upregulated Class III PI3-kinase, the effect of which was augmented by ALDH2 deletion. Accumulation of 4-HNE and autophagic protein markers in DOX-induced cardiomyocytes was significantly reduced by Alda-1. DOX depressed phosphorylated Akt but not AMPK, the effect was augmented by ALDH2 knockout. The autophagy inhibitor 3-MA attenuated, whereas autophagy inducer rapamycin mimicked DOX-induced cardiomyocyte contractile defects. In addition, rapamycin effectively mitigated Alda-1-offered protective action against DOX-induced cardiomyocyte dysfunction. Our data further revealed downregulated ALDH2 and upregulated autophagy levels in the hearts from patients with dilated cardiomyopathy. Taken together, our findings suggest that inhibition of 4-HNE and autophagy may be a plausible mechanism underscoring ALDH2-offered protection against DOX-induced cardiac defect. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". PMID:24434637

  18. Aldehyde dehydrogenase 2 activation in aged heart improves the autophagy by reducing the carbonyl modification on SIRT1

    PubMed Central

    Li, Chen; Yin, Yue; Yang, Jingrun; Wang, Zhifa; Zheng, Qiangsun; Ma, Heng

    2016-01-01

    Cardiac aging is characterized by accumulation of damaged proteins and decline of autophagic efficiency. Here, by forestalling SIRT1 carbonylated inactivation in aged heart, we determined the benefits of activation of aldehyde dehydrogenase 2 (ALDH2) on the autophagy. In this study, the ALDH2 KO mice progressively developed age-related heart dysfunction and showed reduction in the life span, which strongly suggests that ALDH2 ablation leads to cardiac aging. What's more, aged hearts displayed a significant decrease ALDH2 activity, resulting in accumulation of 4-HNE-protein adducts and protein carbonyls, impairment in the autophagy flux, and, consequently, deteriorated cardiac function after starvation. Sustained Alda-1 (selective ALDH2 activator) treatment increased cardiac ALDH2 activity and abrogated these effects. Using SIRT1 deficient heterozygous (Sirt1+/−) mice, we found that SIRT1 was necessary for ALDH2 activation-induced autophagy. We further demonstrated that ALDH2 activation attenuated SIRT1 carbonylation and improved SIRT1 activity, thereby increasing the deacetylation of nuclear LC3 and FoxO1. Sequentially, ALDH2 enhanced SIRT1 regulates LC3-Atg7 interaction and FoxO1 increased Rab7 expression, which were both necessary and sufficient for restoring autophagy flux. These results highlight that both accumulation of proteotoxic carbonyl stress linkage with autophagy decline contribute to heart senescence. ALDH2 activation is adequate to improve the autophagy flux by reducing the carbonyl modification on SIRT1, which in turn plays an important role in maintaining cardiac health during aging. PMID:26741505

  19. Effects on sister chromatid exchange frequency of aldehyde dehydrogenase 2 genotype and smoking in vinyl chloride workers.

    PubMed

    Wong, R H; Wang, J D; Hsieh, L L; Du, C L; Cheng, T J

    1998-12-01

    Vinyl chloride monomer (VCM) is a human carcinogen. However, the exact mechanism of carcinogenesis remains unclear. VCM may be metabolized by cytochrome P450 2E1 (CYP2E1), aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferases (GSTs). Thus workers with inherited variant metabolic enzyme activities may have an altered risk of genotoxicity. This study was designed to investigate which risk factors might affect sister chromatid exchange (SCE) frequency in polyvinyl chloride (PVC) workers. Study subjects were 44 male workers from three PVC factories. Questionnaires were administered to obtain detailed histories of cigarette smoking, alcohol consumption, occupations, and medications. SCE frequency in peripheral lymphocytes was determined using a standardized method, and CYP2E1, GSTM1, GSTT1 and ALDH2 genotypes were identified by the polymerase chain reaction (PCR). Analysis revealed that smoking status and exposure to VCM were significantly associated with increased SCE frequency. The presence of ALDH2 1-2/2-2 genotypes was also significantly associated with an elevation of SCE frequency (9. 5 vs. 8.1, p<0.01). However, CYP2E1, GSTM1 or GSTT1 genotypes were not significantly associated with SCE frequency. When various genotypes were considered together, combination of CYP2E1 c1c2/c2c2 with ALDH2 1-2/2-2 showed an additive effect on SCE frequency. Similar results were also found for the combination of smoking with CYP2E1, or smoking with ALDH2. These results suggest that VCM workers with ALDH2 1-2/2-2 genotypes, who also smoke, may have increased risk of DNA damage.

  20. Increased superoxide production in nitrate tolerance is associated with NAD(P)H oxidase and aldehyde dehydrogenase 2 downregulation.

    PubMed

    Szöcs, Katalin; Lassègue, Bernard; Wenzel, Philip; Wendt, Maria; Daiber, Andreas; Oelze, Matthias; Meinertz, Thomas; Münzel, Thomas; Baldus, Stephan

    2007-06-01

    Chronic administration of nitroglycerin (NTG) induces nitrate tolerance. Among possible underlying mechanisms, increased vascular production of reactive oxygen species (ROS) has emerged as a principal mechanism. Using cell culture and animal models of nitrate tolerance, we aimed to assess the impact of nitrates on NAD(P)H oxidases and aldehyde dehydrogenase 2 (ALDH2) expression. Rats and vascular smooth muscle cells were treated with NTG. Vascular reactivity was assessed by isometric tension studies. Superoxide was detected by dihydroethidium staining. Gene expression was measured by real-time polymerase chain reaction. NAD(P)H oxidase activity was measured using lucigenin-enhanced chemiluminescence. ALDH activity was measured biochemically, and NO consumption electrochemically. Nitrate tolerance was induced in rats by treatment with NTG for 3 days, and detected as impaired endothelium-dependent and -independent relaxation of aortic segments. Although superoxide production was increased in all aortic layers, expression of nox1, nox2 and nox4 was significantly decreased. Similarly, in vascular smooth muscle cells exposed to NTG for 6-24 h, NAD(P)H oxidase activity was increased, in spite of nox1 downregulation. In addition, expression and activity of ALDH-2 was decreased in nitrate-tolerant rings. Furthermore, exogenous addition of ALDH decreased superoxide generation in vitro and attenuated NO consumption in vascular smooth muscle cell homogenates. Our data suggest that in nitrate tolerance, activation of nox enzymes more than compensates for their downregulation, resulting in a net increase in superoxide and NO consumption. Furthermore, reduced ALDH-2 activity and expression leads to decreased NTG bioconversion. Therefore, both mechanisms reduce NO availability and impair vasorelaxation. PMID:17493633

  1. Heterologous expression of betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus confers high salt and heat tolerance to Escherichia coli.

    PubMed

    Yu, Hao-Qiang; Wang, Ying-Ge; Yong, Tai-Ming; She, Yue-Hui; Fu, Feng-Ling; Li, Wan-Chen

    2014-10-01

    Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine, a regulator of osmosis, and therefore BADH is considered to play a significant role in response of plants to abiotic stresses. Here, based on the conserved residues of the deduced amino acid sequences of the homologous BADH genes, we cloned the AnBADH gene from the xerophytic leguminous plant Ammopiptanthus nanus by using reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA is 1,868 bp long without intron, and contains an open reading frame of 1512 bp, and 3'- and 5'-untranslated regions of 294 and 62 bp. It encodes a 54.71 kDa protein of 503 amino acids. The deduced amino acid sequence shares high homology, conserved amino acid residues and sequence motifs crucial for the function with the BADHs in other leguminous species. The sequence of the open reading frame was used to construct a prokaryotic expression vector pET32a-AnBADH, and transform Escherichia coli. The transformants expressed the heterologous AnBADH gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of salt and heat tolerance under the stress conditions of 700 mmol L(-1) NaCl and 55°C high temperature. This result suggests that the AnBADH gene might play a crucial role in adaption of A. nanus to the abiotic stresses, and have the potential to be applied to transgenic operations of commercially important crops for improvement of abiotic tolerance. PMID:25046139

  2. Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99-1 cells based on high aldehyde dehydrogenase activity.

    PubMed

    Wang, Lin; Park, Paul; Zhang, Huina; La Marca, Frank; Lin, Chia-Ying

    2011-01-15

    High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines, of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice, we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH(br) cells) from the OS99-1 xenografts were much less frequent, averaging 3% of the entire tumor population, compared to those isolated directly from the OS99-1 cell line. ALDH(br) cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH(lo) cells), generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH(br) cells illustrated the stem cell characteristics of self-renewal, the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A, Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma.

  3. Plastid-Expressed Betaine Aldehyde Dehydrogenase Gene in Carrot Cultured Cells, Roots, and Leaves Confers Enhanced Salt Tolerance1

    PubMed Central

    Kumar, Shashi; Dhingra, Amit; Daniell, Henry

    2004-01-01

    Salinity is one of the major factors that limits geographical distribution of plants and adversely affects crop productivity and quality. We report here high-level expression of betaine aldehyde dehydrogenase (BADH) in cultured cells, roots, and leaves of carrot (Daucus carota) via plastid genetic engineering. Homoplasmic transgenic plants exhibiting high levels of salt tolerance were regenerated from bombarded cell cultures via somatic embryogenesis. Transformation efficiency of carrot somatic embryos was very high, with one transgenic event per approximately seven bombarded plates under optimal conditions. In vitro transgenic carrot cells transformed with the badh transgene were visually green in color when compared to untransformed carrot cells, and this offered a visual selection for transgenic lines. BADH enzyme activity was enhanced 8-fold in transgenic carrot cell cultures, grew 7-fold more, and accumulated 50- to 54-fold more betaine (93–101 μmol g−1 dry weight of β-Ala betaine and Gly betaine) than untransformed cells grown in liquid medium containing 100 mm NaCl. Transgenic carrot plants expressing BADH grew in the presence of high concentrations of NaCl (up to 400 mm), the highest level of salt tolerance reported so far among genetically modified crop plants. BADH expression was 74.8% in non-green edible parts (carrots) containing chromoplasts, and 53% in proplastids of cultured cells when compared to chloroplasts (100%) in leaves. Demonstration of plastid transformation via somatic embryogenesis utilizing non-green tissues as recipients of foreign DNA for the first time overcomes two of the major obstacles in extending this technology to important crop plants. PMID:15347789

  4. Essential role of aldehyde dehydrogenase 1A3 (ALDH1A3) for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway

    PubMed Central

    Shao, Chunli; Sullivan, James P.; Girard, Luc; Augustyn, Alexander; Yenerall, Paul; Rodriguez, Jaime; Liu, Hui; Behrens, Carmen; Shay, Jerry W.; Wistuba, Ignacio I.; Minna, John D.

    2014-01-01

    Purpose Lung cancer stem cells (CSCs) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. Experimental Design A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ vs. ALDH− cells. RT-PCR, western blot and Aldefluor assay were used to validate identified genes. To explore the function in CSCs we manipulated their expression followed by colony and tumor formation assays. Results We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ vs. ALDH− cells. ShRNA-mediated knockdown of ALDH1A3 in NSCLCs resulted in a dramatic reduction in ALDH activity, clonogenicity and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. By contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated Signal Transducers and Activators of Transcription 3 (STAT3) than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival. Conclusion Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs. PMID:24907115

  5. 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.

  6. 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

  7. 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

    2015-01-01

    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

  8. Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

    PubMed Central

    2014-01-01

    Introduction Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors. Methods Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation. Results Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH+ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH- cells. GD2+ cells showed a 3.9-fold greater capacity than GD2- cells. ALDH+/GD2+cells displayed 12.8-fold greater mammosphere forming ability than ALDH-/GD2- cells. In vivo, the tumor-initiating frequency of ALDH+/GD2+ cells were up to 33-fold higher than that of ALDH+ cells, with as few as 50 ALDH+/GD2+ cells being sufficient for engraftment. Moreover, we

  9. In vivo ethanol elimination in man, monkey and rat: A lack of relationship between the ethanol metabolism and the hepatic activities of alcohol and aldehyde dehydrogenases

    SciTech Connect

    Zorzano, A. ); Herrera, E. )

    1990-01-01

    The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dehydrogenase activity and higher low Km aldehyde dehydrogenase activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.

  10. Pepper aldehyde dehydrogenase CaALDH1 interacts with Xanthomonas effector AvrBsT and promotes effector-triggered cell death and defence responses

    PubMed Central

    Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Xanthomonas type III effector AvrBsT induces hypersensitive cell death and defence responses in pepper (Capsicum annuum) and Nicotiana benthamiana. Little is known about the host factors that interact with AvrBsT. Here, we identified pepper aldehyde dehydrogenase 1 (CaALDH1) as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and co-immunoprecipitation assays confirmed the interaction between CaALDH1 and AvrBsT in planta. CaALDH1:smGFP fluorescence was detected in the cytoplasm. CaALDH1 expression in pepper was rapidly and strongly induced by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) Ds1 (avrBsT) infection. Transient co-expression of CaALDH1 with avrBsT significantly enhanced avrBsT-triggered cell death in N. benthamiana leaves. Aldehyde dehydrogenase activity was higher in leaves transiently expressing CaALDH1, suggesting that CaALDH1 acts as a cell death enhancer, independently of AvrBsT. CaALDH1 silencing disrupted phenolic compound accumulation, H2O2 production, defence response gene expression, and cell death during avirulent Xcv Ds1 (avrBsT) infection. Transgenic Arabidopsis thaliana overexpressing CaALDH1 exhibited enhanced defence response to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis infection. These results indicate that cytoplasmic CaALDH1 interacts with AvrBsT and promotes plant cell death and defence responses. PMID:25873668

  11. Biochemical genetics of opossum aldehyde dehydrogenase 3: evidence for three ALDH3A-like genes and an ALDH3B-like gene.

    PubMed

    Holmes, Roger S

    2010-04-01

    Mammalian ALDH3 isozymes participate in peroxidic and fatty aldehyde metabolism, and in anterior eye tissue UV-filtration. BLAT analyses were undertaken of the opossum genome using rat ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2 amino acid sequences. Two predicted opossum ALDH3A1-like genes and an ALDH3A2-like gene were observed on chromosome 2, as well as an ALDH3B-like gene, which showed similar intron-exon boundaries with other mammalian ALDH3-like genes. Opossum ALDH3 subunit sequences and structures were highly conserved, including residues previously shown to be involved in catalysis and coenzyme binding for rat ALDH3A1. Eleven glycine residues were conserved for all of the opossum ALDH3-like sequences examined, including two glycine residues previously located within the stem of the rat ALDH3A1 active site funnel. Phylogeny studies of human, rat, opossum, and chicken ALDH3-like sequences indicated that the common ancestor for ALDH3A- and ALDH3B-like genes predates the appearance of birds during vertebrate evolution.

  12. Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.

    PubMed

    Wallwey, Christiane; Matuschek, Marco; Li, Shu-Ming

    2010-02-01

    Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. A putative gene fgaDH has been identified in the biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine-type. The deduced gene product FgaDH comprises 261 amino acids with a molecular mass of about 27.8 kDa and contains the conserved motifs of classical short-chain dehydrogenases/reductases (SDRs), but shares no worth mentioning sequence similarity with SDRs and other known proteins. The coding region of fgaDH consisting of two exons was amplified by PCR from a cDNA library of Aspergillus fumigatus, cloned into pQE60 and overexpressed in E. coli. The soluble tetrameric His(6)-FgaDH was purified to apparent homogeneity and characterized biochemically. It has been shown that FgaDH catalyzes the oxidation of chanoclavine-I in the presence of NAD(+) resulting in the formation of chanoclavine-I aldehyde, which was unequivocally identified by NMR and MS analyzes. Therefore, FgaDH functions as a chanoclavine-I dehydrogenase and represents a new group of short-chain dehydrogenases. K (M) values for chanoclavine-I and NAD(+) were determined at 0.27 and 1.1 mM, respectively. The turnover number was 0.38 s(-1). PMID:20039019

  13. Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse.

    PubMed

    Kitagawa, K; Kawamoto, T; Kunugita, N; Tsukiyama, T; Okamoto, K; Yoshida, A; Nakayama, K; Nakayama, K

    2000-07-01

    A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype. PMID:10913633

  14. Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde-alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica.

    PubMed

    Pineda, Erika; Encalada, Rusely; Rodríguez-Zavala, José S; Olivos-García, Alfonso; Moreno-Sánchez, Rafael; Saavedra, Emma

    2010-08-01

    The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O(2) exposure (IC(50) 0.034 mm), whereas short-term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe(2+). CoA and acetyl-CoA prevented, whereas pyruvate exacerbated, inactivation induced by short-term saturating O(2) exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe(2+)-reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe-S clusters. After O(2) exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde-alcohol dehydrogenase (EhADH2), whereas acetyl-CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress. PMID:20629749

  15. Significant improvement of stress tolerance in tobacco plants by overexpressing a stress-responsive aldehyde dehydrogenase gene from maize (Zea mays).

    PubMed

    Huang, Weizao; Ma, Xinrong; Wang, Qilin; Gao, Yongfeng; Xue, Ying; Niu, Xiangli; Yu, Guirong; Liu, Yongsheng

    2008-11-01

    Aldehyde dehydrogenases (ALDHs) play a central role in detoxification processes of aldehydes generated in plants when exposed to the stressed conditions. In order to identify genes required for the stresses responses in the grass crop Zea mays, an ALDH (ZmALDH22A1) gene was isolated and characterized. ZmALDH22A1 belongs to the family ALDH22 that is currently known only in plants. The ZmALDH22A1 encodes a protein of 593 amino acids that shares high identity with the orthologs from Saccharum officinarum (95%), Oryza sativa (89%), Triticum aestivum (87%) and Arabidopsis thaliana (77%), respectively. Real-time PCR analysis indicates that ZmALDH22A1 is expressed differentially in different tissues. Various elevated levels of ZmALDH22A1 expression have been detected when the seedling roots exposed to abiotic stresses including dehydration, high salinity and abscisic acid (ABA). Tomato stable transformation of construct expressing the ZmALDH22A1 signal peptide fused with yellow fluorescent protein (YFP) driven by the CaMV35S-promoter reveals that the fusion protein is targeted to plastid. Transgenic tobacco plants overexpressing ZmALDH22A1 shows elevated stresses tolerance. Stresses tolerance in transgenic plants is accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation.

  16. Human Salivary Aldehyde Dehydrogenase: Purification, Kinetic Characterization and Effect of Ethanol, Hydrogen Peroxide and Sodium Dodecyl Sulfate on the Activity of the Enzyme.

    PubMed

    Alam, Md Fazle; Laskar, Amaj Ahmed; Choudhary, Hadi Hasan; Younus, Hina

    2016-09-01

    Human salivary aldehyde dehydrogenase (hsALDH) enzyme appears to be the first line of defense in the body against exogenous toxic aldehydes. However till date much work has not been done on this important member of the ALDH family. In this study, we have purified hsALDH to homogeneity by diethylaminoethyl-cellulose (DEAE-cellulose) ion-exchange chromatography in a single step. The molecular mass of the homodimeric enzyme was determined to be approximately 108 kDa. Four aromatic substrates; benzaldehyde, cinnamaldehyde, 2-naphthaldehyde and 6-methoxy-2-naphthaldehyde were used for determining the activity of pure hsALDH. K m values for these substrates were calculated to be 147.7, 5.31, 0.71 and 3.31 μM, respectively. The best substrates were found to be cinnamaldehyde and 2-naphthaldehyde since they exhibited high V max /K m values. 6-methoxy-2-naphthaldehyde substrate was used for further kinetic characterization of pure hsALDH. The pH and temperature optima of hsALDH were measured to be pH 8 and 45 °C, respectively. The pure enzyme is highly unstable at high temperatures. Ethanol, hydrogen peroxide and SDS activate hsALDH, therefore it is safe and beneficial to include them in mouthwashes and toothpastes in low concentrations. PMID:27324040

  17. Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus, a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b, and Cytochrome c▿

    PubMed Central

    Gómez-Manzo, S.; Chavez-Pacheco, J. L.; Contreras-Zentella, M.; Sosa-Torres, M. E.; Arreguín-Espinosa, R.; Pérez de la Mora, M.; Membrillo-Hernández, J.; Escamilla, J. E.

    2010-01-01

    Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone. PMID:20802042

  18. Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

    PubMed Central

    2014-01-01

    Background Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s. Results We performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators

  19. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

    PubMed

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-09-30

    Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here

  20. JWH-018 ω-OH, a shared hydroxy metabolite of the two synthetic cannabinoids JWH-018 and AM-2201, undergoes oxidation by alcohol dehydrogenase and aldehyde dehydrogenase enzymes in vitro forming the carboxylic acid metabolite.

    PubMed

    Holm, Niels Bjerre; Noble, Carolina; Linnet, Kristian

    2016-09-30

    Synthetic cannabinoids are new psychoactive substances (NPS) acting as agonists at the cannabinoid receptors. The aminoalkylindole-type synthetic cannabinoid naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) was among the first to appear on the illicit drug market and its metabolism has been extensively investigated. The N-pentyl side chain is a major site of human cytochrome P450 (CYP)-mediated oxidative metabolism, and the ω-carboxylic acid metabolite appears to be a major in vivo human urinary metabolite. This metabolite is, however, not formed to any significant extent in human liver microsomal (HLM) incubations raising the possibility that the discrepancy is due to involvement of cytosolic enzymes. Here we demonstrate in incubations with human liver cytosol (HLC), that JWH-018 ω-OH, but not the JWH-018 parent compound, is a substrate for nicotinamide adenine dinucleotide (NAD(+))-dependent alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. The sole end-product identified in HLC was the JWH-018 ω-COOH metabolite, while trapping tests with methoxyamine proved the presence of the aldehyde intermediate. ADH/ALDH and UDP-glucuronosyl-transferases (UGT) enzymes may therefore both act on the JWH-018 ω-OH substrate. Finally, we note that for [1-(5-fluoropentyl)indol-3-yl]-naphthalen-1-yl-methanone (AM-2201), the ω-fluorinated analog of JWH-018, a high amount of JWH-018 ω-OH was formed in HLM incubated without NADPH, suggesting that the oxidative defluorination is efficiently catalyzed by non-CYP enzyme(s). The pathway presented here may therefore be especially important for N-(5-fluoropentyl) substituted synthetic cannabinoids, because the oxidative defluorination can occur even if the CYP-mediated metabolism preferentially takes place on other parts of the molecule than the N-alkyl side chain. Controlled clinical studies in humans are ultimately required to demonstrate the in vivo importance of the oxidation pathway presented here.

  1. Evidence that the C-terminal domain of a type B PutA protein contributes to aldehyde dehydrogenase activity and substrate channeling.

    PubMed

    Luo, Min; Christgen, Shelbi; Sanyal, Nikhilesh; Arentson, Benjamin W; Becker, Donald F; Tanner, John J

    2014-09-01

    Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH-P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target-decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium. PMID:25137435

  2. Evidence That the C-Terminal Domain of a Type B PutA Protein Contributes to Aldehyde Dehydrogenase Activity and Substrate Channeling

    PubMed Central

    2015-01-01

    Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress curve for the coupled PRODH–P5CDH activity of RcPutA does not exhibit a time lag, implying a substrate channeling mechanism. RcPutA is monomeric in solution, which is unprecedented for PutAs. SAXS rigid body modeling with target–decoy validation is used to build a model of RcPutA. On the basis of homology to aldehyde dehydrogenases (ALDHs), the CTDUF is predicted to consist of a β-hairpin fused to a noncatalytic Rossmann fold domain. The predicted tertiary structural interactions of the CTDUF resemble the quaternary structural interactions in the type A PutA dimer interface. The model is tested by mutagenesis of the dimerization hairpin of a type A PutA and the CTDUF hairpin of RcPutA. Similar functional phenotypes are observed in the two sets of variants, supporting the hypothesis that the CTDUF mimics the type A PutA dimer interface. These results suggest annotation of the CTDUF as an ALDH superfamily domain that facilitates P5CDH activity and substrate channeling by stabilizing the aldehyde-binding site and sealing the substrate-channeling tunnel from the bulk medium. PMID:25137435

  3. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    SciTech Connect

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important component in making biofuels production from lignocellulosic biomass feasible.

  4. Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

    DOE PAGES

    Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; Westpheling, Janet; Elkins, James G.

    2015-07-22

    Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

  5. Mitochondrial Aldehyde Dehydrogenase 2 Plays Protective Roles in Heart Failure After Myocardial Infarction via Suppression of the Cytosolic JNK/p53 Pathway in Mice

    PubMed Central

    Sun, Aijun; Zou, Yunzeng; Wang, Ping; Xu, Danling; Gong, Hui; Wang, Shijun; Qin, Yingjie; Zhang, Peng; Chen, Yunqin; Harada, Mutsuo; Isse, Toyoshi; Kawamoto, Toshihiro; Fan, Huizhi; Yang, Pengyuan; Akazawa, Hiroshi; Nagai, Toshio; Takano, Hiroyuki; Ping, Peipei; Komuro, Issei; Ge, Junbo

    2014-01-01

    Background Increasing evidence suggests a critical role for mitochondrial aldehyde dehydrogenase 2 (ALDH2) in protection against cardiac injuries; however, the downstream cytosolic actions of this enzyme are largely undefined. Methods and Results Proteomic analysis identified a significant downregulation of mitochondrial ALDH2 in the heart of a rat heart failure model after myocardial infarction. The mechanistic insights underlying ALDH2 action were elucidated using murine models overexpressing ALDH2 or its mutant or with the ablation of the ALDH2 gene (ALDH2 knockout) and neonatal cardiomyocytes undergoing altered expression and activity of ALDH2. Left ventricle dilation and dysfunction and cardiomyocyte death after myocardial infarction were exacerbated in ALDH2‐knockout or ALDH2 mutant‐overexpressing mice but were significantly attenuated in ALDH2‐overexpressing mice. Using an anoxia model of cardiomyocytes with deficiency in ALDH2 activities, we observed prominent cardiomyocyte apoptosis and increased accumulation of the reactive aldehyde 4‐hydroxy‐2‐nonenal (4‐HNE). We subsequently examined the impacts of mitochondrial ALDH2 and 4‐HNE on the relevant cytosolic protective pathways. Our data documented 4‐HNE‐stimulated p53 upregulation via the phosphorylation of JNK, accompanying increased cardiomyocyte apoptosis that was attenuated by inhibition of p53. Importantly, elevation of 4‐HNE also triggered a reduction of the cytosolic HSP70, further corroborating cytosolic action of the 4‐HNE instigated by downregulation of mitochondrial ALDH2. Conclusions Downregulation of ALDH2 in the mitochondria induced an elevation of 4‐HNE, leading to cardiomyocyte apoptosis by subsequent inhibition of HSP70, phosphorylation of JNK, and activation of p53. This chain of molecular events took place in both the mitochondria and the cytosol, contributing to the mechanism underlying heart failure. PMID:25237043

  6. Development of Selective Inhibitors for Human Aldehyde Dehydrogenase 3A1 (ALDH3A1) for the Enhancement of Cyclophosphamide Cytotoxicity

    PubMed Central

    Parajuli, Bibek; Georgiadis, Taxiarchis M.; Fishel, Melissa L.; Hurley, Thomas D.

    2014-01-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1) plays an important role in many cellular oxidative processes, including cancer chemo-resistance by metabolizing activated forms of oxazaphosphorine drugs such as cyclophosphamide (CP) and its analogues such as mafosfamide (MF), ifosfamide (IFM), 4-hydroperoxycyclophosphamide (4-HPCP). Compounds that can selectively target ALDH3A1 may permit delineation of its roles in these processes and could restore chemosensitivity in cancer cells that express this isoenzyme. Here we report the detailed kinetic and structural characterization of an ALDH3A1 selective inhibitor, CB29, previously identified in a high throughput screen. Kinetic and crystallographic studies demonstrate that CB29 binds within the aldehyde substrate-binding site of ALDH3A1. Cellular proliferation of ALDH3A1-expressing lung adenocarcinoma (A549) and glioblastoma (SF767) cell lines, as well as the ALDH3A1 non-expressing lung fibroblast cells, CCD-13Lu, is unaffected by treatment with CB29 and its analogues alone. However, the sensitivity toward the anti-proliferative effects of mafosfamide is enhanced by treatment with CB29 and its analogue in the tumour cells. In contrast, the sensitivity of CCD-13Lu cells toward mafosfamide was unaffected by the addition of these same compounds. CB29 is chemically distinct from the previously reported small molecule inhibitors of ALDH isoenzymes and does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1 or ALDH2 isoenzymes at concentrations up to 250 μM. Thus, CB29 is a novel small molecule inhibitor of ALDH3A1, which may be useful as a chemical tool to delineate the role of ALDH3A1 in numerous metabolic pathways, including sensitizing ALDH3A1-positive cancer cells to oxazaphosphorines. PMID:24677340

  7. 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.

  8. 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.

  9. 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. PMID:25772736

  10. 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.

  11. A proposal for nomenclature of aldehyde dehydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes.

    PubMed

    Navarro-Aviño, J P; Prasad, R; Miralles, V J; Benito, R M; Serrano, R

    1999-07-01

    The complete sequencing of the genome of Saccharomyces cerevisiae indicated that this organism contains five genes encoding aldehyde dehydrogenases. YOR374w and YER073w correspond to the mitochondrial isoforms and we propose as gene names ALD4 and ALD5, respectively. YPL061w has been described as the cytoplasmic constitutive isoform and named ALD6. We characterize here the tandem-repeated ORFs YMR170c and YMR169c as the cytoplasmic stress-inducible isoforms, with gene names ALD2 and ALD3, respectively. The expression of ALD2 and ALD3 is dependent on the general-stress transcription factors Msn2,4 but independent of the HOG MAP kinase pathway. ALD3 is induced by a variety of stresses, including osmotic shock, heat shock, glucose exhaustion, oxidative stress and drugs. ALD2 is only induced by osmotic stress and glucose exhaustion. A double null mutant, ald2 ald3, exhibited unchanged sensitivity to any of the above stresses. The only phenotype detected in this mutant was a reduced growth rate in ethanol medium as compared to the wild type. PMID:10407263

  12. 9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia.

    PubMed

    Chowdhury, Suchandra; Chandra, Sarmila; Mandal, Chitra

    2014-10-01

    Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac2-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac2-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH(+)SSC(lo)CD45(hi)Neu5,9Ac2 -GPs(lo)CD34(+)CD38(-)CD90(+)CD117(+)CD133(+)) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH(+)SSC(lo)CD45(lo)Neu5,9Ac2 -GPs(hi)CD34(+)CD38(+)CD90(-)CD117(-)CD133(-)) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy.

  13. Two clinical drugs deubiquitinase inhibitor auranofin and aldehyde dehydrogenase inhibitor disulfiram trigger synergistic anti-tumor effects in vitro and in vivo

    PubMed Central

    Liu, Ningning; Hua, Xianliang; Cai, Jianyu; Yang, Changshan; Long, Huidan; Zhao, Chong; Chen, Xin; Lan, Xiaoying; Zang, Dan; Wu, Jinjie; Li, Xiaofen; Shi, Xianping; Wang, Xuejun; Liu, Jinbao

    2016-01-01

    Inhibition of proteasome-associated deubiquitinases (DUBs) is emerging as a novel strategy for cancer therapy. It was recently reported that auranofin (Aur), a gold (I)-containing compound used clinically to treat rheumatoid arthritis, is a proteasome-associated DUB inhibitor. Disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, is currently in clinical use for treating alcoholism. Recent studies have indicated that DSF can also act as an antitumor agent. We investigated the effect of combining DSF and Aur on apoptosis induction and tumor growth in hepatoma cancer cells. Here we report that (i) the combined treatment of Aur and DSF results in synergistic cytotoxicity to hepatoma cells in vitro and in vivo; (ii) Aur and DSF in combination induces caspase activation, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production; (iii) pan-caspase inhibitor z-VAD-FMK could efficiently block apoptosis but not proteasome inhibition induced by Aur and DSF combined treatment, and ROS is not required for Aur+DSF to induce apoptosis. Collectively, we demonstrate a model of synergism between DSF and proteasome-associated DUB inhibitor Aur in the induction of apoptosis in hepatoma cancer cells, identifying a potential novel anticancer strategy for clinical use in the future. PMID:26625200

  14. Insight into the Structural Requirements of Theophylline-Based Aldehyde Dehydrogenase lAl (ALDHlAl) Inhibitors Through Multi-QSAR Modeling and Molecular Docking Approaches.

    PubMed

    Abdul Amin, Sk; Adhikari, Nilanjan; Gayen, Shovanlal; Jha, Tarun

    2016-01-01

    Over expression of aldehyde dehydrogenase (ALDH1A1) is one of the vital hallmarks of the self-renewal and differentiational cancer stem cells (CSCs). Till now, no selective ALDH1A1 inhibitor is commercially available in the market. So there is an urgent need to explore some novel molecules which can selectively inhibit ALDH1A1 to combat cancer. Presently, our work deals with the development of QSAR models of some theophylline-based molecules by conventional 2D-QSAR, hologram QSAR (HQSAR), and Bayesian classification modeling. The descriptors identified from these QSAR models give avenues to modulate the structure of theophylline-based compounds to a desirable biological end point. Molecular docking study reveals the selectivity of these molecules towards ALDH1A1 (PDB: 4WP7) and important binding residues (GLY 125, 458; THR 129; TRP 178; TYR 297; PHE 171, 466; VAL 174, 460; MET 175; HIS 293 etc.) for the interaction with the receptors. The current study may help to design novel compounds as selective ALDH1A1 inhibitors. PMID:27132720

  15. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    SciTech Connect

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M. . E-mail: Elizabeth.ellis@strath.ac.uk

    2006-01-15

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V {sub max} of 2141 {+-} 500 nmol/min/mg and a K {sub m} of 11 {+-} 4 {mu}M. This enzyme was inhibited by pyrazole with a K {sub I} of 3.1 {+-} 0.57 {mu}M. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V {sub max} of 115 nmol/min/mg and a K {sub m} of 15 {+-} 2 {mu}M and was not inhibited by pyrazole.

  16. Pharmacological recruitment of aldehyde dehydrogenase 3A1 (ALDH3A1) to assist ALDH2 in acetaldehyde and ethanol metabolism in vivo

    PubMed Central

    Chen, Che-Hong; Cruz, Leslie A.; Mochly-Rosen, Daria

    2015-01-01

    Correcting a genetic mutation that leads to a loss of function has been a challenge. One such mutation is in aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2. This mutation is present in ∼0.6 billion East Asians and results in accumulation of toxic acetaldehyde after consumption of ethanol. To temporarily increase metabolism of acetaldehyde in vivo, we describe an approach in which a pharmacologic agent recruited another ALDH to metabolize acetaldehyde. We focused on ALDH3A1, which is enriched in the upper aerodigestive track, and identified Alda-89 as a small molecule that enables ALDH3A1 to metabolize acetaldehyde. When given together with the ALDH2-specific activator, Alda-1, Alda-89 reduced acetaldehyde-induced behavioral impairment by causing a rapid reduction in blood ethanol and acetaldehyde levels after acute ethanol intoxication in both wild-type and ALDH2-deficient, ALDH2*1/*2, heterozygotic knock-in mice. The use of a pharmacologic agent to recruit an enzyme to metabolize a substrate that it usually does not metabolize may represent a novel means to temporarily increase elimination of toxic agents in vivo. PMID:25713355

  17. Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

    PubMed

    Hu, X Y; Fang, Q; Ma, D; Jiang, L; Yang, Y; Sun, J; Yang, C; Wang, J S

    2016-06-10

    Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

  18. Tryptophan in Alcoholism Treatment I:  Kynurenine Metabolites Inhibit the Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase Activity, Elevate Blood Acetaldehyde Concentration and Induce Aversion to Alcohol

    PubMed Central

    Badawy, Abdulla A.-B.; Bano, Samina; Steptoe, Alex

    2011-01-01

    Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of kynurenine metabolites to inhibit the liver mitochondrial low Km aldehyde dehydrogenase (ALDH) activity after administration and in vivo, and to induce aversion to alcohol. Methods: Kynurenic acid (KA), 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) were administered to normal male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring blood acetaldehyde following ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice. Results: ALDH activity was significantly inhibited by all three metabolites by doses as small as 1 mg/kg body wt. Blood acetaldehyde accumulation after ethanol administration was strongly elevated by KA and 3-HK and to a lesser extent by 3-HAA. All three metabolites induced aversion to alcohol in rats and decreased alcohol preference in mice. Conclusions: The above kynurenine metabolites of tryptophan induce aversion to alcohol by inhibiting ALDH activity. An intellectual property covering the use of 3-HK and 3-HAA and derivatives thereof in the treatment of alcoholism by aversion awaits further development. PMID:21896552

  19. The frequency of the mitochondrial aldehyde dehydrogenase I2 (atypical) allele in Caucasian, Oriental and African black populations determined by the restriction profile of PCR-amplified DNA.

    PubMed

    Dandré, F; Cassaigne, A; Iron, A

    1995-06-01

    The aldehyde dehydrogenase I (ALDH I) gene codes for a mitochondrial enzyme which plays a major role in hepatic alcohol detoxication. It has been related to alcohol flushing in Orientals bearing the atypical ALDH I2 gene. The variant protein results from a lysine for glutamate substitution at position 487 (G-->A change in exon 12). A procedure for ALDH I2 detection consisting in a differentiation between the 'atypical' allele and the 'wild' allele has been improved through PCR and subsequent MboII digestion. Blood samples collected on anticoagulant or directly absorbed on blotting paper were used for DNA amplification in the presence of two specific oligonucleotidic primers, each one able to incorporate a restriction site in the amplimer. After MboII digestion, PCR products were separated by polyacrylamide gel electrophoresis and then visualized with ethidium bromide. This technique permits a rapid and non-radioactive detection of atypical ALDH I2 on a PCR product without the use of allele specific oligonucleotides. It was applied to the study of ALDH I2 allele frequency in random population samples of three ethnic groups: Caucasians, Orientals and African blacks.

  20. Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina.

    PubMed

    Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

    2013-01-01

    Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.

  1. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry

    PubMed Central

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76–1.16] in abstainers with *2, 1.00 [0.80–1.26] in *1/*1 drinkers, 0.71 [0.54–0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28–6.13] in abstainers with *2, 1.89 [0.89–4.51] in *1/*1 drinkers, 2.35 [1.06–5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2

  2. Association of Genetically Determined Aldehyde Dehydrogenase 2 Activity with Diabetic Complications in Relation to Alcohol Consumption in Japanese Patients with Type 2 Diabetes Mellitus: The Fukuoka Diabetes Registry.

    PubMed

    Idewaki, Yasuhiro; Iwase, Masanori; Fujii, Hiroki; Ohkuma, Toshiaki; Ide, Hitoshi; Kaizu, Shinako; Jodai, Tamaki; Kikuchi, Yohei; Hirano, Atsushi; Nakamura, Udai; Kubo, Michiaki; Kitazono, Takanari

    2015-01-01

    Aldehyde dehydrogenase 2 (ALDH2) detoxifies aldehyde produced during ethanol metabolism and oxidative stress. A genetic defect in this enzyme is common in East Asians and determines alcohol consumption behaviors. We investigated the impact of genetically determined ALDH2 activity on diabetic microvascular and macrovascular complications in relation to drinking habits in Japanese patients with type 2 diabetes mellitus. An ALDH2 single-nucleotide polymorphism (rs671) was genotyped in 4,400 patients. Additionally, the relationship of clinical characteristics with ALDH2 activity (ALDH2 *1/*1 active enzyme activity vs. *1/*2 or *2/*2 inactive enzyme activity) and drinking habits (lifetime abstainers vs. former or current drinkers) was investigated cross-sectionally (n = 691 in *1/*1 abstainers, n = 1,315 in abstainers with *2, n = 1,711 in *1/*1 drinkers, n = 683 in drinkers with *2). The multiple logistic regression analysis for diabetic complications was adjusted for age, sex, current smoking habits, leisure-time physical activity, depressive symptoms, diabetes duration, body mass index, hemoglobin A1c, insulin use, high-density lipoprotein cholesterol, systolic blood pressure and renin-angiotensin system inhibitors use. Albuminuria prevalence was significantly lower in the drinkers with *2 than that of other groups (odds ratio [95% confidence interval (CI)]: *1/*1 abstainers as the referent, 0.94 [0.76-1.16] in abstainers with *2, 1.00 [0.80-1.26] in *1/*1 drinkers, 0.71 [0.54-0.93] in drinkers with *2). Retinal photocoagulation prevalence was also lower in drinkers with ALDH2 *2 than that of other groups. In contrast, myocardial infarction was significantly increased in ALDH2 *2 carriers compared with that in ALDH2 *1/*1 abstainers (odds ratio [95% CI]: *1/*1 abstainers as the referent, 2.63 [1.28-6.13] in abstainers with *2, 1.89 [0.89-4.51] in *1/*1 drinkers, 2.35 [1.06-5.79] in drinkers with *2). In summary, patients with type 2 diabetes and ALDH2 *2 displayed a

  3. Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

    PubMed Central

    Zheng, Tianyong; Olson, Daniel G.; Tian, Liang; Bomble, Yannick J.; Himmel, Michael E.; Lo, Jonathan; Hon, Shuen; Shaw, A. Joe; van Dijken, Johannes P.

    2015-01-01

    ABSTRACT Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. IMPORTANCE This work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of

  4. Co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of recombinant Klebsiella pneumoniae J2B strain overexpressing aldehyde dehydrogenase.

    PubMed

    Kumar, Vinod; Sankaranarayanan, Mugesh; Jae, Kyeung-Eun; Durgapal, Meetu; Ashok, Somasundar; Ko, Yeounjoo; Sarkar, Ritam; Park, Sunghoon

    2012-10-01

    The co-production of 3-hydroxypropionic acid (3HP) and 1,3-propanediol (PDO) from glycerol was studied using the resting cells of a recombinant Klebsiella pneumoniae J2B strain that overexpresses an aldehyde dehydrogenase (KGSADH). Active biomass was produced in a mineral salt medium containing yeast extract and glycerol under a range of aeration conditions, and shifted to potassium phosphate buffer containing glycerol for bioconversion. The microaerobic or anaerobic conditions were favorable for both the production of active biomass and subsequent bioconversion. At the flask level, the recombinant strain (2.0 g CDW/L) grown under microaerobic conditions produced 43.2 mM 3HP and 59.0 mM PDO from glycerol (117 mM) in 30 min with a cumulative yield of 0.87 (mol/mol). The fed-batch bioconversion, which was performed in a 1.5-L bioreactor with 1.0 g CDW/L at a constant pH 7.0 under anaerobic conditions, resulted in 125.6 mM 3HP and 209.5 mM PDO in 12 h with a cumulative overall productivity, yield, and maximum specific production rate of 27.9 mmol/L/h, 0.71 (mol/mol), and 128.5 mmol/g CDW/h, respectively. Lactate, succinate and 2,3-butanediol were the major by-products, whereas the production of acetate and ethanol was marginal. This is the first report of the simultaneous production of 3HP and PDO from glycerol using a resting cell system.

  5. Effects of Betaine Aldehyde Dehydrogenase-Transgenic Soybean on Phosphatase Activities and Rhizospheric Bacterial Community of the Saline-Alkali Soil

    PubMed Central

    Wang, Da-qing; Yu, Song

    2016-01-01

    The development of transgenic soybean has produced numerous economic benefits; however the potential impact of root exudates upon soil ecological systems and rhizospheric soil microbial diversity has also received intensive attention. In the present study, the influence of saline-alkali tolerant transgenic soybean of betaine aldehyde dehydrogenase on bacterial community structure and soil phosphatase during growth stages was investigated. The results showed that, compared with nontransgenic soybean as a control, the rhizospheric soil pH of transgenic soybean significantly decreased at the seedling stage. Compared to HN35, organic P content was 13.5% and 25.4% greater at the pod-filling stage and maturity, respectively. The acid phosphatase activity of SRTS was significantly better than HN35 by 12.74% at seedling, 14.03% at flowering, and 59.29% at podding, while alkaline phosphatase achieved maximum activity in the flowering stage and was markedly lower than HN35 by 13.25% at pod-filling. The 454 pyrosequencing technique was employed to investigate bacterial diversity, with a total of 25,499 operational taxonomic units (OTUs) obtained from the 10 samples. Notably, the effect of SRTS on microbial richness and diversity of rhizospheric soil was marked at the stage of podding and pod-filling. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla among all samples. Compared with HN35, the relative abundance of Proteobacteria was lower by 2.01%, 2.06%, and 5.28% at the stage of seedling, at pod-bearing, and at maturity. In genus level, the relative abundance of Gp6, Sphingomonas sp., and GP4 was significantly inhibited by SRTS at the stage of pod-bearing and pod-filling. PMID:27689079

  6. Effects of Betaine Aldehyde Dehydrogenase-Transgenic Soybean on Phosphatase Activities and Rhizospheric Bacterial Community of the Saline-Alkali Soil

    PubMed Central

    Wang, Da-qing; Yu, Song

    2016-01-01

    The development of transgenic soybean has produced numerous economic benefits; however the potential impact of root exudates upon soil ecological systems and rhizospheric soil microbial diversity has also received intensive attention. In the present study, the influence of saline-alkali tolerant transgenic soybean of betaine aldehyde dehydrogenase on bacterial community structure and soil phosphatase during growth stages was investigated. The results showed that, compared with nontransgenic soybean as a control, the rhizospheric soil pH of transgenic soybean significantly decreased at the seedling stage. Compared to HN35, organic P content was 13.5% and 25.4% greater at the pod-filling stage and maturity, respectively. The acid phosphatase activity of SRTS was significantly better than HN35 by 12.74% at seedling, 14.03% at flowering, and 59.29% at podding, while alkaline phosphatase achieved maximum activity in the flowering stage and was markedly lower than HN35 by 13.25% at pod-filling. The 454 pyrosequencing technique was employed to investigate bacterial diversity, with a total of 25,499 operational taxonomic units (OTUs) obtained from the 10 samples. Notably, the effect of SRTS on microbial richness and diversity of rhizospheric soil was marked at the stage of podding and pod-filling. Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla among all samples. Compared with HN35, the relative abundance of Proteobacteria was lower by 2.01%, 2.06%, and 5.28% at the stage of seedling, at pod-bearing, and at maturity. In genus level, the relative abundance of Gp6, Sphingomonas sp., and GP4 was significantly inhibited by SRTS at the stage of pod-bearing and pod-filling.

  7. Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.

    PubMed

    Twala, Busisiwe V; Sewell, B Trevor; Jordaan, Justin

    2012-05-10

    The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g⁻¹ displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg⁻¹ and 6.75 U mg⁻¹ for GDH and NOD with respective loading capacities of 51% (0.51 mg mg⁻¹) and 129% (1.29 mg mg⁻¹). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.

  8. Post-Translational Modification as a Potential Explanation of High Levels of Enzyme Polymorphism: Xanthine Dehydrogenase and Aldehyde Oxidase in DROSOPHILA MELANOGASTER

    PubMed Central

    Finnerty, Victoria; Johnson, George

    1979-01-01

    Xanthine dehydrogenase (XDH) and aldehyde oxidase (AO) in Drosophila melanogaster require for their activity the action of another unlinked locus, maroon-like (mal). While the XDH and AO loci are on chromosome 3, mal maps to the X chromosome. Although functional mal gene product is required for XDH and AO activity, it is possible to examine the effects of mutant mal alleles in those cases when pairs of mutants complement to produce a partial restoration of activity. To test whether mal mediates a post-translational modification of the XDH and AO proteins, we constructed several mal heteroallelic complementing stocks of Drosophila in which the third chromosomes were co-isogenic. Since all lines were co-isogenic for the XDH and AO structural genes, any variation in these enzymes seen when comparing these stocks must have been produced by post-translational modification by mal. We examined the XDH and AO proteins in these stocks by gel-sieving electrophoresis, a procedure that permits independent characterization of a protein's charge and shape, and is capable of discriminating many variants not detected in routine electrophoresis. In every mal heteroallelic combination, there is a significant alteration in protein shape, when compared to wild type. The magnitude of differences in shape of XDH and AO is correlated both with differences in their enzyme activities and with differences in their thermal stabilities. As the body of this variation appears heritable, any functional differences resulting from these variants are of real genetic and evolutionary interest. A similar post-translational modification of XDH and AO by yet another locus, lxd, was subsequently documented in an analogous manner. The pattern of electrophoretic differences produced by mal and lxd modification is similar to that reported for electrophoretic "alleles" of XDH in natural populations. The implication is that heritable variation in electrophoretic mobility at these two enzyme loci, and

  9. Structural and functional consequences of coenzyme binding to the inactive asian variant of mitochondrial aldehyde dehydrogenase: roles of residues 475 and 487.

    PubMed

    Larson, Heather N; Zhou, Jianzhong; Chen, Zhiqiang; Stamler, Jonathan S; Weiner, Henry; Hurley, Thomas D

    2007-04-27

    The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2(*)2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k(cat) for these processes and increases the K(m) for NAD(+), as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2(*)2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2(*)2 complexed with coenzyme to 2.5A(.) We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2(*)2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2(*)2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2(*)2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme-binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.

  10. Association between Carotid Intima-media Thickness and Aldehyde Dehydrogenase 2 Glu504Lys Polymorphism in Chinese Han with Essential Hypertension

    PubMed Central

    Ma, Xiao-Xiang; Zheng, Shu-Zhan; Shu, Yan; Wang, Yong; Chen, Xiao-Ping

    2016-01-01

    Background: Aldehyde dehydrogenase 2 (ALDH2) is involved in the pathophysiological processes of cardiovascular diseases. Recent studies showed that mutant ALDH2 could increase oxidative stress and is a susceptible factor for hypertension. In addition, wild-type ALDH2 could improve the endothelial functions, therefore reducing the risk of developing atherosclerosis. The aim of the present study was to explore the frequency of the Glu504Lys polymorphism of the ALDH2 gene and its relation to carotid intima-media thickness (CIMT) in a group of patients with essential hypertension (EH) and to investigate the association between the Glu504Lys polymorphism and CIMT in Chinese Han patients with EH. Methods: In this study, 410 Chinese Han patients with EH who received physical examinations at the People's Hospital of Sichuan Province (China) were selected. DNA microarray chip was used for the genotyping of the Glu504Lys polymorphism of the ALDH2 gene. The differences in CIMT among patients with different Glu504Lys ALDH2 genotypes were analyzed. Results: The mean CIMT of the patients carrying AA/AG and GG genotypes was 1.02 ± 0.31 mm and 0.78 ± 0.28 mm, respectively. One-way ANOVA showed that the CIMT of the patients carrying the AA/AG genotype was significantly higher than in the ones carrying the GG genotype (P < 0.001). Multivariate logistic regression showed that the Glu504Lys AA/AG genotype of the ALDH2 gene was one of the major factors influencing the CIMT in patients with EH (odds ratio = 3.731, 95% confidence interval = 1.589–8.124, P = 0.001). Conclusions: The Glu504Lys polymorphism of the ALDH2 gene is associated with the CIMT of Chinese Han patients with EH in Sichuan, China. PMID:27270535

  11. Structural and Functional Consequences of Coenzyme Binding to the Inactive Asian Variant of Mitochondrial Aldehyde Dehydrogenase: Roles of Residues 475 and 487

    SciTech Connect

    Larson,H.; Zhou, J.; Chen, Z.; Stamler, J.; Weiner, H.; Hurley, T.

    2007-01-01

    The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2*2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k{sub cat} for these processes and increases the K{sub m} for NAD{sup +}, as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2*2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2*2 complexed with coenzyme to 2.5 {angstrom}. We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2*2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2*2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2*2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.

  12. 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

  13. The use of pH-gradient ion-exchange chromatography to separate sheep liver cytoplasmic aldehyde dehydrogenase from mitochondrial enzyme contamination, and observations on the interaction between the pure cytoplasmic enzyme and disulfiram.

    PubMed Central

    Dickinson, F M; Hart, G J; Kitson, T M

    1981-01-01

    1. Sheep liver cytoplasmic aldehyde dehydrogenase can be purified from contamination with the mitochondrial form of the enzyme by pH-gradient ion-exchange chromatography. The method is simple, reproducible and efficient. 2. The purified cytoplasmic enzyme retains about 2% of its original activity in the presence of a large excess of disulfiram. This suggests that the disulfiram-reactive thiol groups are not essential for covalent interaction with the aldehyde substrate during catalysis, as has sometimes been suggested. 3. Between 1.5 and 2.0 molecules of disulfiram per tetrameric enzyme molecule account for the observed loss of activity, suggesting that the enzyme may have only two functional active sites. 4. Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH. PMID:7340819

  14. Differential effects of organic nitrates on arterial diameter among healthy Japanese participants with different mitochondrial aldehyde dehydrogenase 2 genotypes: randomised crossover trial

    PubMed Central

    Sakata, Satoko; Yoshihara, Tatsuya; Arima, Hisatomi; Shiraishi, Fumie; Oniki, Hideyuki; Takahashi-Yanaga, Fumi; Matsumura, Kiyoshi

    2011-01-01

    Objectives To determine whether polymorphisms at codon 487 (*1, GAA=Glu; *2, AAA=Lys) of mitochondrial aldehyde dehydrogenase 2 (ALDH2) influence nitroglycerine (glyceryl trinitrate (GTN))-induced vasodilation, and whether GTN or isosorbide dinitrate (ISDN) is a more effective antianginal agent in each ALDH2 genotype. Design A randomised, open-label, crossover trial with 117 healthy Japanese (20–39 years) whose genotypes were determined (*1/*1, n=47; *1/*2, n=48; *2/*2, n=22) was performed at Kyushu University Hospital, Fukuoka, Japan. Participants were randomly assigned to treatment: sublingual spray of GTN (0.3 mg) or ISDN (1.25 mg). After ≥1 week, measurements were repeated using the other drug. The main outcome measures were the maximal rate of increase in the brachial artery diameter determined by ultrasonography, the time required to attain maximal dilation (Tmax) and the time required to attain 90% maximal dilation (T0.9). Results The maximal artery diameter increase in response to GTN or ISDN did not differ among genotypes. However, GTN Tmax was significantly longer for *2/*2 (299.7 s, 269.0–330.4) than *1/*1 (254.7 s, 238.6–273.4; p=0.0190). GTN T0.9 was significantly longer in the *1/*2 (206.1 s, 191.7–219.3) and *2/*2 (231.4 s, 211.8–251.0) genotypes than *1/*1 (174.9 s, 161.5–188.3; p=0.0068, p<0.0001, respectively). In contrast, the time-course of ISDN-induced vasodilation did not differ among genotypes. GTN Tmax and T0.9 among *1 allele carriers (*1/*1 and *1/*2) were significantly shorter than those of ISDN, whereas the time course of GTN and ISDN vasodilation did not differ among participants carrying *2/*2. Conclusions The amplitude of GTN-induced vasodilation was not influenced by the ALDH2 genotype, but the response was significantly delayed in *2 allele carriers, especially *2/*2. GTN dilated the artery more quickly than ISDN in *1/*1 and *1/*2, but not in *2/*2. Trial registration number UMIN000001492 (UMIN

  15. High ethanol and acetaldehyde impair spatial memory in mouse models: opposite effects of aldehyde dehydrogenase 2 and apolipoprotein E on memory.

    PubMed

    Jamal, Mostofa; Ameno, Kiyoshi; Miki, Takanori; Tanaka, Naoko; Ono, Junichiro; Shirakami, Gotaro; Sultana, Ruby; Yu, Nakamura; Kinoshita, Hiroshi

    2012-05-01

    Aldehyde dehydrogenase 2 deficiency may directly contribute to excess acetaldehyde (AcH) accumulation after ethanol (EtOH) drinking and AcH mediates some of the behavioral effects of EtOH. Apolipoprotein E has been suggested to be involved in the alteration of attention and memory. We have chosen Aldh2-knockout (Aldh2-KO), ApoE-KO, and their wild-type (WT) control mice to examine the effects of EtOH and AcH on spatial memory and to compare the possible relationship between genetic deficiency and memory using two behavioral assessments. Mice were trained for 4 days, with EtOH (0.5, 1.0, 2.0 g/kg) being given intraperitoneally on day 4. A probe trial was given on day 5 in the non-EtOH state in the Morris water maze (MWM). The results showed that 2.0 g/kg EtOH increased errors, indicating memory impairment on the eight-arm radial maze (RAM) for all the mice studied. One gram per kilogram EtOH impaired the performance of Aldh2-KO and ApoE-KO mice, but not WT mice. We found similar effects of EtOH on the MWM performance, with 2.0 g/kg EtOH increasing the latencies. One gram per kilogram EtOH increased the latencies of Aldh2-KO and WT mice, but not ApoE-KO mice. The 2.0 g/kg EtOH-induced memory impairment in Aldh2-KO mice was greater, suggesting an AcH effect. Furthermore, time spent on the probe trial was shorter in mice that had previously received 2.0 g/kg EtOH. ApoE-KO mice learned more slowly, while Aldh2-KO mice learned more quickly. Both the RAM and MWM results suggest that high EtOH and AcH impair spatial memory in mice, while lower doses do not have consistent memory effects. In addition, we conclude that genetic differences might underlie some of EtOH's effects on memory.

  16. The effect of peroxynitrite decomposition catalyst MnTBAP on aldehyde dehydrogenase-2 nitration by organic nitrates: role in nitrate tolerance.

    PubMed

    Mollace, Vincenzo; Muscoli, Carolina; Dagostino, Concetta; Giancotti, Luigino Antonio; Gliozzi, Micaela; Sacco, Iolanda; Visalli, Valeria; Gratteri, Santo; Palma, Ernesto; Malara, Natalia; Musolino, Vincenzo; Carresi, Cristina; Muscoli, Saverio; Vitale, Cristiana; Salvemini, Daniela; Romeo, Francesco

    2014-11-01

    Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event

  17. The novel myxofibrosarcoma cell line MUG-Myx1 expresses a tumourigenic stem-like cell population with high aldehyde dehydrogenase 1 activity

    PubMed Central

    2013-01-01

    Background Myxofibrosarcoma comprises a spectrum of malignant neoplasms withprominent myxoid stromata, cellular pleomorphism, and distinct curvilinear vascular patterns. These neoplasms mainly affect patients in the sixth to eighth decades of life and the overall 5-year survival rate is 60–70%. Methods After the establishment of the novel myxofibrosarcoma cell lines MUG-Myx1, cells were characterized using short tandem repeat (STR), copy number variation (CNV), and genotype/loss-of-heterozygosity (LOH) analyses. The growth behaviour of the cells was analyzed with the xCELLigence system and an MTS assay. The tumourigenicity of MUG-Myx1 was proved in NOD/SCID mice. Additionally, a stem-like cell population with high enzymatic activity of aldehyde dehydrogenase 1 (ALDH1high) was isolated for the first time from myxofibrosarcoma cells using the Aldefluor® assay followed by FACS analysis. Results The frozen primary parental tumour tissue and the MUG-Myx1 cell line showed the same STR profile at the markers D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, D8S1179, TPOX, and FGY. Typically, myxofibrosarcoma gain and/or amplification was mapped to 7p21.3-q31.1, q31.1-q31.33, q33-q36.2, p21.3, p21.2, p14.1-q11.23, q31.33-q33, p21.2-p14.1, q11.23-q21.3, q36.2-q36.3, which, respectively are known to harbour tumour-associated genes, including TIF, BRAF, MLL3, SMO, and MET. Typically an LOH for myxofibrosarcoma on chr5 q21 was found. In addition, MUG-Myx1 ALDH1high cells showed an upregulation of the ABC transporter ABCB1 and ABCG2; higher c-Myc, E-cadherin and SOX-2 expression; and a higher potential for tumourigenicity and proliferation levels. Conclusion The new myxofibrosarcoma cell line MUG-Myx1 was established to enrich the bank of publicly available cell lines, with respect to providing comprehensive genetic and epigenetic characterization. Furthermore, because of their tumourigenicity, the cell line is also

  18. The effect of peroxynitrite decomposition catalyst MnTBAP on aldehyde dehydrogenase-2 nitration by organic nitrates: role in nitrate tolerance.

    PubMed

    Mollace, Vincenzo; Muscoli, Carolina; Dagostino, Concetta; Giancotti, Luigino Antonio; Gliozzi, Micaela; Sacco, Iolanda; Visalli, Valeria; Gratteri, Santo; Palma, Ernesto; Malara, Natalia; Musolino, Vincenzo; Carresi, Cristina; Muscoli, Saverio; Vitale, Cristiana; Salvemini, Daniela; Romeo, Francesco

    2014-11-01

    Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event

  19. Barley peroxidase isozymes

    NASA Astrophysics Data System (ADS)

    Laugesen, Sabrina; Bak-Jensen, Kristian Sass; Hägglund, Per; Henriksen, Anette; Finnie, Christine; Svensson, Birte; Roepstorff, Peter

    2007-12-01

    Thirteen peroxidase spots on two-dimensional gels were identified by comprehensive proteome analysis of the barley seed. Mass spectrometry tracked multiple forms of three different peroxidase isozymes: barley seed peroxidase 1, barley seed-specific peroxidase BP1 and a not previously identified putative barley peroxidase. The presence of multiple spots for each of the isozymes reflected variations in post-translational glycosylation and protein truncation. Complete sequence coverage was achieved by using a series of proteases and chromatographic resins for sample preparation prior to mass spectrometric analysis. Distinct peroxidase spot patterns divided the 16 cultivars tested into two groups. The distribution of the three isozymes in different seed tissues (endosperm, embryo, and aleurone layer) suggested the peroxidases to play individual albeit partially overlapping roles during germination. In summary, a subset of three peroxidase isozymes was found to occur in the seed, whereas products of four other barley peroxidase genes were not detected. The present analysis documents the selective expression profiles and post-translational modifications of isozymes from a large plant gene family.

  20. RNAi-directed downregulation of betaine aldehyde dehydrogenase 1 (OsBADH1) results in decreased stress tolerance and increased oxidative markers without affecting glycine betaine biosynthesis in rice (Oryza sativa).

    PubMed

    Tang, Wei; Sun, Jiaqi; Liu, Jia; Liu, Fangfang; Yan, Jun; Gou, Xiaojun; Lu, Bao-Rong; Liu, Yongsheng

    2014-11-01

    As an important osmoprotectant, glycine betaine (GB) plays an essential role in resistance to abiotic stress in a variety of organisms, including rice (Oryza sativa L.). However, GB content is too low to be detectable in rice, although rice genome possesses several orthologs coding for betaine aldehyde dehydrogenase (BADH) involved in plant GB biosynthesis. Rice BADH1 (OsBADH1) has been shown to be targeted to peroxisome and its overexpression resulted in increased GB biosynthesis and tolerance to abiotic stress. In this study, we demonstrated a pivotal role of OsBADH1 in stress tolerance without altering GB biosynthesis capacity, using the RNA interference (RNAi) technique. OsBADH1 was ubiquitously expressed in different organs, including roots, stems, leaves and flowers. Transgenic rice lines downregulating OsBADH1 exhibited remarkably reduced tolerance to NaCl, drought and cold stresses. The decrease of stress tolerance occurring in the OsBADH1-RNAi repression lines was associated with an elevated level of malondialdehyde content and hydrogen peroxidation. No GB accumulation was detected in transgene-positive and transgene-negative lines derived from heterozygous transgenic T0 plants. Moreover, transgenic OsBADH1-RNAi repression lines showed significantly reduced seed set and yield. In conclusion, the downregulation of OsBADH1, even though not causing any change of GB content, was accounted for the reduction of ability to dehydrogenate the accumulating metabolism-derived aldehydes and subsequently resulted in decreased stress tolerance and crop productivity. These results suggest that OsBADH1 possesses an enzyme activity to catalyze other aldehydes in addition to betaine aldehyde (the precursor of GB) and thus alleviate their toxic effects under abiotic stresses.

  1. The essential function of genes for a hydratase and an aldehyde dehydrogenase for growth of Pseudomonas sp. strain Chol1 with the steroid compound cholate indicates an aldolytic reaction step for deacetylation of the side chain.

    PubMed

    Holert, Johannes; Jagmann, Nina; Philipp, Bodo

    2013-08-01

    In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD(+) as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

  2. Human {gamma}-aminobutyraldehyde dehydrogenase (ALDH9): cDNA sequence, genomic organization, polymorphism, chromosomal localization, and tissue expression

    SciTech Connect

    Lin, S.W.; Chen, J.C.; Hsu, L.C.

    1996-06-15

    The cDNA and the gene (ALDH9) for a human aldehyde dehydrogenase isozyme, which has a high activity for oxidation of {gamma}-aminobutyraldehyde and other amino aldehydes, were cloned and characterized. The cDNA has an open reading frame of 1479 bp encoding 493 amino acid residues. The gene is about 45 kb and consists of 10 coding exons interrupted by nine introns. The gene was assigned to chromosome 1q22-q23, using fluorescence in situ hybridization. Northern blot hybridization indicated that the size of the mRNA is about 2.4 kb and that the gene is expressed at high levels in adult liver, skeletal muscle, and kidney and low levels in heart, pancreas, lung, and brain. The gene is polymorphic, i.e., C or T at nt 327 and C or G at nt 344. 17 refs., 4 figs., 1 tab.

  3. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells

    PubMed Central

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-01-01

    Background Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. Methods We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. Results We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by ≥ 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. Conclusion These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer. PMID:19025616

  4. Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola)

    PubMed Central

    Huss, M. J.; Campbell, C. L.; Jennings, D. B.; Leslie, J. F.

    1996-01-01

    Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven

  5. Biliverdin reductase isozymes in metabolism.

    PubMed

    O'Brien, Luke; Hosick, Peter A; John, Kezia; Stec, David E; Hinds, Terry D

    2015-04-01

    The biliverdin reductase (BVR) isozymes BVRA and BVRB are cell surface membrane receptors with pleiotropic functions. This review compares, for the first time, the structural and functional differences between the isozymes. They reduce biliverdin, a byproduct of heme catabolism, to bilirubin, display kinase activity, and BVRA, but not BVRB, can act as a transcription factor. The binding motifs present in the BVR isozymes allow a wide range of interactions with components of metabolically important signaling pathways such as the insulin receptor kinase cascades, protein kinases (PKs), and inflammatory mediators. In addition, serum bilirubin levels have been negatively associated with abdominal obesity and hypertriglyceridemia. We discuss the roles of the BVR isozymes in metabolism and their potential as therapeutic targets. PMID:25726384

  6. [Effects of panthenol and carnitine on aldehyde metabolic enzymes in rats with tetrachloromethane-induced liver injury].

    PubMed

    Satanovskaia, V I; Pron'ko, P S; Gaĭshmanova, A V; Miskevich, D A

    2009-01-01

    Tetrachloromethane (2 g/kg, intragastric) produced a decrease in the activity of NAD- and NADH- dependent aldehyde dehydrogenases with high Km for aldehydes in rat liver. Panthenol and L-carnitine administered separately normalized the activity of aldehyde dehydrogenases, while a combination of the drugs did not produce any significant effect. PMID:19441727

  7. Purification, crystallization and preliminary X-ray analysis of recombinant betaine aldehyde dehydrogenase 2 (OsBADH2), a protein involved in jasmine aroma, from Thai fragrant rice (Oryza sativa L.)

    PubMed Central

    Kuaprasert, Buabarn; Silprasit, Kun; Horata, Natharinee; Khunrae, Pongsak; Wongpanya, Ratree; Boonyalai, Nonlawat; Vanavichit, Apichart; Choowongkomon, Kiattawee

    2011-01-01

    Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD+. Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N2 immediately prior to X-ray diffraction experiments. The crystals belonged to space group C2221, with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress. PMID:22102032

  8. [The PQQ-dehydrogenases. A novel example of bacterial quinoproteins].

    PubMed

    Flores-Encarnación, Marcos; Sánchez-Cuevas, Mariano; Ortiz-Gutiérrez, Felipe

    2004-01-01

    The word "quinoprotein" describes four groups of different enzymes which have cofactors containing o-quinones. Pyrrolo-quinoline quinone (PQQ) is not covalently attached. PQQ is the cofactor of several quinoprotein bacterial dehydrogenases including glucose dehydrogenase (G-DH), alcohol dehydrogenase (A-DH) and aldehyde dehydrogenase (AL-DH). These dehydrogenases are located in the periplasm of Gram-negative bacteria. This report summarises the structural properties of quinoprotein dehydrogenases, such as the biological functions and biotechnological aspects more important.

  9. 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...

  10. The Crystal Structure of a Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight Into the Reaction Mechansim and Shows A Novel Binding Mode of the 2'-Phosphate of NADP+ and A Novel Cation Binding Site

    SciTech Connect

    Gonzalez-Segura, L.; Rudino-Pinera, E; Munoz-Clares, R; Horjales, E

    2009-01-01

    In the human pathogen Pseudomonas aeruginosa, the NAD(P)+-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors-abundant at infection sites-and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP+ and one of the even fewer that require K+ ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP+ and K+ ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the 'oxyanion hole.' The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2?-phosphate of the NADP+, thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K+ binding sites per subunit. One is in an

  11. The crystal structure of a ternary complex of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa Provides new insight into the reaction mechanism and shows a novel binding mode of the 2'-phosphate of NADP+ and a novel cation binding site.

    PubMed

    González-Segura, Lilian; Rudiño-Piñera, Enrique; Muñoz-Clares, Rosario A; Horjales, Eduardo

    2009-01-16

    In the human pathogen Pseudomonas aeruginosa, the NAD(P)(+)-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP(+) and one of the even fewer that require K(+) ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP(+) and K(+) ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the "oxyanion hole." The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2'-phosphate of the NADP(+), thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K(+) binding sites per subunit

  12. Plant Triose Phosphate Isomerase Isozymes 1

    PubMed Central

    Pichersky, Eran; Gottlieb, Leslie D.

    1984-01-01

    We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16663420

  13. On the distribution and characteristics of isozyme expression in Mycoplasma, Acholeplasma, and Ureaplasma species.

    PubMed Central

    O'Brien, S. J.; Simonson, J. M.; Razin, S.; Barile, M. F.

    1983-01-01

    A summary of a survey of three genera of mycoplasmatales (Mycoplasma, Acholeplasma, and Ureaplasma) for isozyme expression is presented. Isozyme analysis of mycoplasmas has been employed in at least three distinct areas: (1) as genetic markers for identification, individualization, and taxonomic classification; (2) as markers for cell culture contamination; and (3) as a qualitative measure of the operative metabolic pathways in the diverse species. We have found five ubiquitous enzymes: purine nucleoside phosphorylase, adenylate kinase, inorganic pyrophosphatase, dipeptidase, and esterase. Three enzymes, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, and superoxide dismutase, were restricted to Acholeplasma species and were not detected in Mycoplasma or Ureaplasma. Four glycolytic enzymes, glucose phosphate isomerase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase, were restricted to those species of Mycoplasma and Acholeplasma capable of glucose fermentation. Two of these glycolytic enzymes, glucose phosphate isomerase and lactate dehydrogenase, were detected in serovars I and II of U. urealyticum, which is inconsistent with the non-glycolytic activity in this genus. Images FIG. 1 FIG. 2 FIG. 3 PMID:6679151

  14. New Ideas for an Old Enzyme: A Short, Question-Based Laboratory Project for the Purification and Identification of an Unknown LDH Isozyme

    ERIC Educational Resources Information Center

    Coleman, Aaron B.

    2010-01-01

    Enzyme purification projects are an excellent way to introduce many aspects of protein biochemistry, but can be difficult to carry out under the constraints of a typical undergraduate laboratory course. We have designed a short laboratory project for the purification and identification of an "unknown" lactate dehydrogenase (LDH) isozyme that can…

  15. Purification and some properties of peroxidase isozymes from pineapple stem.

    PubMed

    Sung, H Y; Yu, R H; Chang, C T

    1993-01-01

    The enzyme peroxidase is widely distributed among the higher plants. Isozymes of peroxidase are known to occur in a variety of tissues in a large number of plant species. In this study, peroxidase isozymes were purified from the extract of pineapple stem through successive steps of ammonium sulfate fractionation, CM-Sepharose CL-6B chromatographies and DEAE-Sepharose CL-6B chromatographies. By these steps, twelve isozymes of peroxidase were obtained. Some properties of the isozymes were studied and compared.

  16. Isozyme variation in wild and cultivated pineapple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isozyme variation was studied in 161 accessions of pineapple including four species of Ananas and one of Pseudananas. Six enzyme systems (ADH, GPI, PGM, SKDH, TPI, UGPP) involving seven putative loci revealed 35 electromorphs . Considerable variation exists within and between species of Ananas. Sixt...

  17. Kynurenine Aminotransferase Isozyme Inhibitors: A Review

    PubMed Central

    Nematollahi, Alireza; Sun, Guanchen; Jayawickrama, Gayan S.; Church, W. Bret

    2016-01-01

    Kynurenine aminotransferase isozymes (KATs 1–4) are members of the pyridoxal-5’-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies. PMID:27314340

  18. Fatty Aldehydes in Cyanobacteria Are a Metabolically Flexible Precursor for a Diversity of Biofuel Products

    PubMed Central

    Kaiser, Brett K.; Carleton, Michael; Hickman, Jason W.; Miller, Cameron; Lawson, David; Budde, Mark; Warrener, Paul; Paredes, Angel; Mullapudi, Srinivas; Navarro, Patricia; Cross, Fred; Roberts, James M.

    2013-01-01

    We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis. PMID:23505484

  19. [Activity of aldehyde scavenger enzymes in the heart of rats of different age during immobilized stress].

    PubMed

    Grabovetskaia, E R; Davydov, V V

    2009-01-01

    This study was made to determine the activity of aldehyde scavenger enzymes in the heart's postmitochondrial fraction of rats of different age during immobilization stress. Our study demonstrated, that immobilization of 1.5-, 2- and 12-month rats was accompanied by inhibiting activity of aldehyde dehydrogenase and aldehyde reductase. At the same time we observed an increase in glutathione transferase activity in immobilized 1.5-month-old rats and that in reductase activity in 24-month-old rats. The revealed changes can lead to a decrease in the rate of endogenous aldehyde utilization in the heart during stress at puberty.

  20. Possible role of alteration of aldehyde's scavenger enzymes during aging.

    PubMed

    Davydov, Vadim V; Dobaeva, Nataly M; Bozhkov, Anatoly I

    2004-01-01

    Apoptosis in tissues is induced by different kind of signals including endogenous aldehydes, such as 4-hydroxy-2, 3-nonenal. The accumulation rate of aldehydes in the cell is affected by conditions of oxidative stress. In the cell, aldehydes can be metabolized by various isoforms of aldehyde dehydrogenase, aldehyde reductase, and glutathione-S-transferase. There is evidence suggesting that the catalytic properties of these enzymes change during ontogenesis, and that aging is accompanied by their reduced activities. These functional changes may contribute substantially to the alteration in the organism sensitivity to damaging action of stress factors during aging, to age-related modulation of the action of endogenous aldehydes as a signal for apoptosis, and finally, to the origin of diseases associated with aging. In this context, the stimulation of enzymes' expression, and the activation of the catalytic properties of enzymes responsible for catabolism of endogenous aldehydes could become a perspective direction in increasing the organism resistance to the action of damaging factors during aging.

  1. Microbial Engineering for Aldehyde Synthesis

    PubMed Central

    Kunjapur, Aditya M.

    2015-01-01

    Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity. PMID:25576610

  2. Betaine aldehyde, betaine, and choline levels in rat livers during ethanol metabolism.

    PubMed

    Chern, M K; Gage, D A; Pietruszko, R

    2000-12-01

    Betaine aldehyde levels were determined in rat livers following 4 weeks of ethanol feeding, employing the Lieber-De Carli liquid diet. The results showed that the levels of betaine aldehyde are unaffected by alcohol feeding to rats. These levels in both experimental and control animals were found to be quite low, 5.5 nmol/g liver. Betaine aldehyde levels have not been determined previously in mammalian liver because of methodological difficulties. This investigation employed fast atom bombardment-mass spectroscopy to determine the levels of betaine aldehyde, betaine, and choline. The decrease in betaine levels following ethanol administration confirmed the results of other investigators. Choline levels determined during this investigation were lower than previously reported. The reason for starting this investigation was the fact that the enzyme that catalyzes betaine aldehyde dehydrogenation to betaine, which is distributed in both mitochondria and the cytoplasm, was found to also metabolize acetaldehyde with K(m) and V(max) values lower than those for betaine aldehyde. Thus, it appeared likely that the metabolism of acetaldehyde during ethanol metabolism might inhibit betaine aldehyde conversion to betaine and thereby result in decreased betaine levels (Barak et al., Alcohol 13: 395-398, 1996). The fact that betaine aldehyde levels in alcohol-fed animals were similar to those in controls demonstrates that competition between acetaldehyde and betaine aldehyde for the same enzyme does not occur. This complete lack of competition suggests that betaine aldehyde dehydrogenase in the mitochondrial matrix may totally metabolize betaine aldehyde to betaine without any involvement of cytoplasmic betaine aldehyde dehydrogenase. PMID:11077045

  3. Molecular evolutionary genetics of isozymes: pattern, theory, and application.

    PubMed

    Nevo, E

    1990-01-01

    Isozyme studies at the population genetics-ecology interface conducted at the Institute of Evolution, University of Haifa, during 15 years, 1974-1989, are reviewed in terms of the evidence, theoretical, and practical implications. These studies involve numerous individuals, populations, species, and higher taxa in nature of plants, animals, and humans tested for variation at 15 to 50 primary isozyme loci. The isozyme studies have been conducted mainly in individuals sampled in natural populations at the local, regional, and global levels. Two of the species studied were wild cereals, the progenitors of wheat and barley in the Near East Fertile Crescent. These studies have been complemented by laboratory controlled a priori experimentation of inorganic and organic pollution biology. The human genetics laboratory compared isozyme structure of Jewish and non-Jewish populations. Our results indicate that: (i) isozyme diversity in nature in abundant, at least partly adaptive, and is oriented and maintained primarily by ecological factors. (ii) Natural selection in action is highlighted by stresses involving among others thermal, chemical, and climatic factors. (iii) Speciation can occur with little change in isozyme diversity. (iv) Jews from diverse countries, and in spite of 2,000 years of Diaspora, retain in the frequencies of some isozymes their Near Eastern origins. (v) Wild cereals harbor rich genetic resources exploitable in breeding either directly as adaptive structures, or indirectly as genetic markers for genotypic production of elite agronomic traits. (vi) Isozymes have been utilized as genetic monitors of marine pollution thereby contributing to environmental quality and conservation. (vii) Isozymes can substantially contribute to conservation biology. (viii) Isozymes have been successfully utilized in constructing molecular phylogenies and in revealing new sibling species. (ix) Future theoretical and practical directions of isozyme studies at the protein

  4. Molecular evolutionary genetics of isozymes: pattern, theory, and application.

    PubMed

    Nevo, E

    1990-01-01

    Isozyme studies at the population genetics-ecology interface conducted at the Institute of Evolution, University of Haifa, during 15 years, 1974-1989, are reviewed in terms of the evidence, theoretical, and practical implications. These studies involve numerous individuals, populations, species, and higher taxa in nature of plants, animals, and humans tested for variation at 15 to 50 primary isozyme loci. The isozyme studies have been conducted mainly in individuals sampled in natural populations at the local, regional, and global levels. Two of the species studied were wild cereals, the progenitors of wheat and barley in the Near East Fertile Crescent. These studies have been complemented by laboratory controlled a priori experimentation of inorganic and organic pollution biology. The human genetics laboratory compared isozyme structure of Jewish and non-Jewish populations. Our results indicate that: (i) isozyme diversity in nature in abundant, at least partly adaptive, and is oriented and maintained primarily by ecological factors. (ii) Natural selection in action is highlighted by stresses involving among others thermal, chemical, and climatic factors. (iii) Speciation can occur with little change in isozyme diversity. (iv) Jews from diverse countries, and in spite of 2,000 years of Diaspora, retain in the frequencies of some isozymes their Near Eastern origins. (v) Wild cereals harbor rich genetic resources exploitable in breeding either directly as adaptive structures, or indirectly as genetic markers for genotypic production of elite agronomic traits. (vi) Isozymes have been utilized as genetic monitors of marine pollution thereby contributing to environmental quality and conservation. (vii) Isozymes can substantially contribute to conservation biology. (viii) Isozymes have been successfully utilized in constructing molecular phylogenies and in revealing new sibling species. (ix) Future theoretical and practical directions of isozyme studies at the protein

  5. Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus.

    PubMed Central

    Gillooly, D J; Robertson, A G; Fewson, C A

    1998-01-01

    The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases. PMID:9494109

  6. Aldehyde-stabilized cryopreservation.

    PubMed

    McIntyre, Robert L; Fahy, Gregory M

    2015-12-01

    We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at -135 °C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays. PMID:26408851

  7. Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

    PubMed

    Kuo, D J; Dikdan, G; Jordan, F

    1986-03-01

    A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.

  8. General and Versatile Autoinhibition of PLC Isozymes

    SciTech Connect

    Hicks, Stephanie N.; Jezyk, Mark R.; Gershburg, Svetlana; Seifert, Jason P.; Harden, T. Kendall; Sondek, John

    2008-10-31

    Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-{beta}2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-{beta}2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.

  9. Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C.

    PubMed

    Kusakabe, T; Motoki, K; Sugimoto, Y; Hori, K

    1998-08-01

    To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172-7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677-83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387-93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243-306) may modulate the interaction of IGS units with the alpha/beta barrel of the aldolase molecule.

  10. Phylogeny congruence analysis and isozyme classification: the pyruvate kinase system.

    PubMed

    Guderley, H; Fournier, P; Auclair, J C

    1989-09-22

    As the isozymes of pyruvate kinase (PK) are best known in rats, the characteristics of the rat isozymes are generally used to classify the PK isozymes in other species. Given the discrepancies generated by this classification by analogy, we evaluated a classification using a phylogeny congruence analysis of the compositional relatedness of vertebrate PK's. While our phylogenetic analysis confirmed the well established separation of the L and R isozymes from the K and M isozymes, its power became most evident in the identification of non-orthologous (or variant) forms of PK. Our analysis emphasized the uniqueness of chicken liver PK which cannot be classified either as a K or an L isozyme, confirmed that tumors express a variety of forms of PK, and indicated that lungs systematically express PK's which are not orthologous with PK's from other tissues. The determination of orthology by the phylogeny congruence analysis assumes that the structural data from different sources are subject to similar methodological error. However, we cannot reject the possibility that an apparent lack of orthology be due to artifacts during purification and analysis. PMID:2615396

  11. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-11-05

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease.

  12. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  13. Glutamic oxaloacetic transaminase isozymes from rat liver. Purification and physicochemical characterization.

    PubMed

    Huynh, Q K; Sakakibara, R; Watanabe, T; Wada, H

    1980-07-01

    Glutamic oxaloacetic transaminase isozymes were purified simultaneously to homogeneity from rat liver with high yields. Three subforms of mitochondrial isozyme and three subforms of cytosolic isozyme were separated by chromatography on CM-Sephadex and electrophoresis on polyacrylamide gel. The general enzymatic properties of the purified isozymes such as their kinetic parameters, isoelectric points, molecular weights, amino acid compositions, NH2-terminal amino acid sequences and COOH-terminal amino acids were determined. Most of these properties of the isozymes are similar to those of the corresponding isozymes from other sources, such as rat brain and pig and human heart. In amino acid compositions, cytosolic isozyme from rat liver has more proline and glycine and less arginine, threonine and leucine than pig heart cytosolic isozyme; the mitochondrial isozyme has more glutamic acid and glycine and less serine than the corresponding pig heart isozyme. The NH2-terminal amino acid sequences of GOT isozymes from rat liver were identical with those of the GOT isozymes from pig heart up to the 10th residues except for the 5th residues. The subforms of mitochondrial isozyme from rat liver were generated on storage at 4 degrees C for 4-8 weeks.

  14. Antioxidant and isozyme features of two strains of Laminaria japonica (Phaeophyceae)

    NASA Astrophysics Data System (ADS)

    Wang, You; Tang, Xuexi; Li, Yongqi; Yu, Zhiming

    2007-01-01

    Healthy sporophytes of two gametophyte mutants of Laminaria japonica with different heat resistances: kelp 901 ( 901, with comparatively stronger heat-resistance) and Rongcheng No.1 ( RC, sensitive to heat stress), were respectively collected during October to December 2002 from Yantai and Rongcheng Sea Farm in the Shandong Peninsula of China. The contents of some biochemical materials and antioxidant capacity were analyzed under controlled laboratory conditions to identify if there is any relation between the overall antioxidant capacity and the heat-resistance in L. japonica and to understand possible mechanism of heat-resistance. Results show that: (1) the overall antioxidant capacity in healthy sporophyte of 901, such as vitamin E, polyphenol, and ascorbic acid contents and the enzymatic activity of SOD, POD, CAT, Gpx, PPO, and PAL, were not always higher than that of RC under controlled laboratory conditions, and no significance ( P>0.05) was shown in total antioxidant capacity (T-AOC) in 901 and RC. Result suggested that the difference in antioxidant capacity was not a decisive factor for different heat-resistances in L. japonica; (2) the simultaneous assay on isozymes was carried out using vertical polyacrylamide gel electrophoresis (PAGE). Considerable differences in peroxide (PRX), malate dehydrogenase (MDH), malic enzyme (ME), polyphenol oxidase (PPO) and glutamate dehydrogenase (GDH) were obtained in 901 and RC from either the band number, relative mobility ( R f ), or staining intensity, and ME could be used as an indicator to distinguish healthy sporophyte of 901 and RC under controlled laboratory conditions.

  15. Fatty Aldehyde and Fatty Alcohol Metabolism: Review and Importance for Epidermal Structure and Function

    PubMed Central

    Rizzo, William B.

    2014-01-01

    Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren-Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. PMID:24036493

  16. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-06-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.

  17. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed Central

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-01-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity. Images PMID:6286971

  18. Benzene toxicity: emphasis on cytosolic dihydrodiol dehydrogenases

    SciTech Connect

    Bolcsak, L.E.

    1982-01-01

    Blood dyscrasias such as leukopenia and anemia have been clearly identified as consequences of chronic benzene exposure. The metabolites, phenol, catechol, and hydroquinone produced inhibition of /sup 59/Fe uptake in mice which followed the same time course as that produced by benzene. The inhibitor of benzene oxidation, 3-amino-1,2,4-triazole, mitigated the inhibitory effects of benzene and phenol only. These data support the contention that benzene toxicity is mediated by a metabolite and suggest that the toxicity of phenol is a consequence of its metabolism to hydroquinone and that the route of metabolism to catechol may also contribute to the production of toxic metabolite(s). The properties of mouse liver cytosolic dihydrodiol dehydrogenases were examined. These enzymes catalyze the NADP/sup +/-dependent oxidation of trans-1,2-dihydro-1,2-dihydroxybenzene (BDD) to catechol, a possible toxic metabolite of benzene produced via this metabolic route. Four distinct dihydrodiol dehydrogenases (DD1, DD2, DD3, and DD4) were purified to apparent homogeneity as judged by SDS polyacrylamide gel electrophoresis and isoelectric focusing. DD1 appeared to be identical to the major ketone reductase and 17..beta..-hydroxysteroid dehydrogenase activity in the liver. DD2 exhibited aldehyde reductase activity. DD3 and DD4 oxidized 17..beta..-hydroxysteroids, but no carbonyl reductase activity was detected. These relationships between BDD dehydrogenases and carbonyl reductase and/or 17..beta..-hydroxysteroid dehydrogenase activities were supported by several lines of evidence.

  19. [Polymorphism of Glucose-6-phosphate Dehydrogenase in the Chronically Irradiated Scots Pine Populations].

    PubMed

    Kazakova, E A; Volkova, P Yu; Geras'kin, S A; Pomelova, D O

    2015-01-01

    Polymorphism of glucose-6-phosphate dehydrogenase enzyme was studied in the Scots pine populations growing in the sites of Bryansk region which were radioactively contaminated as a result of the Chernobyl accident. It was revealed that the frequency of mutations in isozyme loci increased along with the level of a dose rate (7-130 mGy/year) in the sites under the study. Significant changes in the activity of this enzyme did not depend on the level of radiation exposure.

  20. Developmental changes in the pyruvate kinase isozymes of coho salmon.

    PubMed

    Guderley, H E; Cardenas, J M

    1979-04-01

    Pyruvate kinase exists as two major isozymes in coho salmon. As in mammals and birds, one form is present in the early embryo and maintains a wide tissue distribution in adults. This salmonid type K shows anodal migration during electrophoresis at pH 7.5. The appearence of functional musculature in the developing embryos. In adult animals this second form is the only pyruvate kinase in muscle. Brain, kidney, liver and gill contain primarily the type K pyruvate kinase while heart contains both major forms along with three intermediate forms which presumably constitute a hybrid set. Since there is no additional isozyme restricted to gluconeogenic tissues, we conclude that a type L isozyme has not developed in these animals. The two major isozymes are immunologically distincy. Both forms are dubject to fructose 1,6-bisphosphate activation of phosphoenolpyruvate binding, but the magnitude of the effect is small. The affinities for phosphoenolpyruvate are similar, but salmon type K has hyperbolic saturation curves with this substrate and type M has sigmoidal saturation curves. While the immunological data indicates considerable divergence in structure, the kinetic parameters of the two forms have remained relatively similar. PMID:469475

  1. Subunit structure and isozymic forms of gamma-glutamyl transpeptidase.

    PubMed

    Tate, S S; Meister, A

    1976-08-01

    gamma-Glutamyl transpeptidase is associated with the membranes of a number of epithelial and lymphoid cells. When the enzyme is isolated from rat kidney by a method involving detergent extraction and affinity chromatography, an aggregate of molecular weight greater than 200,000 (heavy form) is obtained. Treatment of the heavy form with bromelain yields a light form of the enzyme (molecular weight of approximately 68,000), which is separable by isoelectric focusing into 12 enzymatically active isozymes which are very similar with respect to catalytic behavior, content of amino acids, hexoses, and aminohexoses, but which differ significantly in sialic acid content. Treatment with neuraminidase converts the acidic isozymes to more basic forms. Each isozyme dissociates in sodium dodecyl sulfate into two nonidentical glycopeptides (molecular weights of 46,000 and 22,000) which can be cross-linked with dimethylsuberimidate to yield a species with an apparent molecular weight of 70,000, which indicates that the isozymes are dimers. Physical and immunological studies indicate that the heavy form of the enzyme contains the dimeric light form as well as other membrane proteins.

  2. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  3. Intraspecific isozyme polymorphism of Anopheles gambiae in relation to environment, behavior, and malaria transmission in southwestern Burkina Faso.

    PubMed

    Coosemans, M; Smits, A; Roelants, P

    1998-01-01

    A cellulose acetate electrophoresis system was used to study the isozyme polymorphism of the Anopheles gambiae complex in a rural village and a city in southwestern Burkina Faso. In both areas A. gambiae Giles was the dominant species (95%) whereas A. arabiensis Patton represented only 5%. Both species were separated readily by octanol dehydrogenase Odh and mannose phosphate isomerase (Mpi) even if they shared some alleles at these two loci. Polymorphism analysis (13 loci) at the intraspecific level of A. gambiae showed a significant difference between the specimens collected in the city from those collected in the village in their allelic and genotypic frequencies of isocitrate dehydrogenase-1 and malate dehydrogenase-1 and in their allelic frequencies for Mpi. No genetic difference was observed between the human biting A. gambiae collected inside or outside the houses in either the village or the city. The Plasmodium falciparum-infected A. gambiae differed from the noninfected ones in their allelic and genotypic frequencies at Mpi and acid phosphatase (Acp). A two-fold difference in infection rate was found for the genotype Mpi130/130 and Acp110/100 compared with other genotypes. However, infected anophelines were found in all genotypes that belonged to these two enzyme systems. Consequently, no refractory mechanism occurs in these natural populations.

  4. Glucose-6-phosphate dehydrogenase

    MedlinePlus

    ... this 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 type of ...

  5. GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass.

    PubMed

    Wang, Xu; Ma, Menggen; Liu, Z Lewis; Xiang, Quanju; Li, Xi; Liu, Na; Zhang, Xiaoping

    2016-08-01

    Scheffersomyces (Pichia) stipitis is one of the most promising yeasts for industrial bioethanol production from lignocellulosic biomass. S. stipitis is able to in situ detoxify aldehyde inhibitors (such as furfural and 5-hydroxymethylfurfural (HMF)) to less toxic corresponding alcohols. However, the reduction enzymes involved in this reaction remain largely unknown. In this study, we reported that an uncharacterized open reading frame PICST_72153 (putative GRE2) from S. stipitis was highly induced in response to furfural and HMF stresses. Overexpression of this gene in Saccharomyces cerevisiae improved yeast tolerance to furfural and HMF. GRE2 was identified as an aldehyde reductase which can reduce furfural to FM with either NADH or NADPH as the co-factor and reduce HMF to FDM with NADPH as the co-factor. This enzyme can also reduce multiple aldehydes to their corresponding alcohols. Amino acid sequence analysis indicated that it is a member of the subclass "intermediate" of the short-chain dehydrogenase/reductase (SDR) superfamily. Although GRE2 from S. stipitis is similar to GRE2 from S. cerevisiae in a three-dimensional structure, some differences were predicted. GRE2 from S. stipitis forms loops at D133-E137 and T143-N145 locations with two α-helices at E154-K157 and E252-A254 locations, different GRE2 from S. cerevisiae with an α-helix at D133-E137 and a β-sheet at T143-N145 locations, and two loops at E154-K157 and E252-A254 locations. This research provided guidelines for the study of other SDR enzymes from S. stipitis and other yeasts on tolerant mechanisms to aldehyde inhibitors derived from lignocellulosic biomass. PMID:27003269

  6. Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes

    PubMed Central

    Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.; Pisithkul, Tippapha; Amador-Noguez, Daniel

    2016-01-01

    ABSTRACT NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. IMPORTANCE Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more

  7. Pyruvate kinases of salmon: purification and comparison with the isozymes from birds and mammals.

    PubMed

    Guderley, H; Cardenas, J M

    1980-02-01

    Pyruvate kinase occurs as two major forms in coho salmon; the type M isozyme occurs primarily in muscle and heart, but type K has a more generalized tissue distribution, in parallel with the type K isozyme in other vertebrate systems. In order to assess the evolutionary relationships among the fish, avian, and mammalian isozymes of pyruvate kinase, we have purified the two isozymes from fish, have examined some of their physical properties, and have studied their immunological relationships to the avian and mammalian isozymes. Salmon type K is at least partially inactivated by antibody to bivine type L pyruvate kinase as well as by antibodies produced against chicken, bovine, and salmon type M isozymes. Salmon type M pyruvate kinase, on the other hand, is not significantly corss-reactive with the bovine type L isozyme, but is at least partially inactivated by antibodies produced against bovine or chicken type M isozymes. Mammalian type L pyruvate kinase is immunologically distinct from either mammalian type K or type M, but salmon type K has some structural features in common with all three mammalian isozymes. Thus, salmon fish type K pyruvate kinase could be similar to a primordial form that was antecedent to the three major differentiated isozymes of higher vertebrates. PMID:7373271

  8. Synthesis of 5'-Aldehyde Oligonucleotide.

    PubMed

    Lartia, Rémy

    2016-01-01

    Synthesis of oligonucleotide ending with an aldehyde functional group at their 5'-end (5'-AON) is possible for both DNA (5'-AODN) and RNA (5'-AORN) series irrespectively of the nature of the last nucleobase. The 5'-alcohol of on-support ODN is mildly oxidized under Moffat conditions. Transient protection of the resulting aldehyde by N,N'-diphenylethylenediamine derivatives allows cleavage, deprotection, and RP-HPLC purification of the protected 5'-AON. Finally, 5'-AON is deprotected by usual acetic acid treatment. In the aggregates, 5'-AON can be now synthesized and purified as routinely as non-modified ODNs, following procedures similar to the well-known "DMT-On" strategy. PMID:26967469

  9. Comparative analysis of peroxidase profiles in Chinese kale (Brassica alboglabra L.): evaluation of leaf growth related isozymes.

    PubMed

    Tang, Lei; Wang, Chenchen; Huang, Jiabao; Zhang, Jianhua; Mao, Zhonggui; Wang, Haiou

    2013-01-15

    Plant peroxidases (EC 1.11.1.7) with different isoforms catalyze various reactions in plant growth and development. However, it is difficult to elucidate the function of each isozyme in one plant. Here, we compared profiles of entire isozyme in young seedling and mature leaves of Chinese kale (Brassica alboglabra L.) on zymogram and ion exchange chromatography in order to investigate leaf growth related peroxidase isozymes. The results showed that four isozymes were constitutively expressed in kale leaves, whereas other two isozymes were induced in the mature leaves. The Mono Q ion exchange chromatography separated the six isozymes into two major groups due to the difference in their isoelectric points. The results suggested that although there were several isozymes in the leaves of Chinese kale, one isozyme functioned mainly through the leaf development. Two anionic isozymes with molecular weights lower than 32 kDa were considered mature related.

  10. Isozyme gene expression in potato tumors incited by Agrobacterium.

    PubMed

    Oliver, J L

    1986-06-01

    Two plant tumors (crown galls and hairy roots) were experimentally provoked on potato cv. 'Désirée' by oncogenic strains of Agrobacterium tumefaciens and A. rhizogenes. A marked shift in the expression of some organ-specific genes occurred in crown galls derived from the central zone of tubers: two novel isozyme genes (Est-B and Pox-E) were expressed, two others (Est-C and Pox-F) were suppressed and the remaining ones were maintained in the original state. When the starting tissue was the stem segment, a smaller shift occurred, namely the activation of Adh-A and the suppression of Pox-F. In all cases, the isozyme profiles characterizing all crown galls, whatever their origin, were identical. Under normal aeration conditions, Adh-A was not expressed in either tumoral or non-tumoral roots. However, under the relative anaerobic conditions of in vitro cultures, a difference existed between both types of roots: Adh-A was expressed in normal but not in tumoral roots. This means that hairy roots can tolerate higher levels of anaerobiosis without giving rise to an anaerobic response. For the remaining isozymes, no alteration occurred in either organized (hairy root) or unorganized (crown gall) tumors, as compared to the corresponding non-tumoral tissues (normal root and callus, respectively). PMID:24247945

  11. An isozyme of earthworm serine proteases acts on hydrolysis of triacylglycerol.

    PubMed

    Nakajima, Nobuyoshi; Sugimoto, Manabu; Tsuboi, Sadao; Tsuji, Hideaki; Ishihara, Kohji

    2005-10-01

    An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.

  12. The thiocarbamate disulphide drug, disulfiram induces osteopenia in rats by inhibition of osteoblast function due to suppression of acetaldehyde dehydrogenase activity.

    PubMed

    Mittal, Monika; Khan, Kainat; Pal, Subhashis; Porwal, Konica; China, Shyamsundar Pal; Barbhuyan, Tarun K; Baghel, Khemraj S; Rawat, Tara; Sanyal, Sabyasachi; Bhadauria, Smrati; Sharma, Vishnu L; Chattopadhyay, Naibedya

    2014-05-01

    Dithiocarbamates (DTC), a sulfhydryl group containing compounds, are extensively used by humans that include metam and thiram due to their pesticide properties, and disulfiram (DSF) as an alcohol deterrent. We screened these DTC in an osteoblast viability assay. DSF exhibited the highest cytotoxicity (IC50 488nM). Loss in osteoblast viability and proliferation was due to induction of apoptosis via G1 arrest. DSF treatment to osteoblasts reduced glutathione (GSH) levels and exogenous addition of GSH prevented DSF-induced reactive oxygen species generation and osteoblast apoptosis. DSF also inhibited osteoblast differentiation in vitro and in vivo, and the effect was associated with inhibition of aldehyde dehydrogenase (ALDH) activity. Out of various ALDH isozymes, osteoblasts expressed only ALDH2 and DSF downregulated its transcript as well as activity. Alda-1, a specific activator of ALDH2, stimulated osteoblast differentiation. Subcutaneous injection of DSF over the calvarium of new born rats reduced the differentiation phenotype of calvarial osteoblasts but increased the mRNA levels of Runx-2 and osteocalcin. DSF treatment at a human-equivalent dose of 30 mg/kg p.o. to adult Sprague Dawley rats caused trabecular osteopenia and suppressed the formation of mineralized nodule by bone marrow stromal cells. Moreover, DSF diminished bone regeneration at the fracture site. In growing rats, DSF diminished growth plate height, primary and secondary spongiosa, mineralized osteoid and trabecular strength. Substantial decreased bone formation was also observed in the cortical site of these rats. We conclude that DSF has a strong osteopenia inducing effect by impairing osteoblast survival and differentiation due to the inhibition of ALDH2 function. PMID:24496638

  13. Evaluation of the toxicity of stress-related aldehydes to photosynthesis in chloroplasts.

    PubMed

    Mano, Jun'ichi; Miyatake, Fumitaka; Hiraoka, Eiji; Tamoi, Masahiro

    2009-09-01

    Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO(2) photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. alpha,beta-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO(2)-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H(2)O --> ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.

  14. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed. PMID:11451442

  15. Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

    PubMed

    Gerdemann, C; Eicken, C; Magrini, A; Meyer, H E; Rompel, A; Spener, F; Krebs, B

    2001-07-01

    The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.

  16. Process for producing furan from furfural aldehyde

    DOEpatents

    Diebold, J.P.; Evans, R.J.

    1987-04-06

    A process of producing furan and derivatives thereof as disclosed. The process includes generating furfural aldehyde vapors and then passing those vapors over a zeolite catalyst at a temperature and for a residence time effective to decarbonylate the furfural aldehydes to form furans and derivatives thereof. The resultant furan vapors and derivatives are then separated. In a preferred form, the furfural aldehyde vapors are generated during the process of converting biomass materials to liquid and gaseous fuels.

  17. Process for producing furan from furfural aldehyde

    DOEpatents

    Diebold, James P.; Evans, Robert J.

    1988-01-01

    A process of producing furan and derivatives thereof is disclosed. The process includes generating furfural aldehyde vapors and then passing those vapors over a zeolite catalyst at a temperature and for a residence time effective to decarbonylate the furfural aldehydes to form furans and derivatives thereof. The resultant furan vapors and derivatives are then separated. In a preferred form, the furfural aldehyde vapors are generated during the process of converting biomass materials to liquid and gaseous fuels.

  18. Microsphere coated substrate containing reactive aldehyde groups

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Richard C. K. (Inventor)

    1984-01-01

    A synthetic organic resin is coated with a continuous layer of contiguous, tangential, individual microspheres having a uniform diameter preferably between 100 Angstroms and 2000 Angstroms. The microspheres are an addition polymerized polymer of an unsaturated aldehyde containing 4 to 20 carbon atoms and are covalently bonded to the substrate by means of high energy radiation grafting. The microspheres contain reactive aldehyde groups and can form conjugates with proteins such as enzymes or other aldehyde reactive materials.

  19. DIFFERENTIATING THE TOXICITY OF CARCINOGENIC ALDEHYDES FROM NONCARCINOGENIC ALDEHYDES IN THE RAT NOSE USING CDNA ARRAYS

    EPA Science Inventory

    Differentiating the Toxicity of Carcinogenic Aldehydes from Noncarcinogenic Aldehydes in the Rat Nose Using cDNA Arrays.

    Formaldehyde is a widely used aldehyde in many industrial settings, the tanning process, household products, and is a contaminant in cigarette smoke. H...

  20. Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes.

    PubMed

    Stivers, J T; Washabaugh, M W

    1993-12-14

    Catalysis of C(alpha)-proton transfer from 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the steady-state kinetics of the reaction of [1-L]acetaldehyde (L = H, D, or T) to form acetoin and the primary kinetic isotope effects on the reaction. The PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) have different steady-state kinetic parameters and isotope effects for acetoin formation in the presence and absence of the nonsubstrate allosteric effector pyruvamide: pyruvamide activation occurs by stabilization of the acetaldehyde/PDC ternary complex. The magnitudes of primary L(V/K)-type (L = D or T) isotope effects on C(alpha)-proton transfer from alpha 4-PDC-bound HETDP provide no evidence for significant breakdown of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP and products. The substrate concentration dependence of the deuterium primary kinetic isotope effects provides evidence for an intrinsic isotope effect of 4.1 for C(alpha)-proton transfer from alpha 4-PDC-bound HETDP. A 1.10 +/- 0.02-fold 14C isotope discrimination against [1,2-14C]acetaldehyde in acetoin formation is inconsistent with a stepwise mechanism, in which the addition step occurs after rate-limiting formation of the C(alpha)-carbanion/enamine as a discrete enzyme-bound intermediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transition state.

  1. Gaseous aliphatic aldehydes in Chinese incense smoke

    SciTech Connect

    Lin, J.M.; Wang, L.H. )

    1994-09-01

    Aliphatic aldehydes were found during the combustion of materials. Tobacco smoke contains aldehydes. Fire fighters were exposed to aldehydes when they conducted firefighting. Aldehydes in ambient air come mainly from the incomplete combustion of hydrocarbons and from photochemical reaction. Most aldehydes in ambient air are formaldehyde and acetaldehyde. Formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and benzaldehyde were found in the atmosphere in Los Angeles. Burning Chinese incense for worshipping deities is a Chinese daily routine. It was suspected to be a factor causing nasopharynegeal cancer. Epidemiological studies correlated it with the high risk of childhood brain tumor and the high risk of childhood leukemia. Ames test identified the mutagenic effect of the smoke from burning Chinese incense. The smoke had bee proved to contain polycyclic aromatic hydrocarbons and aromatic aldehydes. Suspicion about formaldehyde and other alphatic aldehydes was evoked, when a survey of indoor air pollution was conducted in Taipei city. This study determined the presence of aliphatic aldehydes in the smoke from burning Chinese incense under a controlled atmosphere. 12 refs., 5 figs., 2 tabs.

  2. Aldehyde-containing urea-absorbing polysaccharides

    NASA Technical Reports Server (NTRS)

    Mueller, W. A.; Hsu, G. C.; Marsh, H. E., Jr. (Inventor)

    1977-01-01

    A novel aldehyde containing polymer (ACP) is prepared by reaction of a polysaccharide with periodate to introduce aldehyde groups onto the C2 - C3 carbon atoms. By introduction of ether and ester groups onto the pendant primary hydroxyl solubility characteristics are modified. The ACP is utilized to absorb nitrogen bases such as urea in vitro or in vivo.

  3. EMISSIONS OF ODOROUS ALDEHYDES FROM ALKYD PAINT

    EPA Science Inventory

    Aldehyde emissions are widely held responsible for the acrid after-odor of drying alkyd-based paint films. The aldehyde emissions from three different alkyd paints were measured in small environmental chambers. It was found that, for each alkyd paint applied, more than 2 mg of ...

  4. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    PubMed

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  5. Association of a specific cationic peroxidase isozyme with maize stress and disease resistance responses, genetic identification, and identification of a cDNA coding for the isozyme.

    PubMed

    Dowd, Patrick F; Johnson, Eric T

    2005-06-01

    The presence of a pI 9.0 cationic peroxidase isozyme from milk stage pericarp of six susceptible and five resistant inbreds was correlated significantly with previously reported field data on percentage infection by Aspergillus flavus in the inbreds and their hybrids. The isozyme was constitutively expressed in some additional maize tissues and lines examined, and frequently induced by mechanical damage, heat shock, Fusarium proliferatum, and/or Bacillus subtilis in other lines tested. Native/IEF two-dimensional electrophoresis identified the isozyme as the previously genetically identified px5. A cDNA clone expressed in black Mexican sweet (BMS) maize cell cultures produced the pI 9.0 isozyme. In addition to potential use in marker-assisted breeding, enhanced expression of this cationic peroxidase through breeding or genetic engineering may lead to enhanced disease or insect resistance.

  6. Emissions of odorous aldehydes from alkyd paint

    NASA Astrophysics Data System (ADS)

    Chang, John C. S.; Guo, Zhishi

    Aldehyde emissions are widely held responsible for the acrid after-odor of drying alkyd-based paint films. The aldehyde emissions from three different alkyd paints were measured in small environ-mental chambers. It was found that, for each gram of alkyd paint applied, more than 2 mg of aldehydes (mainly hexanal) were emitted during the curing (drying) period. Since no measurable hexanal was found in the original paint, it is suspected that the aldehydes emitted were produced by autoxidation of the unsaturated fatty acid esters in the alkyd resins. The hexanal emission rate was simulated by a model assuming that the autoxidation process was controlled by a consecutive first-order reaction mechanism. Using the emission rate model, indoor air quality simulation indicated that the hexanal emissions can result in prolonged (several days) exposure risk to occupants. The occupant exposure to aldehydes emitted from alkyd paint also could cause sensory irritation and other health concerns.

  7. ISSR and isozyme characterization of androgenetic dihaploids reveals tetrasomic inheritance in tetraploid somatic hybrids between Solanum melongena and Solanum aethiopicum group Gilo.

    PubMed

    Toppino, Laura; Mennella, Giuseppe; Rizza, Fulvia; D'Alessandro, Antonietta; Sihachakr, Darasinh; Rotino, Giuseppe L

    2008-01-01

    Gene exchanges between Solanum melongena and its allied relative Solanum aethiopicum are a crucial prerequisite for introgression of useful traits from the allied species into the cultivated eggplant. In order to evaluate the extent of genetic recombination between the 2 species, biochemical and molecular markers were employed. A dihaploid population obtained through anther culture of the corresponding tetraploid somatic hybrids was genetically analyzed. The extent of disomic/tetrasomic inheritance and segregation ratios of 3 isozyme systems and intersimple sequence repeat (ISSR) markers were evaluated. The dihaploids, being derived from microspores, allowed for simple, complete, and accurate analyses. The segregation of 280 ISSR markers (110 aethiopicum-specific, 104 melongena-specific, and 66 monomorphic) were evaluated in 71 dihaploids. According to the genetic constitution (simplex/duplex/triplex), almost 64% of the fragments revealed the tetrasomic and/or disomic inheritance. With regard to the assigned species-specific fragments, 68% and 4% were unambiguously the result of tetrasomic and disomic inheritance, respectively. Twenty-four of the 66 monomorphic ISSRs were inherited according to random chromatid segregation. The phenotypes of glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and shikimate dehydrogenase (SKDH) were studied in 70 dihaploids and inferences were made about the allelic state of their 5 loci. The isozyme markers segregated in the dihaploids in a distorted manner, their segregations did not fit in with any of the expected segregation ratios. However, tetrasomic inheritance might be suggested for G-6-PDH 2 and SKDH 1 loci. Our results demonstrated that gene exchanges occurred readily in the somatic hybrids between S. melongena and S. aethiopicum gr. Gilo.

  8. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  9. Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M.; Atsumi, Shota

    2015-01-01

    Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90–99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2–C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. PMID:25108218

  10. Enzymatic conversion of atmospheric aldehydes into alcohol in a phospholipid polymer film.

    PubMed

    Tanaka, Naoki; Watari, Akihiro; Tada, Tomoko; Asada, Tomoko; Kunugi, Shigeru; Lee, Yin-Fai; Yamada, Satoshi; Shuto, Kenshiro; Sakaki, Shujiro

    2009-02-01

    We developed a unique method for converting atmospheric aldehyde into alcohol using formaldehyde dehydrogenase from Pseudomonas putida (PFDH) doped in a polymer film. A film of poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate) (PMB), which has a chemical structure similar to that of a biological membrane, was employed for its biocompatibility. A water-incorporated polymer film entrapping PFDH and its cofactor NAD(+) was obtained by drying a buffered solution of PMB, PFDH, and NAD(+). The aldehydes in the air were absorbed into the polymer film and then enzymatically oxidized by PFDH doped in the PMB film. Interestingly, alcohol and carboxylic acid were produced by the enzymatic reaction, indicating that PFDH catalyzes dismutation of aldehyde in the PMB film. Importantly, a PFDH-PMB film catalyzes aldehyde degradation without consuming the nucleotide cofactor, thereby allowing repeated use of the film. The activity of PFDH in the PMB film was higher than that in other common water-soluble polymers, suggesting that the hydrational state in a phospholipid polymer matrix is suitable for enzymatic activity.

  11. Tissue distribution and ontogeny of steroid 5 alpha-reductase isozyme expression.

    PubMed Central

    Thigpen, A E; Silver, R I; Guileyardo, J M; Casey, M L; McConnell, J D; Russell, D W

    1993-01-01

    The synthesis of dihydrotestosterone is catalyzed by steroid 5 alpha-reductase isozymes, designated types 1 and 2. Mutation of type 2 results in male pseudohermaphroditism, in which the external genitalia are phenotypically female at birth. Two striking and unexplained features of this disorder are that external genitalia of affected males undergo virilization during puberty and that these individuals have less temporal hair regression. The tissue-specific and developmental expression patterns of the 5 alpha-reductase isozymes were investigated by immunoblotting. The type 1 isozyme is not detectable in the fetus, is transiently expressed in newborn skin and scalp, and permanently expressed in skin from the time of puberty. There was no qualitative difference in 5 alpha-reductase type 1 expression between adult balding vs. nonbalding scalp. The type 2 isozyme is transiently expressed in skin and scalp of newborns. Type 2 is the predominant isozyme detectable in fetal genital skin, male accessory sex glands, and in the prostate, including benign prostatic hyperplasia and prostate adenocarcinoma tissues. Both isozymes are expressed in the liver, but only after birth. These results are consistent with 5 alpha-reductase type 1 being responsible for virilization in type 2-deficient subjects during puberty, and suggest that the type 2 isozyme may be an initiating factor in development of male pattern baldness. Images PMID:7688765

  12. Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

    PubMed

    Jadhav, Swati B; Bankar, Sandip B; Granström, Tom; Ojamo, Heikki; Singhal, Rekha S; Survase, Shrikant A

    2015-09-01

    Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

  13. Plant Formate Dehydrogenase

    SciTech Connect

    John Markwell

    2005-01-10

    The research in this study identified formate dehydrogenase, an enzyme that plays a metabolic role on the periphery of one-carbon metabolism, has an unusual localization in Arabidopsis thaliana and that the enzyme has an unusual kinetic plasticity. These properties make it possible that this enzyme could be engineered to attempt to engineer plants with an improved photosynthetic efficiency. We have produced transgenic Arabidopsis and tobacco plants with increased expression of the formate dehydrogenase enzyme to initiate further studies.

  14. Diacylglycerol kinase η1 is a high affinity isozyme for diacylglycerol.

    PubMed

    Komenoi, Suguru; Takemura, Fumika; Sakai, Hiromichi; Sakane, Fumio

    2015-05-01

    Diacylglycerol kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of diacylglycerol are supplied.

  15. Serum amylase isozymes in patients with chronic pancreatitis with hyperamylasemia.

    PubMed

    Wakabayashi, A; Saeki, M; Mori, R; Oshiba, S

    1977-01-01

    In order to clarify the relationship between hyperamylasemia and clinical states in chronic pancreatitis, serum amylase isozymes were studied in 39 cases of chronic pancreatitis including 13 cases of alcoholic pancreatitis. Hyperamylasemia in chronic pancreatitis is generally due to high pancreatic type isoamylase (P-amylase) activity in acute exacerbation, sometimes accompanied by a transient elevation in salivary type isoamylase (S-amylase). On remission, however, hyperamylasemia due to high S-amylase activity has been found. These were cases of advanced alcoholic pancreatitis, which exhibited a characteristic pattern of low serum P-amylase and high serum S-amylase activities while the clearance ratio (Cam/Ccr) was normal despite high S-amylase activity. It should be noted that hyperamylasemia in chronic pancreatitis may be caused by high S-amylase activity in addition to high P-amylase activity, especially in alcoholic pancreatitis.

  16. Towards isozyme-selective HDAC inhibitors for interrogating disease.

    PubMed

    Gupta, Praveer; Reid, Robert C; Iyer, Abishek; Sweet, Matthew J; Fairlie, David P

    2012-01-01

    Histone deacetylase (HDAC) enzymes have emerged as promising targets for the treatment of a wide range of human diseases, including cancers, inflammatory and metabolic disorders, immunological, cardiovascular, and infectious diseases. At present, such applications are limited by the lack of selective inhibitors available for each of the eighteen HDAC enzymes, with most currently available HDAC inhibitors having broad-spectrum activity against multiple HDAC enzymes. Such broad-spectrum activity maybe useful in treating some diseases like cancers, but can be detrimental due to cytotoxic side effects that accompany prolonged treatment of chronic diseased states. Here we summarize progress towards the design and discovery of HDAC inhibitors that are selective for some of the eleven zinc-containing classical HDAC enzymes, and identify opportunities to use such isozyme-selective inhibitors as chemical probes for interrogating the biological roles of individual HDAC enzymes in diseases.

  17. Gel Separation of Isozymes To Study Relatedness of Common Bean Cultivars from ARound the World.

    ERIC Educational Resources Information Center

    Hammond, Paul; Oxley, Mary; Ealing, Ben

    2002-01-01

    Explains a three week laboratory experiment on the gel separation of peroxidase, which is the most well-known isozyme, from beans. Includes suggestions on the laboratory management. (Contains 13 references.) (YDS)

  18. Identification and expression of a novel carbonic anhydrase isozyme in the pufferfish Takifugu vermicularis.

    PubMed

    Sumi, Kanij Rukshana; Nou, Ill-Sup; Kho, Kang Hee

    2016-08-22

    Carbonic anhydrase (CA) is a key element for maintaining acid base balance in fish. In our present experiment, novel CA isozymes were identified from the pear puffer (Takifugu vermicularis). Based on the high homology of two predicted CA sequences of the tiger puffer (Takifugu rubripes), a 1715bp novel cDNA was obtained from T. vermicularis. The open reading frame showed a complete coding sequence of 552bp with a deduced peptide sequence of 183 amino acids that exhibited highest (97%) identity with pufferfish putative CA III and CA IV-like sequences. In addition, this translated protein sequence showed 36-37% identity with zebrafish CA IV-like, CA XVa, CA XVb, and CA XVc proteins. Phylogenetic analysis revealed that the pufferfish novel protein (pCAn) was a membrane-bound CA protein. Alignment of multiple CA sequences illustrated that most of the putative active site residues of the pCAn isozyme were situated at highly conserved regions of the CA sequences. Examination of motif distribution suggested that the pCAn isozyme was very similar to the puffer predicted CA IV-like isozyme. Reverse transcription-polymerase chain reaction (PCR) analysis showed highly differential expression in the brain, gills, kidney, and muscle, whereas CA mRNA expression was almost absent in heart, liver, and intestine. Quantitative PCR expression of CA mRNA abundance suggested several-fold higher expression of pCAn isozymes in the gills compared to other tissues tested. Our results suggest that the pCAn isozyme might be related to CA IV-like isozymes. Further functional studies are needed to investigate the function of the pCAn isozyme in T. vermicularis. PMID:27188255

  19. Isolation and characterization of two cDNA clones of anaerobically induced lactate dehydrogenase from barley roots

    SciTech Connect

    Hondred, D.; Hanson, A.D. )

    1990-05-01

    In barley roots during hypoxia, five lactate dehydrogenase (LDH) isozymes accumulate with a concomitant increase in enzyme activity ({approximately}20-fold). These isozymes are thought to be tetramers resulting from the random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH have been isolated from a {lambda}gt11 library using antiserum raised against barley LDH purified {approximately}3,000-fold and using nucleic acid probes synthesized by the polymerase chain reaction. Two cDNA clones were obtained (1,305 and 1,166 bp). The deduced amino acid sequences of the two barley LDHs are 96% identical to each other, and 50% and 40% identical to vertebrate and bacterial LDHs, respectively. Northern blots identified a single mRNA band ({approximately}1.5 kb) whose level rose 8-fold during hypoxia.

  20. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

    PubMed

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. PMID:25656103

  1. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

    PubMed

    Moon, Jaewoong; Liu, Z Lewis

    2015-04-01

    The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production.

  2. First comparative phenetic studies of Argentinean species of Acacia (Fabaceae), using morphometric, isozymal, and RAPD approaches.

    PubMed

    Casiva, Paola V; Saidman, Beatriz O; Vilardi, Juan C; Cialdella, Ana M

    2002-05-01

    Morphological and genetic diversity among Acacia aroma, A. macracantha, A. caven, and A. furcatispina were studied with morphometric, isozymal, and RAPD approaches. The analysis of seven isozyme systems revealed 21 loci, and RAPD analysis showed 34 loci. Most of these loci allowed us to differentiate the species, with the exception of A. aroma and A. macracantha, the two most similar species. The levels of genetic variability estimated by isozymes were higher than those obtained from RAPD analyses. Morphometric characters showed highly significant differences among the species, although A. aroma and A. macracantha are differentiated only by thorn length. The phenogram obtained from isozyme data is consistent with morphological data. The RAPD phenogram based on allelic frequencies showed agreement with morphological and isozymal approaches only at the intraspecific levels, while the RAPD phenogram based on Nei and Li's similarity measures agreed with the phenograms constructed from isozyme and morphological data. High similarities and high indirect gene flow were found between A. aroma and A. macracantha, results that call the relationship between them into question. PMID:21665685

  3. HIV-1 production is specifically associated with human NMT1 long form in human NMT isozymes.

    PubMed

    Takamune, Nobutoki; Gota, Kayoko; Misumi, Shogo; Tanaka, Kenzo; Okinaka, Shigetaka; Shoji, Shozo

    2008-02-01

    The N-myristoylation of the N-terminal of human immunodeficiency virus type-1 (HIV-1) Pr55(gag) by human N-myristoyltransferase (hNMT) is a prerequisite modification for HIV-1 production. hNMT consists of multiple isozymes encoded by hNMT1 and hNMT2. The hNMT1 isozyme consists of long, medium, and short forms. Here, we investigated which isozyme is crucial for HIV-1 production. Human embryonic kidney (HEK) 293 cells transfected with infectious HIV-1 vectors were used as models of HIV-1-infected cells in this study. The significant reduction in HIV-1 production and the failure of the specific localization of Pr55(gag) in a detergent-resistant membrane fraction were dependent on the knockdown of the different forms of the hNMT1 isozyme but not of the hNMT2 isozyme. Additionally, the coexpression of an inactive mutant hNMT1 isozyme, namely the hNMT1 long form (hNMT1(L)), but not that of other hNMT mutants resulted in a significant reduction in HIV-1 production. These results strongly suggest that HIV-1 production is specifically associated with hNMT1, particularly hNMT1(L), but not with hNMT2 in vivo, contributing to the understanding of a step in HIV-1 replication.

  4. Evolution of the functional properties of pyruvate kinase isozymes: pyruvate kinase L from Rana pipiens.

    PubMed

    Fournier, P; Guderley, H

    1986-01-01

    The regulatory properties of type L pyruvate kinase from Rana pipiens are intermediate between those of the mammalian K and L isozymes. As with mammalian type L, the levels of the frog isozyme are affected by the animal's nutritional state. The mammalian and amphibian isozymes show similar sensitivities to fructose 1,6-bisphosphate activation and amino acid inhibition. By contrast, the frog L isozyme shares several properties of the K class: i.e. irreversible inactivation by oxidized glutathione and lack of response to a cyclic AMP stimulated phosphorylation. Furthermore, as for some mammalian K isozymes, frog type L shows a high PEP affinity and a low cooperativity of PEP binding. Insofar as the properties of this present day enzyme reflect those of its counterpart in the amphibian ancestor of higher vertebrates, our results suggest that at its first expression, the type L resembled the type K. Many important regulatory properties of the L isozyme, especially the sensitivity to phosphorylation, were acquired more recently perhaps in association with an increased importance of constant blood glucose. PMID:3489743

  5. Isozymes of antioxidative enzymes during ripening and storage of ber ( Ziziphus mauritiana Lamk.).

    PubMed

    Kumar, Sunil; Yadav, Praduman; Jain, Veena; Malhotra, Sarla P

    2014-02-01

    Isozyme profile of antioxidative enzymes viz. superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) was studied during ripening and storage of two cultivars of ber fruit (Ziziphus mauritiana Lamk.) differing in their shelf-lives viz. Umran (shelf-life, 8-9 d) and Kaithali (shelf-life, 4-5 d). The profile revealed that Umran variety exhibited three bands each of SOD and POX while in Kaithali, these enzymes had two isoenzymes throughout ripening. CAT and APX, however, showed two isozymes each during ripening of both the varieties and the pattern remained the same at all the stages of ripening except at the initial stage i.e immature green stage where single CAT isozyme was visible. During storage, one extra band each of SOD and POX present only in Umran got disappeared at later stages of storage, whereas in Kaithali, the pattern remained unchanged. Also, there was no change in the pattern of CAT and APX isozymes during storage of both the varieties. One isozyme of CAT could be considered as ripening related while one isozyme each of SOD and POX could be related to higher shelf life of fruits. PMID:24493891

  6. Scavenger receptor for aldehyde-modified proteins.

    PubMed

    Horiuchi, S; Murakami, M; Takata, K; Morino, Y

    1986-04-15

    This paper describes an unexpectedly broad ligand specificity of a scavenger receptor of sinusoidal liver cells that is responsible for endocytic uptake of formaldehyde-treated bovine serum albumin (f-Alb). Binding of 125I-f-Alb to the isolated cells was effectively inhibited by bovine serum albumin (BSA) modified with aliphatic aldehydes such as glycolaldehye, DL-glyceraldehyde, and propionaldehyde whereas albumin preparations modified by aromatic aldehydes such as pyridoxal, pyridoxal phosphate, salicylaldehyde, and benzaldehyde did not affect this binding process. Binding of 125I-glycolaldehyde-treated BSA to the cells exhibited a saturation kinetics with an apparent Kd = 3.3 micrograms of the ligand/ml. This binding process was inhibited by unlabeled f-Alb as well as by the antibody raised against the f-Alb receptor. Indeed, 125I-glycolaldehyde-treated BSA underwent a rapid plasma clearance (t1/2 approximately 2 min) which was markedly retarded by unlabeled f-Alb. Upon treatment by these aldehydes, other proteins such as ovalbumin, soybean trypsin inhibitor, and hemoglobin were also converted to active ligands for the f-Alb receptor, while no ligand activity was generated with gamma-globulin and RNase A. These results clearly show that the f-Alb receptor, originally described as being specific for f-Alb, exhibits a broad ligand specificity in terms of both aldehydes and proteins and, hence, should be described as a scavenger receptor for aldehyde-modified proteins.

  7. Differential effects of polyamine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases.

    PubMed

    Murakami, Keiko; Haneda, Miyako; Iwata, Shouko; Yoshino, Masataka

    2012-01-01

    Two isozymes of NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) exist in mammalian tissues: mitochondrial (ICD1) and cytosolic (ICD2). Effects of polyamines such as spermine, spermidine, and putrescine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases were analyzed kinetically. Spermine activated ICD2, the cytosolic NADP-isocitrate dehydrogenase from rat liver with the increase in the maximal velocity and the decrease in the affinity for the substrates isocitrate and NADP. The activating action of spermine can be explained by "the uncompetitive effect," and the dissociation constant of spermine for the enzyme-substrate complex was determined to be 1.68 mM. Spermidine and putrescine showed little or no effect. ICD1, the mitochondrial form of NADP-isocitrate dehydrogenase from rat and porcine heart was inhibited by spermine effectively, and by spermidine and putrescine to a lesser extent. Spermine inhibited the enzyme competitively with respect to NADP, and noncompetitively with respect to isocitrate. K(i) value of the enzyme for spermine was 1.3 mM. These results suggest that activation by spermine of cytosolic NADP-isocitrate dehydrogenase can enhance the antioxidant activity by regeneration of GSH, and further is responsible for the stimulation of lipid biosynthesis in cytosol. Spermine may contribute to NADPH supply by enhancing transhydrogenase (EC1.6.1.2) activity through the spermine-dependent activation of Ca(2+) -incorporation to mitochondria.

  8. The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.

    PubMed

    Lamb, A L; Newcomer, M E

    1999-05-11

    Retinoic acid, a hormonally active form of vitamin A, is produced in vivo in a two step process: retinol is oxidized to retinal and retinal is oxidized to retinoic acid. Retinal dehydrogenase type II (RalDH2) catalyzes this last step in the production of retinoic acid in the early embryo, possibly producing this putative morphogen to initiate pattern formation. The enzyme is also found in the adult animal, where it is expressed in the testis, lung, and brain among other tissues. The crystal structure of retinal dehydrogenase type II cocrystallized with nicotinamide adenine dinucleotide (NAD) has been determined at 2.7 A resolution. The structure was solved by molecular replacement using the crystal structure of a mitochondrial aldehyde dehydrogenase (ALDH2) as a model. Unlike what has been described for the structures of two aldehyde dehydrogenases involved in the metabolism of acetaldehyde, the substrate access channel is not a preformed cavity into which acetaldehyde can readily diffuse. Retinal dehydrogenase appears to utilize a disordered loop in the substrate access channel to discriminate between retinaldehyde and short-chain aldehydes.

  9. Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)-isocitrate dehydrogenase subunit I-deficient mutant of Saccharomyces cerevisiae.

    PubMed

    Johnson, C H; McEwen, J E

    1999-06-30

    A cDNA library was prepared from Histoplasma capsulatum strain G-217B yeast cells and an apparently full-length cDNA for a subunit of the citric acid cycle enzyme NAD(+)-isocitrate dehydrogenase was identified by sequence analysis. Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S. cerevisiae NAD(+)-isocitrate dehydrogenase than to the IDH2 catalytic subunit. After expression in S. cerevisiae from an S. cerevisiae promoter, it was shown to functionally complement an S. cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)-isocitrate dehydrogenase enzyme activity. These results confirm that the H. capsulatum cDNA encodes a homologue of subunit I of the S. cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle.

  10. The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity.

    PubMed

    Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M

    2005-10-18

    Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes. PMID:16216081

  11. In Silico Analysis of Arabidopsis thaliana Peroxisomal 6-Phosphogluconate Dehydrogenase

    PubMed Central

    Fernández-Fernández, Álvaro D.; Corpas, Francisco J.

    2016-01-01

    NADPH, whose regeneration is critical for reductive biosynthesis and detoxification pathways, is an essential component in cell redox homeostasis. Peroxisomes are subcellular organelles with a complex biochemical machinery involved in signaling and stress processes by molecules such as hydrogen peroxide (H2O2) and nitric oxide (NO). NADPH is required by several peroxisomal enzymes involved in β-oxidation, NO, and glutathione (GSH) generation. Plants have various NADPH-generating dehydrogenases, one of which is 6-phosphogluconate dehydrogenase (6PGDH). Arabidopsis contains three 6PGDH genes that probably are encoded for cytosolic, chloroplastic/mitochondrial, and peroxisomal isozymes, although their specific functions remain largely unknown. This study focuses on the in silico analysis of the biochemical characteristics and gene expression of peroxisomal 6PGDH (p6PGDH) with the aim of understanding its potential function in the peroxisomal NADPH-recycling system. The data show that a group of plant 6PGDHs contains an archetypal type 1 peroxisomal targeting signal (PTS), while in silico gene expression analysis using affymetrix microarray data suggests that Arabidopsis p6PGDH appears to be mainly involved in xenobiotic response, growth, and developmental processes. PMID:27034898

  12. Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme.

    PubMed

    Duarte-Vázquez, Miguel A; García-Almendárez, Blanca E; Rojo-Domínguez, Arturo; Whitaker, John R; Arroyave-Hernández, C; Regalado, Carlos

    2003-01-01

    A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability. PMID:12475613

  13. Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

    PubMed

    Rozeboom, Henriëtte J; Yu, Shukun; Mikkelsen, Rene; Nikolaev, Igor; Mulder, Harm J; Dijkstra, Bauke W

    2015-12-01

    The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed β-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

  14. Influence of Tris(hydroxymethyl)aminomethane on kinetic mechanism of yeast alcohol dehydrogenase.

    PubMed

    Trivić, S; Leskovac, V; Zeremski, J; Stancić, B; Anderson, B M

    1998-02-01

    Acetaldehyde, propionaldehyde, glyceraldehyde-3-P and 4-dimethylaminocinnamaldehyde form Schiff bases in Tris. HCl buffers; the rates of formation and dissociation of Schiff bases, and equilibrium constants for their formation are very similar for the first three aldehydes. The steady-state kinetic constants for the yeast alcohol dehydrogenase-catalyzed reaction, propan-1-ol + NAD+ reversible propionaldehyde + NADH + H+, have been determined in several Tris. HCl buffers of increasing concentration at pH 8.1. In the forward direction, oxidation of alcohol, most kinetic constants are increased by increasing concentrations of Tris. In the reverse direction, reduction of aldehyde, substrate, NADH, Tris and Schiff base were equilibrated before enzyme reaction was started. It was found that Schiff base, rather than Tris, binds to free enzyme competitively with respect to NADH. Tris and Schiff base do not influence the binding of aldehyde to enzyme in any way. PMID:9879514

  15. Purification and characterization of the dog hepatic cytochrome P-450 isozyme(s) responsible for the metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl

    SciTech Connect

    Duignan, D.B.; Sipes, I.G.; Leonard, T.B.; Halpert, J.R.

    1986-03-01

    Pretreatment of dogs and rats with phenobarbital (PB) enhances the in vitro metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB); however, in control dog microsomes there is an approximately 5-fold greater rate of metabolism than that observed in PB-induced rat microsomes. At least two PB-induced isozymes of cytochrome P-450 are detected in dog microsomes, and by use of Octylamino-Sepharose. Hydroxylapatite, and DEAE-Sephacel column chromatography, one of these isozymes (which the authors call PBD-2) has been purified to >95%, as determined by SDS-PAGE. In a reconstituted system, PBD-2 can metabolize 245-HCB at a rate similar to that seen for PB-B, the major PB-induced isozyme in the rat. In addition, antibodies raised against PB-B cross-react with PBD-2, and the NH/sub 2/-terminal amino acid sequence of PBD-2 is nearly 70% homologous to that of PB-B. These results suggest that a cytochrome P-450 isozyme capable of metabolizing 245-HCB in the dog (PBD-2) is similar to PB-B. The importance of PBD-2 will be further elucidated by conducting inhibition studies with anti-PBD-2 antibodies in control and PB-induced dog microsomes.

  16. Reciprocal regulation of transcription factors and PLC isozyme gene expression in adult cardiomyocytes.

    PubMed

    Singal, Tushi; Dhalla, Naranjan S; Tappia, Paramjit S

    2010-06-01

    By employing a pharmacological approach, we have shown that phospholipase C (PLC) activity is involved in the regulation of gene expression of transcription factors such as c-Fos and c-Jun in cardiomyocytes in response to norepinephrine (NE). However, there is no information available regarding the identity of specific PLC isozymes involved in the regulation of c-Fos and c-Jun or on the involvement of these transcription factors in PLC isozyme gene expression in adult cardiomyocytes. In this study, transfection of cardiomyocytes with PLC isozyme specific siRNA was found to prevent the NE-mediated increases in the corresponding PLC isozyme gene expression, protein content and activity. Unlike PLC gamma(1) gene, silencing of PLC beta(1), beta(3) and delta(1) genes with si RNA prevented the increases in c-Fos and c-Jun gene expression in response to NE. On the other hand, transfection with c-Jun si RNA suppressed the NE-induced increase in c-Jun as well as PLC beta(1), beta(3) and delta(1) gene expression, but had no effect on PLC gamma(1) gene expression. Although transfection of cardiomyocytes with c-Fos si RNA prevented NE-induced expression of c-Fos, PLC beta(1) and PLC beta(3) genes, it did not affect the increases in PLC delta(1) and PLC gamma(1) gene expression. Silencing of either c-Fos or c-Jun also depressed the NE-mediated increases in PLC beta(1), beta(3) and gamma(1) protein content and activity in an isozyme specific manner. Furthermore, silencing of all PLC isozymes as well as of c-Fos and c-Jun resulted in prevention of the NE-mediated increase in atrial natriuretic factor gene expression. These findings, by employing gene silencing techniques, demonstrate that there occurs a reciprocal regulation of transcription factors and specific PLC isozyme gene expression in cardiomyocytes.

  17. New studies of the alcohol dehydrogenase cline in D. melanogaster from Mexico.

    PubMed

    Pipkin, S B; Franklin-Springer, E; Law, S; Lubega, S

    1976-01-01

    An altitudinal cline of frequencies of alcohol dehydrogenase alleles occurs in D. melanogaster populations of southeastern Mexico. A similar cline of two aldehyde oxidase alleles is present, but frequencies of esterase-6 alleles are not distributed clinically. Collections were made from small dispersed populations. Some gene flow occurred throughout the lowlands according to the distribution of two moderately endemic autosomal inversions and five previously described inversions. The clines are believed dependent on a limited gene flow between temperature races of D. melanogaster.

  18. Characterization of alcohol dehydrogenase 1 and 3 from Neurospora crassa FGSC2489.

    PubMed

    Park, Yong-Cheol; San, Ka-Yiu; Bennett, George N

    2007-08-01

    Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD(+)-binding motif and showed 54-75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 +/- 9 mU/mg using ethanol and NAD(+) as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3-C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD(+) preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes. PMID:17516063

  19. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    DOE PAGES

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacylmore » intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.« less

  20. Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action

    SciTech Connect

    Huo, Lu; Davis, Ian; Liu, Fange; Andi, Babak; Esaki, Shingo; Iwaki, Hiroaki; Hasegawa, Yoshie; Orville, Allen M.; Liu, Aimin

    2015-01-07

    Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation.

  1. Volatile aldehydes in libraries and archives

    NASA Astrophysics Data System (ADS)

    Fenech, Ann; Strlič, Matija; Kralj Cigić, Irena; Levart, Alenka; Gibson, Lorraine T.; de Bruin, Gerrit; Ntanos, Konstantinos; Kolar, Jana; Cassar, May

    2010-06-01

    Volatile aldehydes are produced during degradation of paper-based materials. This may result in their accumulation in archival and library repositories. However, no systematic study has been performed so far. In the frame of this study, passive sampling was carried out at ten locations in four libraries and archives. Despite the very variable sampling locations, no major differences were found, although air-filtered repositories were found to have lower concentrations while a non-ventilated newspaper repository exhibited the highest concentrations of volatile aldehydes (formaldehyde, acetaldehyde, furfural and hexanal). Five employees in one institution were also provided with personal passive samplers to investigate employees' exposure to volatile aldehydes. All values were lower than the presently valid exposure limits. The concentration of volatile aldehydes, acetic acid, and volatile organic compounds (VOCs) in general was also compared with that of outdoor-generated pollutants. It was evident that inside the repository and particularly inside archival boxes, the concentration of VOCs and acetic acid was much higher than the concentration of outdoor-generated pollutants, which are otherwise more routinely studied in connection with heritage materials. This indicates that further work on the pro-degradative effect of VOCs on heritage materials is necessary and that monitoring of VOCs in heritage institutions should become more widespread.

  2. Oxidation of Aromatic Aldehydes Using Oxone

    ERIC Educational Resources Information Center

    Gandhari, Rajani; Maddukuri, Padma P.; Thottumkara, Vinod K.

    2007-01-01

    The experiment demonstrating the feasibility of using water as a solvent for organic reactions which highlights the cost and environmental benefits of its use is presented. The experiment encourages students to think in terms of the reaction mechanism of the oxidation of aldehydes knowing that potassium persulfate is the active oxidant in Oxone…

  3. Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius.

    PubMed

    Extance, Jonathan; Crennell, Susan J; Eley, Kirstin; Cripps, Roger; Hough, David W; Danson, Michael J

    2013-10-01

    Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

  4. Aldehyde Reduction by Cytochrome P450

    PubMed Central

    Amunom, Immaculate; Srivastava, Sanjay; Prough, Russell A.

    2011-01-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model α,β-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  5. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN...

  6. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN...

  7. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Amine aldehyde condensate. 721.639... Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN...

  8. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for...

  9. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for...

  10. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for...

  11. 40 CFR 721.5762 - Aromatic aldehyde phenolic resin (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Aromatic aldehyde phenolic resin... Specific Chemical Substances § 721.5762 Aromatic aldehyde phenolic resin (generic). (a) Chemical substance... aromatic aldehyde phenolic resin (PMN P-01-573) is subject to reporting under this section for...

  12. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  13. The human liver glycogen synthase isozyme gene is located on the short arm of chromosome 12

    SciTech Connect

    Nuttall, F.Q.; Gannon, M.C. ); Kubic, V.L.; Hoyt, K.J. )

    1994-01-15

    Glycogen synthase catalyzes the rate-limiting step in glycogen synthesis. Its activity is regulated by a complex phosphorylation-dephosphorylation mechanism and by allosteric stimulators and inhibitors. Two isozymes of synthase, a skeletal muscle type and liver type, have been identified in rabbit and rat tissues using specific polyclonal antibodies. The skeletal muscle type isozyme is present in several organs in addition to skeletal muscle; the liver isozyme has been identified only in liver. Recently, we have purified and characterized the human liver synthase isozyme. We also have cloned and sequenced the gene from a human liver cDNA library. Using the entire cDNA coding sequence as a probe, we report here the localization of the liver synthase isozyme gene to the short arm of chromosome 12. These studies revealed a centromeric signal on chromosome 12 together with signal to glycogen synthase on the short arm of this chromosome in the p11.2-p12.2 region. Measurements of the relative distance from the midpoint of the centromere to the signal corresponding to glycogen synthase suggests that the locus is in the p12.2 band rather than in the more centromeric location.

  14. Na,K-ATPase Isozymes in Colorectal Cancer and Liver Metastases

    PubMed Central

    Baker Bechmann, Marc; Rotoli, Deborah; Morales, Manuel; Maeso, María del Carmen; García, María del Pino; Ávila, Julio; Mobasheri, Ali; Martín-Vasallo, Pablo

    2016-01-01

    The goal of this study was to define Na,K-ATPase α and β subunit isoform expression and isozyme composition in colorectal cancer cells and liver metastases. The α1, α3, and β1 isoforms were the most highly expressed in tumor cells and metastases; in the plasma membrane of non-neoplastic cells and mainly in a cytoplasmic location in tumor cells. α1β1 and α3β1 isozymes found in tumor and metastatic cells exhibit the highest and lowest Na+ affinity respectively and the highest K+ affinity. Mesenchymal cell isozymes possess an intermediate Na+ affinity and a low K+ affinity. In cancer, these ions are likely to favor optimal conditions for the function of nuclear enzymes involved in mitosis, especially a high intra-nuclear K+ concentration. A major and striking finding of this study was that in liver, metastasized CRC cells express the α3β1 isozyme. Thus, the α3β1 isozyme could potentially serve as a novel exploratory biomarker of CRC metastatic cells in liver. PMID:26858653

  15. Bioinformatic scaling of allosteric interactions in biomedical isozymes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-09-01

    Allosteric (long-range) interactions can be surprisingly strong in proteins of biomedical interest. Here we use bioinformatic scaling to connect prior results on nonsteroidal anti-inflammatory drugs to promising new drugs that inhibit cancer cell metabolism. Many parallel features are apparent, which explain how even one amino acid mutation, remote from active sites, can alter medical results. The enzyme twins involved are cyclooxygenase (aspirin) and isocitrate dehydrogenase (IDH). The IDH results are accurate to 1% and are overdetermined by adjusting a single bioinformatic scaling parameter. It appears that the final stage in optimizing protein functionality may involve leveling of the hydrophobic limits of the arms of conformational hydrophilic hinges.

  16. Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes.

    PubMed

    Kuo, Ting-Chun; Shaw, Jei-Fu; Lee, Guan-Chiun

    2015-09-01

    The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1-CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24h for a total reaction time of 48 h at 37 °C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil.

  17. Isozyme expression of Chinese and Japanese populations of Chlamys farreri and their reciprocal hybrids

    NASA Astrophysics Data System (ADS)

    Li, Taiwu; Liu, Yan; Song, Linsheng; Sun, Xiuqin

    2005-06-01

    Chinese and Japanese population of Chlamys farreri and their reciprocal hybrids were surveyed in isozyme variability at 13 loci by polyacrytamide gel electrophoresis (PAGE). Isozyme banding patterns indicated these hybrids were diploid. Loci that were observed as being monomorphic in inbred populations of C. farreri were also found to be monomorphic in filial progeny; loci that observed to be polymorphic in parental type generations were also polymorphic in hybrid generations. Differences existed among allelic frequency of the four types of cross. Within the reciprocal hybrids the expression of malic enzyme (ME) isozyme was sufficient to distinguishing individual hybrids because of the band, Rf=0.38. However, there were no noticeable variations among all the samples to differentiate one from another. Inbreeding was likely to be the main problem in aquaculture. The introduction of new broodstock can improve the genetic diversity. Hybrid vigor has manifested to a certain extent in the present study.

  18. Regulated Expression of Three Alcohol Dehydrogenase Genes in Barley Aleurone Layers 1

    PubMed Central

    Hanson, Andrew D.; Jacobsen, John V.; Zwar, John A.

    1984-01-01

    Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity. Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O2 deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O2 levels were reduced below 5%, and were most intense at 0% O2. In vivo35S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O2, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O2, the synthesis of all three ADH peptides increased markedly. Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O2 deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O2, and about 100-fold after 1 day at 0% O2. We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O2, that regulation of mRNA level is likely to be a major controlling factor, and

  19. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

    PubMed Central

    Palosaari, N R; Rogers, P

    1988-01-01

    The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases. Images PMID:3384801

  20. Differential expression of 5-alpha reductase isozymes in the prostate and its clinical implications

    PubMed Central

    Wang, Kai; Fan, Dong-Dong; Jin, Song; Xing, Nian-Zeng; Niu, Yi-Nong

    2014-01-01

    The development of human benign or malignant prostatic diseases is closely associated with androgens, primarily testosterone (T) and dihydrotestosterone (DHT). T is converted to DHT by 5-alpha reductase (5-AR) isozymes. Differential expression of 5-AR isozymes is observed in both human benign and malignant prostatic tissues. 5-AR inhibitors (5-ARI) are commonly used for the treatment of benign prostatic hyperplasia (BPH) and were once promoted as chemopreventive agents for prostate cancer (PCa). This review discusses the role of the differential expression of 5-AR in the normal development of the human prostate and in the pathogenesis and progression of BPH and PCa. PMID:24457841

  1. Elusive transition state of alcohol dehydrogenase unveiled

    PubMed Central

    Roston, Daniel; Kohen, Amnon

    2010-01-01

    For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (> 1) 2° kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (< 1), this KIE eludes the traditional interpretation of 2° KIEs. It does, however, enable the development of a comprehensive model for the “tunneling ready state” (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate. PMID:20457944

  2. Cyanobacterial NADPH dehydrogenase complexes

    SciTech Connect

    Ogawa, Teruo; Mi, Hualing

    2007-07-01

    Cyanobacteria possess functionally distinct multiple NADPH dehydrogenase (NDH-1) complexes that are essential to CO2 uptake, photosystem-1 cyclic electron transport and respiration. The unique nature of cyanobacterial NDH-1 complexes is the presence of subunits involved in CO2 uptake. Other than CO2 uptake, chloroplastic NDH-1 complex has similar role as cyanobacterial NDH-1 complexes in photosystem-1 cyclic electron transport and respiration (chlororespiration). In this mini-review we focus on the structure and function of cyanobacterial NDH-1 complexes and their phylogeny. The function of chloroplastic NDH-1 complex and characteristics of plants defective in NDH-1 are also described forcomparison.

  3. Neurotoxicity of reactive aldehydes: the concept of "aldehyde load" as demonstrated by neuroprotection with hydroxylamines.

    PubMed

    Wood, Paul L; Khan, M Amin; Kulow, Sarah R; Mahmood, Siddique A; Moskal, Joseph R

    2006-06-20

    The concept of "oxidative stress" has become a mainstay in the field of neurodegeneration but has failed to differentiate critical events from epiphenomena and sequalae. Furthermore, the translation of current concepts of neurodegenerative mechanisms into effective therapeutics for neurodegenerative diseases has been meager and disappointing. A corollary of current concepts of "oxidative stress" is that of "aldehyde load". This relates to the production of reactive aldehydes that covalently modify proteins, nucleic acids, lipids and carbohydrates and activate apoptotic pathways. However, reactive aldehydes can also be generated by mechanisms other than "oxidative stress". We therefore hypothesized that agents that can chemically neutralize reactive aldehydes should demonstrate superior neuroprotective actions to those of free radical scavengers. To this end, we evaluated hydroxylamines as aldehyde-trapping agents in an in vitro model of neurodegeneration induced by the reactive aldehyde, 3-aminopropanal (3-AP), a product of polyamine oxidase metabolism of spermine and spermidine. In this model, the hydroxylamines N-benzylhydroxylamine, cyclohexylhydroxylamine and t-butylhydroxylamine were shown to protect, in a concentration-dependent manner, against 3-AP neurotoxicity. Additionally, a therapeutic window of 3 h was demonstrated for delayed administration of the hydroxylamines. In contrast, the free radical scavengers TEMPO and TEMPONE and the anti-oxidant ascorbic acid were ineffective in this model. Extending these tissue culture findings in vivo, we examined the actions of N-benzylhydroxylamine in the trimethyltin (TMT) rat model of hippocampal CA3 neurodegeneration. This model involves augmented polyamine metabolism resulting in the generation of reactive aldehydes that compromise mitochondrial integrity. In the rat TMT model, NBHA (50 mg/kg, sc, daily) provided 100% protection against neurodegeneration, as reflected by measurements of KCl-evoked glutamate

  4. Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis.

    PubMed

    Ngo, Ho-Phuong-Thuy; Hong, Seung-Hye; Hong, Myoung-Ki; Pham, Tan-Viet; Oh, Deok-Kun; Kang, Lin-Woo

    2013-09-01

    Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

  5. Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis

    PubMed Central

    Ngo, Ho-Phuong-Thuy; Hong, Seung-Hye; Hong, Myoung-Ki; Pham, Tan-Viet; Oh, Deok-Kun; Kang, Lin-Woo

    2013-01-01

    Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD+ to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD+. The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding V M of 2.55 Å3 Da−1 and a solvent content of 51.8%. PMID:23989158

  6. Affinity chromatography of nicotinamide-adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide.

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems

  7. One-Pot Amide Bond Formation from Aldehydes and Amines via a Photoorganocatalytic Activation of Aldehydes.

    PubMed

    Papadopoulos, Giorgos N; Kokotos, Christoforos G

    2016-08-19

    A mild, one-pot, and environmentally friendly synthesis of amides from aldehydes and amines is described. Initially, a photoorganocatalytic reaction of aldehydes with di-isopropyl azodicarboxylate leads to an intermediate carbonyl imide, which can react with a variety of amines to afford the desired amides. The initial visible light-mediated activation of a variety of monosubstituted or disubstituted aldehydes is usually fast, occurring in a few hours. Following the photocatalytic reaction, addition of the primary amine at room temperature or the secondary amine at elevated temperatures leads to the corresponding amide from moderate to excellent yields without epimerization. This methodology was applied in the synthesis of Moclobemide, a drug against depression and social anxiety. PMID:27227271

  8. Genetics Home Reference: pyruvate dehydrogenase deficiency

    MedlinePlus

    ... control the activity of the complex: pyruvate dehydrogenase phosphatase turns on (activates) the complex, while pyruvate dehydrogenase ... binding protein (the PDHX gene), and pyruvate dehydrogenase phosphatase (the PDP1 gene) have been identified in people ...

  9. Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi.

    PubMed Central

    Roeder, D L; Collmer, A

    1985-01-01

    The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate-limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric-focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis. Images PMID:2995324

  10. Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila.

    PubMed Central

    Williams, T. G.; Colman, B.

    1993-01-01

    External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions. PMID:12231991

  11. Cloning, expression, and purification of choline dehydrogenase from the moderate halophile Halomonas elongata.

    PubMed

    Gadda, Giovanni; McAllister-Wilkins, Elien Elizabeth

    2003-04-01

    Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 micro mol of O(2) min(-1) mg(-1) mM(-1) at pH 7 and 25 degrees C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V(max) values for choline and betaine-aldehyde were 10.9 and 5.7 micro mol of O(2) min(-1) mg(-1), respectively. These V(max) values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.

  12. Two-Step biocatalytic conversion of an ester to an aldehyde in reverse micelles.

    PubMed

    Yang, F; Russell, A J

    1994-02-01

    Lipases from Candida cyclindracea (L-1754) and wheat germ (L-3001) have been used to hydrolyze esters to their corresponding alcohols and acids in reverse micelles. Alcohol dehydrogenase from baker's yeast (YADH) was subsequently used to reduce the alcohol products to aldehydes. Cofactor recycling in the redox reaction was achieved using a sacrificial cosubstrate, as described previously. Four surfactants (sodium dioctylsulfosuccinate, Nonidet P-40 with Triton X-35, polyoxyethylene, 10-cetyl-ether, polyoxyethylene sorbitan trioleate) were employed to determine the effect of amphiphile on ester hydrolysis and redox reaction rates separately. The effect of type of organic solvent, W(0) [(water]/[surfactant)], and substrate concentration on separte enzyme activity were also investigated. A brief investigation of a single phase, two-step reaction catalyzed by the combination of lipase and YADH in reverse micelles is also reported. The activities of the enzymes are significantly different when used together instead of independently. (c) 1994 John Wiley & Sons, Inc.

  13. Substrate Specificity of the Purified Primary Alcohol Dehydrogenases from Methanol-Oxidizing Bacteria

    PubMed Central

    Sperl, George T.; Forrest, Hugh S.; Gibson, David T.

    1974-01-01

    Hyphomicrobium strain WC, Pseudomonas strain TP-1, and Pseudomonas strain W1 are capable of growth on methanol as the sole source of carbon and energy. Methanol-grown cells of each organism contain a primary alcohol dehydrogenase that has been purified to homogeneity. Each enzyme has a molecular weight of 120,000 and shows an in vitro requirement for phenazine methosulfate and ammonium ions for enzymatic activity. Normal aliphatic alcohols are oxidized rapidly by each enzyme. The presence of a methyl group on the carbon atom adjacent to the primary alcohol group lowers the enzymatic activity. This effect is reduced as the methyl substituent is moved further away from the hydroxyl group. The effect of other substituents on enzymatic activity is reported. Methanol, formaldehyde, and to a limited extent acetaldehyde are oxidized by the primary alcohol dehydrogenases. Higher aldehydes are not oxidized. A possible explanation for this specificity, with regard to aldehydes, is presented in terms of degree of hydration of the aldehyde. Images PMID:4828309

  14. Purification and characterization of benzyl alcohol- and benzaldehyde- dehydrogenase from Pseudomonas putida CSV86.

    PubMed

    Shrivastava, Rahul; Basu, Aditya; Phale, Prashant S

    2011-08-01

    Pseudomonas putida CSV86 utilizes benzyl alcohol via catechol and methylnaphthalenes through detoxification pathway via hydroxymethylnaphthalenes and naphthaldehydes. Based on metabolic studies, benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) were hypothesized to be involved in the detoxification pathway. BADH and BZDH were purified to apparent homogeneity and were (1) homodimers with subunit molecular mass of 38 and 57 kDa, respectively, (2) NAD(+) dependent, (3) broad substrate specific accepting mono- and di-aromatic alcohols and aldehydes but not aliphatic compounds, and (4) BADH contained iron and magnesium, while BZDH contained magnesium. BADH in the forward reaction converted alcohol to aldehyde and required NAD(+), while in the reverse reaction it reduced aldehyde to alcohol in NADH-dependent manner. BZDH showed low K (m) value for benzaldehyde as compared to BADH reverse reaction. Chemical cross-linking studies revealed that BADH and BZDH do not form multi-enzyme complex. Thus, the conversion of aromatic alcohol to acid is due to low K (m) and high catalytic efficiency of BZDH. Phylogenetic analysis revealed that BADH is a novel enzyme and diverged during the evolution to gain the ability to utilize mono- and di-aromatic compounds. The wide substrate specificity of these enzymes enables strain to detoxify methylnaphthalenes to naphthoic acids efficiently.

  15. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective

    PubMed Central

    2015-01-01

    Aldehydes are electrophilic compounds to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivatives and corresponding differences in chemical reactions and biological targets. To gain mechanistic insight, we have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biological nucleophilic targets. Our analyses indicate that short chain aldehydes and longer chain saturated alkanals are hard electrophiles that cause toxicity by forming adducts with hard biological nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsaturated carbonyl derivatives, alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and molecular mechanisms of toxicity. As we will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochemical variables that limit target accessibility, e.g., steric hindrance and solubility. The unsaturated aldehydes are also members of the conjugated type-2 alkene chemical class that includes α,β-unsaturated amide, ketone, and ester derivatives. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixture of unsaturated carbonyl derivatives could cause “type-2 alkene toxicity” through additive interactions

  16. Molecular mechanisms of aldehyde toxicity: a chemical perspective.

    PubMed

    LoPachin, Richard M; Gavin, Terrence

    2014-07-21

    Aldehydes are electrophilic compounds to which humans are pervasively exposed. Despite a significant health risk due to exposure, the mechanisms of aldehyde toxicity are poorly understood. This ambiguity is likely due to the structural diversity of aldehyde derivatives and corresponding differences in chemical reactions and biological targets. To gain mechanistic insight, we have used parameters based on the hard and soft, acids and bases (HSAB) theory to profile the different aldehyde subclasses with respect to electronic character (softness, hardness), electrophilic reactivity (electrophilic index), and biological nucleophilic targets. Our analyses indicate that short chain aldehydes and longer chain saturated alkanals are hard electrophiles that cause toxicity by forming adducts with hard biological nucleophiles, e.g., primary nitrogen groups on lysine residues. In contrast, α,β-unsaturated carbonyl derivatives, alkenals, and the α-oxoaldehydes are soft electrophiles that preferentially react with soft nucleophilic thiolate groups on cysteine residues. The aldehydes can therefore be grouped into subclasses according to common electronic characteristics (softness/hardness) and molecular mechanisms of toxicity. As we will discuss, the toxic potencies of these subgroups are generally related to corresponding electrophilicities. For some aldehydes, however, predictions of toxicity based on electrophilicity are less accurate due to inherent physicochemical variables that limit target accessibility, e.g., steric hindrance and solubility. The unsaturated aldehydes are also members of the conjugated type-2 alkene chemical class that includes α,β-unsaturated amide, ketone, and ester derivatives. Type-2 alkenes are electrophiles of varying softness and electrophilicity that share a common mechanism of toxicity. Therefore, exposure to an environmental mixture of unsaturated carbonyl derivatives could cause "type-2 alkene toxicity" through additive interactions

  17. Isozyme variation in germplasm accessions of the wild oat Avena sterilis L.

    PubMed

    Phillips, T D; Murphy, J P; Goodman, M M

    1993-03-01

    Optimal exploitation of crop genetic resources requires a knowledge of the range and structure of the variation present in the gene pool of interest. Avena sterilis L., the cultivated oat progenitor, contains a store of genetic diversity that is readily accessible to the oat breeder. The objectives of the present paper were: (1) to evaluate isozyme polymorphisms in a sample of A. sterilis accessions from the U.S. National Small Grains Collection, (2) to analyze the distribution of isozyme diversity across the geographic range of the accessions, (3) to classify the accessions into groups based on isozyme variation, and (4) to suggest strategies for efficient sampling of this germplasm collection. One thousand and five accessions from 23 countries and 679 collection sites were screened for variation using 23 enzyme systems. Due to limited information about the genetic relationship among individual members of families of isozymes in hexaploid oat species, data were recorded solely for band presence. The frequencies of bands in accessions from the various countries were used to calculate the probability of genotypic identity (Ix.y), the probability of a unique genotype (Ux.y), and an adjusted polymorphic index (Hx). Accessions from Turkey and Lebanon had the largest polymorphic index values, Turkish and Moroccan accessions displayed the greatest numbers of bands. Accessions from Iran, Turkey, Iraq, and Lebanon had the largest mean probabilities of containing unique genotypes. Based on isozyme data, Turkey appeared to represent the center of diversity in this germplasm collection. Band frequencies calculated among countries were used in a principal component analysis. Accessions from Israel and Morocco clustered together; accessions from Iran, Iraq, Turkey, and Ethiopia formed another group; and Algerian accessions formed an outlying group. Several isozyme bands had a regional distribution. These results suggested that choosing accessions from countries based on their

  18. Medium-chain and short-chain dehydrogenases/reductases in retinoid metabolism

    PubMed Central

    Parés, X.; Farrés, J.; Kedishvili, N.; Duester, G.

    2009-01-01

    Retinoic acid (RA), the most active retinoid, is synthesized in two steps from retinol. The first step, oxidation of retinol to retinaldehyde, is catalyzed by cytosolic alcohol dehydrogenases (ADHs) of the medium-chain dehydrogenase/reductase (MDR) superfamily and microsomal retinol dehydrogenases (RDHs) of the short-chain dehydrogenase/reductase (SDR) superfamily. The second step, oxidation of retinaldehyde to RA, is catalyzed by several aldehyde dehydrogenases. ADH1 and ADH2 are the major MDR enzymes in liver retinol detoxification, while ADH3 (less active) and ADH4 (most active) participate in RA generation in tissues. Several NAD+- and NADP+-dependent SDRs are retinoid active. Their in vivo contribution has been demonstrated in the visual cycle (RDH5, RDH12), adult retinoid homeostasis (RDH1) and embryogenesis (RDH10). Km values for most retinoid-active ADHs and RDHs are close to 1 μM or lower, suggesting that they participate physiologically in retinol/retinaldehyde interconversion. Probably none of these enzymes uses retinoids bound to cellular retinol-binding protein, but only free retinoids. The large number of enzymes involved in the two directions of this step, also including aldoketo reductases, suggests that retinaldehyde levels are strictly regulated. PMID:19011747

  19. Appearance of aldehydes in the surface layer of lake waters.

    PubMed

    Dąbrowska, Agata; Nawrocki, Jacek; Szeląg-Wasielewska, Elżbieta

    2014-07-01

    The paper presents results concerning the changes in the content of aldehydes in samples of lake water collected near the lake surface. The study of lake waters was undertaken to explain which physicochemical parameters of the environment have the greatest influence on the level of aldehydes, which of the aldehydes are most often met in surface water and in what concentrations. We observed that formaldehyde, acetaldehyde, propanal, glyoxal, methylglyoxal and acetone were commonly present in surface water samples, while semi-volatile and poorly soluble aldehydes such as nonanal and decanal were observed seasonally. The contents of total aldehydes varied in a wide range, from 55 to 670 μg/l, and the concentration of total organic carbon varied significantly from 3 to 18 mg /l, but there was no evident correlation between them in all of samples. The total content of aldehydes did not depend on the meteorological parameters such as air temperature, UV radiation and ozone concentration; however, it was noted that the level of carbonyl concentration is related to the period of intense precipitation: in the period of very low precipitations, the highest contents of total aldehydes were determined in all of the water samples, and in the periods of intense precipitations, the content of total aldehydes was drastically smaller.

  20. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN...

  1. 40 CFR 721.639 - Amine aldehyde condensate.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.639 Amine aldehyde condensate. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as an amine aldehyde condensate (PMN...

  2. Alcohol Dehydrogenase from Methylobacterium organophilum

    PubMed Central

    Wolf, H. J.; Hanson, R. S.

    1978-01-01

    The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10−5 M for methanol and 8.2 × 10−5 M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum. Images PMID:80974

  3. Yeast Alcohol Dehydrogenase Structure and Catalysis

    PubMed Central

    2015-01-01

    Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1) is the constitutive enzyme that reduces acetaldehyde to ethanol during the fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino acid residues. A structure for ADH1 was determined by X-ray crystallography at 2.4 Å resolution. The asymmetric unit contains four different subunits, arranged as similar dimers named AB and CD. The unit cell contains two different tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C subunits in each dimer are structurally similar, with a closed conformation, bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. In contrast, the B and D subunits have an open conformation with no bound coenzyme, and the catalytic zinc has an alternative, inverted coordination with Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the dimeric subunits of the tetramer provides two structures that appear to be relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure. PMID:25157460

  4. Modulation of superoxide dismutase (SOD) isozymes by organ development and high long-term salinity in the halophyte Cakile maritima.

    PubMed

    Houmani, Hayet; Rodríguez-Ruiz, Marta; Palma, José M; Abdelly, Chedly; Corpas, Francisco J

    2016-05-01

    Superoxide dismutase (SOD) activity catalyzes the disproportionation of superoxide radicals into hydrogen peroxide and oxygen. This enzyme is considered to be a first line of defense for controlling the production of reactive oxygen species (ROS). In this study, the number and type of SOD isozymes were identified in the principal organs (roots, stems, leaves, flowers, and seeds) of Cakile maritima. We also analyzed the way in which the activity of these SOD isozymes is modulated during development and under high long-term salinity (400 mM NaCl) stress conditions. The data indicate that this plant contains a total of ten SOD isozymes: two Mn-SODs, one Fe-SOD, and seven CuZn-SODs, with the Fe-SOD being the most prominent isozyme in the different organs analyzed. Moreover, the modulation of SOD isozymes, particularly CuZn-SODs, was only detected during development and under severe salinity stress conditions. These data suggest that, in C. maritima, the occurrence of these CuZn-SODs in roots and leaves plays an adaptive role since this CuZn-SOD isozyme might replace the diminished Fe-SOD activity under salinity stress to overcome this adverse environmental condition. PMID:26159565

  5. Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

    PubMed

    Kim, Sun-Ki; Min, Won-Ki; Park, Yong-Cheol; Seo, Jin-Ho

    2015-11-01

    Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins.

  6. Application of repeated aspartate tags to improving extracellular production of Escherichia coli L-asparaginase isozyme II.

    PubMed

    Kim, Sun-Ki; Min, Won-Ki; Park, Yong-Cheol; Seo, Jin-Ho

    2015-11-01

    Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins. PMID:26320714

  7. A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in Thermoanaerobacter pseudethanolicus 39E

    DOE PAGES

    Clarkson, Sonya M.; Hamilton-Brehm, Scott D.; Giannone, Richard J.; Engle, Nancy L.; Tschaplinski, Timothy J.; Hettich, Robert L.; Elkins, James G.

    2014-12-03

    Background: Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria is important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains such as Thermoanaerobacter spp. may also enable improvements in candidate CBP microorganisms. Results: T. pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30more » mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated vs. 73 downregulated. Only 86 proteins exhibited a 2-fold change in abundance in either direction. Of these, 53 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least 2-fold by furfural and were targeted for further investigation: Teth39_1597, encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. AKR also showed significant activity with NADPH, but only with four carbon butyr

  8. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP.

  9. Cloning and comparative protein modeling of two purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas).

    PubMed

    Durmus, A; Eicken, C; Spener, F; Krebs, B

    1999-09-14

    The sequence of cDNA fragments of two isozymes of the purple acid phosphatase from sweet potato (spPAP1 and spPAP2) has been determined by 5' and 3' rapid amplification of cDNA ends protocols using oligonucleotide primers based on amino acid information. The encoded amino acid sequences of these two isozymes show an equidistance of 72-77% not only to each other, but also to the primary structure of the purple acid phosphatase from red kidney bean (kbPAP). A three-dimensional model of the active site has been constructed for spPAP2 on the basis of the kbPAP crystallographic structure that helps to explain the reported differences in the visible and EPR spectra of spPAP2 and kbPAP. PMID:10556574

  10. Morphology and isozyme band-profile as sexual determinant of nutmeg plant (Myristica fragrants Houttj).

    NASA Astrophysics Data System (ADS)

    Karmanah; Maslahat, M.; Nurhayati, dan L.

    2016-01-01

    Sex information in nutmeg plant is important to distinguish between male and female species. The recommendation of optimum sex-ratio for a nutmeg plantation is 1:10. This research is aimed to discover the morphological characteristic of nutmeg plant and the difference of leaf pattern as sexual determinant between male and female plant with the comparison of isozyme analysis. The result of morphological identification provided from 3 research farm location indicated that female nutmeg was higher, with longer and wider leaf, more vein, bigger diameter of stem and branching angle as compared to male nutmeg. Two parameters which were widht of leaf and branching angle showed significant differences, where female nutmeg plants have wider leaves and branching angle. Isozyme analysis suggested that the analysis of peroxidase enzyme (PER) was better and able to provide more information than aspartarte amino transferase (AAT) and acid phospatase (ACP) Enzymes.

  11. Comparative characterization of pulmonary surfactant aggregates and alkaline phosphatase isozymes in human lung carcinoma tissue.

    PubMed

    Iino, Nozomi; Matsunaga, Toshiyuki; Harada, Tsuyoshi; Igarashi, Seiji; Koyama, Iwao; Komoda, Tsugikazu

    2007-05-01

    Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.

  12. Shifts in the myosin heavy chain isozymes in the mouse heart result in increased energy efficiency

    PubMed Central

    Hoyer, Kirsten; Krenz, Maike; Robbins, Jeffrey; Ingwall, Joanne S.

    2007-01-01

    Cardiac-specific transgenesis in the mouse is widely used to study the basic biology and chemistry of the heart and to model human cardiovascular disease. A fundamental difference between mouse and human hearts is the background motor protein: mouse hearts contain predominantly the αα-myosin heavy chain (MyHC) isozyme while human hearts contain predominantly the ββ-MyHC isozyme. Although the intrinsic differences in mechanical and enzymatic properties of the αα- and ββ-MyHC molecules are well known, the consequences of isozyme shifts on energetic of the intact beating heart remain unknown. Therefore, we compared the free energy of ATP hydrolysis (|ΔG~ATP|) determined by 31P NMR spectroscopy in isolated perfused littermate mouse hearts containing the same amount of myosin comprised of either >95% αα-MyHC or ~83% ββ-MyHC. |ΔG~ATP| was ~2 kJ mol−1 higher in the ββ-MyHC hearts at all workloads. Furthermore, upon inotropic challenge, hearts containing predominantly ββ-MyHC hearts increased developed pressure more than αα-MyHC hearts whereas heart rate increased more in αα-MyHC hearts. Thus, hearts containing predominantly the ββ-MyHC isozyme are more energy efficient than αα-MyHC hearts. We suggest that these fundamental differences in the motor protein energy efficiency at the whole heart level should be considered when interpreting results using mouse-based cardiovascular modeling of normal and diseased human heart. PMID:17054980

  13. Distinct 1-monoacylglycerol and 2-monoacylglycerol kinase activities of diacylglycerol kinase isozymes.

    PubMed

    Sato, Yuriko; Murakami, Chiaki; Yamaki, Atsumi; Mizuno, Satoru; Sakai, Hiromichi; Sakane, Fumio

    2016-09-01

    Diacylglycerol kinase (DGK) consists of ten isozymes and is involved in a wide variety of patho-physiological events. However, the enzymological properties of DGKs have not been fully understood. In this study, we performed a comprehensive analysis on the 1-monoacylglycerol kinase (MGK) and 2-MGK activities of ten DGK isozymes. We revealed that type I (α, β and γ), type II (δ, η and κ) and type III (ε) DGKs have 7.9-19.2% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were <3.0%. Both the 1-MGK and 2-MGK activities of the type IV DGKs (ζ and ι) were <1% relative to their DGK activities. Intriguingly, type V DGKθ has approximately 6% 1-MGK activity and <2% 2-MGK activity compared to its DGK activity. Purified DGKθ exhibited the same results, indicating that its 1-MGK activity is intrinsic. Therefore, DGK isozymes are categorized into three types with respect to their 1-MGK and 2-MGK activities: those having (1) 2-MGK activity relatively stronger than their 1-MGK activity (types I-III), (2) only negligible 1-MGK and 2-MGK activities (type IV), and (3) 1-MGK activity stronger than its 2-MGK activity (type V). The 1-MGK activity of DGKθ and the 2-MGK activity of DGKα were stronger than those of the acylglycerol kinase reported as 1-MGK and 2-MGK to date. The presence or absence of 1-MGK and 2-MGK activities may be essential to the patho-physiological functions of each DGK isozyme.

  14. New tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates.

    PubMed

    Kim, Young-Jin; Chung, Joo Eun; Kurisawa, Motoichi; Uyama, Hiroshi; Kobayashi, Shiro

    2004-01-01

    In this study, new tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates, have been developed. Tyrosinase is a copper-containing enzyme that catalyzes the hydroxylation of a monophenol (monophenolase activity) and the oxidation of an o-diphenol (diphenolase activity). In the measurement of tyrosinase inhibition activity, (+)-catechin acted as substrate and cofactor of tyrosinase. On the other hand, the polycondensates inhibited the tyrosine hydroxylation and L-DOPA oxidation by chelation to the active site of tyrosinase. The UV-visible spectrum of a mixture of tyrosinase and the polycondensate exhibited a characteristic shoulder peak ascribed to the chelation of the polycondensate to the active site of tyrosinase. Furthermore, circular dichroism measurement showed a small red shift of the band due to the interaction between tyrosinase and the polycondensate. These data support that the polycondensate acts as an inhibitor of tyrosinase. PMID:15003008

  15. Conversion of crude Jatropha curcas seed oil into biodiesel using liquid recombinant Candida rugosa lipase isozymes.

    PubMed

    Kuo, Ting-Chun; Shaw, Jei-Fu; Lee, Guan-Chiun

    2015-09-01

    The versatile Candida rugosa lipase (CRL) has been widely used in biotechnological applications. However, there have not been feasibility reports on the transesterification of non-edible oils to produce biodiesel using the commercial CRL preparations, mixtures of isozymes. In the present study, four liquid recombinant CRL isozymes (CRL1-CRL4) were investigated to convert various non-edible oils into biodiesel. The results showed that recombinant CRL2 and CRL4 exhibited superior catalytic efficiencies for producing fatty acid methyl ester (FAME) from Jatropha curcas seed oil. A maximum 95.3% FAME yield was achieved using CRL2 under the optimal conditions (50 wt% water, an initial 1 equivalent of methanol feeding, and an additional 0.5 equivalents of methanol feeding at 24h for a total reaction time of 48 h at 37 °C). We concluded that specific recombinant CRL isozymes could be excellent biocatalysts for the biodiesel production from low-cost crude Jatropha oil. PMID:26011691

  16. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7

    PubMed Central

    Kouyama, Tsutomu

    2016-01-01

    Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS) in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region.

  17. Carbonic anhydrase isozymes of osteoclasts and erythrocytes of osteopetrotic microphthalmic mice.

    PubMed

    Jilka, R L; Rogers, J I; Khalifah, R G; Vaananen, H K

    1985-01-01

    The content of carbonic anhydrase isozymes I (CA I) and II (CA II) in red blood cells and bone of osteopetrotic microphthalmic (mi/mi) mice was analyzed. Monospecific rabbit polyclonal antibodies against purified rat carbonic anhydrase I or II detected both isozymes in hemolysates of both normal and mi/mi mice. Total carbonic anhydrase activity measurements of hemolysates from normal or mi/mi mice were identical. A procedure based on bromopyruvate inactivation was devised to measure the relative contributions of CA I and II isozymes to the carbon dioxide hydration activity of hemolyzates. CA II dominates the observed activity of hemolysates of normal and mi/mi mice. Immunohistochemical studies showed that CA II was present in osteoclasts of tibial and calvarial bones of both normal and mi/mi mice. Thus, in contrast to the several cases of inherited osteopetrosis in humans, there is no lack of active CA II in erythrocytes of mi/mi mice. The absence of CA II, therefore, does not appear to play a role in the etiology of osteopetrosis in the mi/mi mouse.

  18. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7.

    PubMed

    Murakami, Midori; Kouyama, Tsutomu

    2016-01-01

    Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS) in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region. PMID:27656296

  19. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7

    PubMed Central

    Kouyama, Tsutomu

    2016-01-01

    Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS) in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region. PMID:27656296

  20. Isozyme analysis of progeny derived from (Allium fistulosum ×Allium cepa) ×Allium cepa.

    PubMed

    Cryder, C M; Corgan, J N; Urquhart, N S; Clason, D

    1991-09-01

    Relatively large quantities of seed were obtained from the interspecific backcross (A. fistulosum xA. cepa) ×A. cepa allowing, for the first time, an extensive study of the heritable traits exhibited by backcross progeny. Two backcross populations, BC1034 and BC1040, distinguished by differentA. fistulosum parents, were characterized for the isozyme markersIdh-1, Adh-1, andPgi-1. Statistical methods are described to calculate cell probabilities for a mixed population of F2 and BC1 progeny, using an estimate of the fraction of F2 progeny in the population derived from the isozyme data. Cell probability distributions were calculated for a mixed population with independent pairs of loci and a mixed population with nonindependent pairs of loci. The isozyme lociIdh-1 andPgi-1 appear to be linked, with a map distance estimated at 33 centimorgans (cM) in BC1034 and 42 cM in BC1040. The probability distribution model for linked loci did not account for all of the distorted segregation ratios inIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The cytological literature does not support linkage betweenIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The distorted segregation ratios for these pairs of loci are likely the result of genetic incompatibilities between the two species.

  1. Selection of allelic isozyme polymorphisms in marine organisms: pattern, theory, and application.

    PubMed

    Nevo, E; Lavie, E; Ben-Shlomo, R

    1983-01-01

    The evolutionary significance of allelic isozyme polymorphisms in several Mediterranean marine organisms was tested initially by post-hoc gene frequency analyses at 11-15 gene loci in natural populations of barnacles, Balanus amphitrite, under thermal [Nevo et al, 1977] and chemical [Nevo et al, 1978] pollutions. We next carried out pre-hoc controlled laboratory experiments to test the effects of heavy metal pollution (Hg, Zn, Cd) on genotypic frequencies of 15 phosphoglucomutase (PGM) genotypes in thousands of individuals of the shrimp Palaemon elegans [Nevo et al, 1980, 1981a, and the present study]. Similarly, we tested the effects of Hg, Zn, Cd, Pb, Cu pollutions on the genotypic and allelic frequencies of five phosphoglucose isomerase (PGI) genotypes in the two close species of marine gastropods, Monodonta turbinata and M turbiformis [Lavie and Nevo, 1982, and the present study]. In both the thermal and chemical pollution studies, we established in repeated experiments statistically significant differences of allele frequencies at 8 out of 11 (73%) and 10 out of 15 (67%) gene loci, respectively, between the contrasting environments in each. While no specific function could be singled out in the post-hoc chemical study due to the complex nature of polluted marine water, temperature could be specified as the primary selective agent in the thermal study. The strongest direct and specific evidence for significant differential survivorship among allelic isozyme genotypes was obtained in the pre-hoc studies in Palaemon and Monodonta. Their differential viability was probably associated with the different degree of heavy metal inhibition uniquely related to each specific pollutant. Furthermore, we demonstrated in the two closely related Monodonta species parallel genotypic differentiation as a response to pollution. Our results are inconsistent with the neutral theory of allelic isozyme polymorphisms and appear to reflect the adaptive nature of the allelic isozyme

  2. Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

    PubMed Central

    2014-01-01

    The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated. PMID:24949265

  3. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed Central

    Burdette, D; Zeikus, J G

    1994-01-01

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

  4. Identification and analysis of drug-responsive expression of UDP-glucuronosyltransferase family 1 (UGT1) isozyme in rat hepatic microsomes using anti-peptide antibodies.

    PubMed

    Ikushiro, S; Emi, Y; Iyanagi, T

    1995-12-20

    Expression of rat hepatic UDP-glucuronosyltransferase family 1 (UGT1) isozymes has been examined using anti-peptide antibodies raised against a conserved carboxyl-terminal portion of all isozymes and variable amino-terminal portions of each isozyme of the phenol cluster (UGT1A) and bilirubin cluster (UGT1B). Among the isozymes expressed in rat hepatic microsomes, UGT1B1 (54 kDa) of bilirubin cluster was found to be a major form and minor forms were identified as UGT1A1 (53 kDa), UGT1B2 (56 kDa), and UGT1B5 (57 kDa). Using a combination of 2D sodium dodecyl sulfate gel electrophoresis and immunoblotting, all the isozymes were found to be simultaneously lacked in Gunn rat hepatic microsomes. The effects of various drugs as inducer on the expression of each UGT1 isozyme were analyzed. The UGT1A1 and UGT1A2 of the phenol cluster isozymes were significantly induced in 3-methylcholanthrene-treated rats. The expression of UGT1B1 and the glucuronidation activity toward bilirubin in rat hepatic microsomes were induced two- to threefold by clofibrate and dexamethasone administration. On the other hand, the regulation of UGT1B2 and UGT1B5 expression was different from that of UGT1B1. These results clearly show the drug-responsive expression of each UGT1 isozyme using isozyme-specific antibodies for the first time. PMID:8554318

  5. NADH dehydrogenase: a new molecular marker for homoeology group 4 in Triticeae. A map of the 4RS chromosome arm in rye.

    PubMed

    Figueiras, A M; Zaragoza, C; Gallego, F J; Benito, C

    1991-12-01

    Structural gene loci encoding the monomeric isozymes nicotin adenin dinucleotide dehydrogenase (NADH dehydrogenase or NDH) have been located on the 4AL, 4Bα, and 4DS chromosome arms of Triticum aestivum cv "Chinese Spring", on the 4RS chromosome arm of Secale cereale cultivars "Imperial", "King II", "Dakold," and "Ailes," on the 4S(1) S/7S(1) chromosome of Aegilops longissima, the 4E of Elytrigia elongata, and the CSU-A of Aegilops umbellulata. All the results support the homoeologous relationships among these chromosomes in the five species studied. In addition, a map of the 4RS chromosome arm in cv "Ailes" has been realized, linking loci Pgm-1 (located on the 4RS chromosome arm) and Ndh-1 (17.91 cM), with an estimated distance between both loci and the centromere of 20.00 cM and 32.12 cM, respectively. PMID:24202354

  6. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the fermentation of wine and then returned to the distilled spirits plant from which distillates were... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are...

  7. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the fermentation of wine and then returned to the distilled spirits plant from which distillates were... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are...

  8. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... the fermentation of wine and then returned to the distilled spirits plant from which distillates were... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are...

  9. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the fermentation of wine and then returned to the distilled spirits plant from which distillates were... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are...

  10. 27 CFR 24.183 - Use of distillates containing aldehydes.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the fermentation of wine and then returned to the distilled spirits plant from which distillates were... fermentation of wine made from a different kind of fruit. Distillates containing aldehydes which are...

  11. Molecular Structure and Reactivity in the Pyrolysis of Aldehydes

    NASA Astrophysics Data System (ADS)

    Sias, Eric; Cole, Sarah; Sowards, John; Warner, Brian; Wright, Emily; McCunn, Laura R.

    2016-06-01

    The effect of alkyl chain structure on pyrolysis mechanisms has been investigated in a series of aldehydes. Isovaleraldehyde, CH_3CH(CH_3)CH_2CHO, and pivaldehyde, (CH_3)_3CCHO, were subject to thermal decomposition in a resistively heated SiC tubular reactor at 800-1200 °C. Matrix-isolation FTIR spectroscopy was used to identify pyrolysis products. Carbon monoxide and isobutene were major products from each of the aldehydes, which is consistent with what is known from previous studies of unbranched alkyl-chain aldehydes. Other products observed include vinyl alcohol, propene, acetylene, and ethylene, revealing complexities to be considered in the pyrolysis of large, branched-chain aldehydes.

  12. Silver-catalyzed synthesis of amides from amines and aldehydes

    DOEpatents

    Madix, Robert J; Zhou, Ling; Xu, Bingjun; Friend, Cynthia M; Freyschlag, Cassandra G

    2014-11-18

    The invention provides a method for producing amides via the reaction of aldehydes and amines with oxygen adsorbed on a metallic silver or silver alloy catalyst. An exemplary reaction is shown in Scheme 1: (I), (II), (III). ##STR00001##

  13. 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 ...

  14. Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae.

    PubMed

    Tibbetts, A S; Appling, D R

    2000-07-01

    The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.

  15. Amino Acid Residues Critical for the Specificity for Betaine Aldehyde of the Plant ALDH10 Isoenzyme Involved in the Synthesis of Glycine Betaine1[W][OA

    PubMed Central

    Díaz-Sánchez, Ángel G.; González-Segura, Lilian; Mújica-Jiménez, Carlos; Rudiño-Piñera, Enrique; Montiel, Carmina; Martínez-Castilla, León P.; Muñoz-Clares, Rosario A.

    2012-01-01

    Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant Km(BAL) increases and Vmax/Km(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the Vmax/Km(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants. PMID:22345508

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

    PubMed Central

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

    2015-01-01

    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 CO2-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 CO2 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 CO2 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 %. PMID:26113881

  17. Reducing Isozyme Competition Increases Target Fatty Acid Accumulation in Seed Triacylglycerols of Transgenic Arabidopsis1[OPEN

    PubMed Central

    van Erp, Harrie; Shockey, Jay; Zhang, Meng; Adhikari, Neil D.; Browse, John

    2015-01-01

    One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies. PMID:25739701

  18. Comparative electron paramagnetic resonance study of radical intermediates in turnip peroxidase isozymes.

    PubMed

    Ivancich, A; Mazza, G; Desbois, A

    2001-06-12

    The occurrence of isozymes in plant peroxidases is poorly understood. Turnip roots contain seven season-dependent isoperoxidases with distinct physicochemical properties. In the work presented here, multifrequency electron paramagnetic resonance spectroscopy has been used to characterize the Compound I intermediate obtained by the reaction of turnip isoperoxidases 1, 3, and 7 with hydrogen peroxide. The broad (2500 G) Compound I EPR spectrum of all three peroxidases was consistent with the formation of an exchange-coupled oxoferryl-porphyrinyl radical species. A dramatic pH dependence of the exchange interaction of the [Fe(IV)=O por(*+)] intermediate was observed for all three isoperoxidases and for a pH range of 4.5-7.7. This result provides substantial experimental evidence for previous proposals concerning the protein effect on the ferro- or antiferromagnetic character of the exchange coupling of Compound I based on model complexes. Turnip isoperoxidase 7 exhibited an unexpected pH effect related to the nature of the Compound I radical. At basic pH, a narrow radical species ( approximately 50 G) was formed together with the porphyrinyl radical. The g anisotropy of the narrow radical Delta(g) = 0.0046, obtained from the high-field (190 and 285 GHz) EPR spectrum, was that expected for tyrosyl radicals. The broad g(x) edge of the Tyr* spectrum centered at a low g(x) value (2.00660) strongly argues for a hydrogen-bonded tyrosyl radical in a heterogeneous microenvironment. The relationship between tyrosyl radical formation and the higher redox potential of turnip isozyme 7, as compared to that of isozyme 1, is discussed. PMID:11389600

  19. [Effect of Fuzheng Huayu recipe on CYP450 isozymes in normal and liver fibrosis rats].

    PubMed

    Zheng, Tian-hui; Liu, Wei; Li, Shu-ping; Yang, Tao; Wang, Chang-hong; Liu, Cheng-hai

    2015-03-01

    To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats. PMID:26226765

  20. Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1†

    PubMed Central

    Gonçalves, Edmilson R.; Hara, Hirofumi; Miyazawa, Daisuke; Davies, Julian E.; Eltis, Lindsay D.; Mohn, William W.

    2006-01-01

    Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes. PMID:16957245

  1. Soybean roots retain the seed urease isozyme synthesized during embryo development. [Glycine max (L. ) Merr

    SciTech Connect

    Torisky, R.S.; Polacco, J.C. )

    1990-10-01

    Roots of young soybean (Glycine max (L.) Merr.) plants (up to 25 days old) contain two distinct urease isozymes, which are separable by hydroxyapatite chromatography. These two urease species (URE1 and URE2) differ in: (a) electrophoretic mobility in native gels, (b) pH dependence, and (c) recognition by a monoclonal antibody specific for the seed (embryo-specific) urease. By these parameters root URE1 urease is similar to the abundant embryo-specific urease isozyme, while root URE2 resembles the ubiquitous urease which has previously been found in all soybean tissues examined (leaf, embryo, seed coat, and cultured cells). The embryo-specific and ubiquitous urease isozymes are products of the Eu1 and Eu4 structural genes, respectively. Roots of the eu1-sun/eu1-sun genotype, which lacks the embryo-specific urease (i.e. seed urease-null), contain no URE1 urease activity. Roots of eu4/eu4, which lacks ubiquitous urease, lack the URE2 (leaflike) urease activity. From these genetic and biochemical criteria, then, we conclude that URE1 and URE2 are the embryo-specific and ubiquitous ureases, respectively. Adventitious roots generated from cuttings of any urease genotype lack URE1 activity. In seedling roots the seedlike (URE1) activity declines during development. Roots of 3-week-old plants contain 5% of the total URE1 activity of the radicle of 4-day-old seedlings, which, in turn, has approximately the same urease activity level as the dormant embryonic axis. The embryo-specific urease incorporates label from ({sup 35}S)methionine during embryo development but not during germination, indicating that there is no de novo synthesis of the embryo-specific (URE1) urease in the germinating root.

  2. Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

    PubMed

    Howard, Michael J; Karasik, Agnes; Klemm, Bradley P; Mei, Christine; Shanmuganathan, Aranganathan; Fierke, Carol A; Koutmos, Markos

    2016-05-01

    Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N-1of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10(4)-10(5)M(-1)s(-1)) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3'-discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes. PMID:26966150

  3. Beta-hexosaminidase isozymes and replacement therapy in Gm2 gangliosidosis.

    PubMed

    Rattazzi, M C

    1983-01-01

    The problem of cell targeting of lysosomal enzymes is a critical one in the development of strategies for therapeutic enzyme replacement in lysosomal storage diseases. In principle, posttranscriptional isozymes with different carbohydrate-chain composition may be helpful in exploiting existing glycosyl-specific receptors on target cells, if the receptor specificities are known and match the glycosyl composition of available isozymes. In practice, however, the choice is limited to isozymes that can be obtained from tissues available in abundance, such as placenta or blood plasma. Our early experiments show that one can interfere with the interaction between hepatic (RES) receptor and enzyme glycosyl chain, to obtain extrahepatic targeting of beta-hexosaminidase, with catabolic effects. This approach, of course, does not have an immediate therapeutic application, as it involves injection of large amounts of foreign material in order to inhibit hepatic uptake. Modification of the glycosyl chain may be the method of choice in selected instances [Furbish et al. 1981], but is applicability again depends on the knowledge of receptor specificity on target cells and on composition of the glycosyl chain of the enzyme in question. Our recent experiments are a first step toward obtaining enzyme forms that can be endocytosed efficiently by mechanisms that are independent of glycosyl-specific receptors. Charge-mediated, absorptive endocytosis can be obtained by covalent coupling of cationic PLL to beta-hexosaminidase. Given the abundance of negative surface charges on most cell types [Weiss, 1969], this approach may be applicable to different target cells and organs, and possibly also to lysosomal enzymes other than beta-hexosaminidase. The existence of glycosyl recognition signals on beta-hexosaminidase can be obviated by simple chemical manipulations, such as Na-metaperiodate oxidation, which effectively prevents hepatic RES uptake [Rattazzi et al, 1982]. In combination with

  4. RS-Predictor models augmented with SMARTCyp reactivities: Robust metabolic regioselectivity predictions for nine CYP isozymes

    PubMed Central

    Zaretzki, Jed; Rydberg, Patrik; Bergeron, Charles; Bennett, Kristin P.; Olsen, Lars

    2012-01-01

    RS-Predictor is a tool for creating pathway-independent, isozyme-specific site of metabolism (SOM) prediction models using any set of known cytochrome P450 substrates and metabolites. Until now, the RS-Predictor method was only trained and validated on CYP 3A4 data, but in the present study we report on the versatility the RS-Predictor modeling paradigm by creating and testing regioselectivity models for substrates of the nine most important CYP isozymes. Through curation of source literature, we have assembled 680 substrates distributed among CYPs 1A2, 2A6, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1 and 3A4, which we believe is the largest publicly accessible collection of P450 ligands and metabolites ever released. A comprehensive investigation into the importance of different descriptor classes for predicting the regioselectivity of each isozyme is made through the generation of multiple independent RS-Predictor models for each set of isozyme substrates. Two of these models include a DFT reactivity descriptor derived from SMARTCyp. Optimal combinations of RS-Predictor and SMARTCyp are shown to have stronger performance than either method alone, while also exceeding the accuracy of the commercial regioselectivity prediction methods distributed by StarDrop and Schrödinger, correctly identifying a large proportion of the metabolites in each substrate set within the top two rank-positions: 1A2(83.0%), 2A6(85.7%), 2B6(82.1%), 2C19(86.2%), 2C8(83.8%), 2C9(84.5%), 2D6(85.9%), 2E1(82.8%), 3A4(82.3%) and merged(86.0%). Comprehensive datamining of each substrate set and careful statistical analyses of the predictions made by the different models revealed new insights into molecular features that control metabolic regioselectivity and enable accurate prospective prediction of likely SOMs. PMID:22524152

  5. Selectivity of pyripyropene derivatives in inhibition toward acyl-CoA:cholesterol acyltransferase 2 isozyme.

    PubMed

    Ohshiro, Taichi; Ohte, Satoshi; Matsuda, Daisuke; Ohtawa, Masaki; Nagamitsu, Tohru; Sunazuka, Toshiaki; Harigaya, Yoshihiro; Rudel, Lawrence L; Omura, Satoshi; Tomoda, Hiroshi

    2008-08-01

    Selectivity of 96 semisynthetic derivatives prepared from fungal pyripyropene A, originally isolated as a potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), toward ACAT1 and ACAT2 isozymes was investigated in the cell-based assay using ACAT1- and ACAT2-expressing CHO cells. Eighteen derivatives including PR-71 (7-O-isocaproyl derivative) showed much more potent ACAT2 inhibition (IC50: 6.0 to 62 nM) than pyripyropene A (IC50: 70 nM). Among them, however, natural pyripyropene A showed the highest selectivity toward ACAT2 with a selectivity index (SI) of >1000, followed by PR-71 (SI, 667). PMID:18997389

  6. Kinetic and docking studies of cytosolic/tumor-associated carbonic anhydrase isozymes I, II and IX with some hydroxylic compounds.

    PubMed

    Durdagi, Serdar; Korkmaz, Neslihan; Işık, Semra; Vullo, Daniela; Astley, Demet; Ekinci, Deniz; Salmas, Ramin E; Senturk, Murat; Supuran, Claudiu T

    2016-12-01

    A series of hydroxylic compounds (1-10, NK-154 and NK-168) have been assayed for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic isozymes I, II and tumor-associated isozyme IX. The investigated compounds showed inhibition constants in the range of 0.068-4003, 0.012-9.9 and 0.025-115 μm at the hCA I, hCA II and hCA IX enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are calculated using scoring algorithms, namely Glide/induced fit docking. The inhibitory potencies of the novel compounds were analyzed at the human isoforms hCA I, hCA II and hCA IX as targets and the KI values were calculated.

  7. Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity[W

    PubMed Central

    Shumskaya, Maria; Bradbury, Louis M.T.; Monaco, Regina R.; Wurtzel, Eleanore T.

    2012-01-01

    Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zea mays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis. PMID:23023170

  8. Kinetic and docking studies of cytosolic/tumor-associated carbonic anhydrase isozymes I, II and IX with some hydroxylic compounds.

    PubMed

    Durdagi, Serdar; Korkmaz, Neslihan; Işık, Semra; Vullo, Daniela; Astley, Demet; Ekinci, Deniz; Salmas, Ramin E; Senturk, Murat; Supuran, Claudiu T

    2016-12-01

    A series of hydroxylic compounds (1-10, NK-154 and NK-168) have been assayed for the inhibition of three physiologically relevant carbonic anhydrase isozymes, the cytosolic isozymes I, II and tumor-associated isozyme IX. The investigated compounds showed inhibition constants in the range of 0.068-4003, 0.012-9.9 and 0.025-115 μm at the hCA I, hCA II and hCA IX enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are calculated using scoring algorithms, namely Glide/induced fit docking. The inhibitory potencies of the novel compounds were analyzed at the human isoforms hCA I, hCA II and hCA IX as targets and the KI values were calculated. PMID:26634620

  9. Purification and characterization of two distinct aldehyde-oxidizing enzymes from the liver of black seabream.

    PubMed

    Fong, Wing-Ping; Cheng, Christopher H K; Tang, Wai-Kwan

    2003-02-01

    Two aldehyde dehydrogenases (ALDH) were purified from the liver of black seabream (Acanthopagrus schlegeli). Chromatography of the liver homogenate on an alpha-cyanocinnamate-Sepharose affinity column results in two activity peaks using acetaldehyde as the substrate. The eluate was subjected to another affinity chromatography on p-hydroxyacetophenone-Sepharose. The final preparation showed a single band on SDS-PAGE with a subunit M.W. of 56,000. N-terminal amino acid sequencing of the first 29 residues followed by blastp analysis on the NCBI database revealed this protein as ALDH-2, as it exhibited 69% identity with human mitochondrial ALDH-2. Chromatography of the alpha-cyanocinnamate-Sepharose column flow-through fractions on Affi-gel Blue agarose yielded another ALDH. The purified protein, with a subunit M.W. of 57,500, was identified as antiquitin (turgor ALDH) by its first 18 N-terminal amino acid residues, which showed 83% identity with the deduced amino acid sequence of human antiquitin. Kinetically, both ALDHs showed maximal activity at pH around 8.5-9.0. They differed, however, in their catalytic efficiency towards the oxidation of acetaldehyde. Antiquitin had much lower affinity towards acetaldehyde; the Km value being approximately 220-fold higher than that of ALDH-2. The Vmax of antiquitin was only approximately 12% of ALDH-2. Antiquitin is believed to be involved in the regulation of cellular turgor pressure. However, all previous studies on antiquitin have been confined to the nucleotide level and the protein has never been isolated from any source. The development of an effective purification procedure and the demonstration that this protein is an enzyme with aldehyde-oxidizing activity would be highly valuable for further investigations on the physiological significance of this evolutionarily conserved protein. PMID:12604200

  10. Structure and evolution of vertebrate aldehyde oxidases: from gene duplication to gene suppression.

    PubMed

    Kurosaki, Mami; Bolis, Marco; Fratelli, Maddalena; Barzago, Maria Monica; Pattini, Linda; Perretta, Gemma; Terao, Mineko; Garattini, Enrico

    2013-05-01

    Aldehyde oxidases (AOXs) and xanthine dehydrogenases (XDHs) belong to the family of molybdo-flavoenzymes. Although AOXs are not identifiable in fungi, these enzymes are represented in certain protists and the majority of plants and vertebrates. The physiological functions and substrates of AOXs are unknown. Nevertheless, AOXs are major drug metabolizing enzymes, oxidizing a wide range of aromatic aldehydes and heterocyclic compounds of medical/toxicological importance. Using genome sequencing data, we predict the structures of AOX genes and pseudogenes, reconstructing their evolution. Fishes are the most primitive organisms with an AOX gene (AOXα), originating from the duplication of an ancestral XDH. Further evolution of fishes resulted in the duplication of AOXα into AOXβ and successive pseudogenization of AOXα. AOXβ is maintained in amphibians and it is the likely precursors of reptilian, avian, and mammalian AOX1. Amphibian AOXγ is a duplication of AOXβ and the likely ancestor of reptilian and avian AOX2, which, in turn, gave rise to mammalian AOX3L1. Subsequent gene duplications generated the two mammalian genes, AOX3 and AOX4. The evolution of mammalian AOX genes is dominated by pseudogenization and deletion events. Our analysis is relevant from a structural point of view, as it provides information on the residues characterizing the three domains of each mammalian AOX isoenzyme. We cloned the cDNAs encoding the AOX proteins of guinea pig and cynomolgus monkeys, two unique species as to the evolution of this enzyme family. We identify chimeric RNAs from the human AOX3 and AOX3L1 pseudogenes with potential to encode a novel microRNA.

  11. Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

    PubMed Central

    Wu, Gang; Fiser, András; ter Kuile, Benno; Šali, Andrej; Müller, Miklós

    1999-01-01

    Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently. PMID:10339579

  12. Novel dehydrogenase catalyzes oxidative hydrolysis of carbon-nitrogen double bonds for hydrazone degradation.

    PubMed

    Itoh, Hideomi; Suzuta, Tetsuya; Hoshino, Takayuki; Takaya, Naoki

    2008-02-29

    Hydrazines and their derivatives are versatile artificial and natural compounds that are metabolized by elusive biological systems. Here we identified microorganisms that assimilate hydrazones and isolated the yeast, Candida palmioleophila MK883. When cultured with adipic acid bis(ethylidene hydrazide) as the sole source of carbon, C. palmioleophila MK883 degraded hydrazones and accumulated adipic acid dihydrazide. Cytosolic NAD+- or NADP+-dependent hydrazone dehydrogenase (Hdh) activity was detectable under these conditions. The production of Hdh was inducible by adipic acid bis(ethylidene hydrazide) and the hydrazone, varelic acid ethylidene hydrazide, under the control of carbon catabolite repression. Purified Hdh oxidized and hydrated the C=N double bond of acetaldehyde hydrazones by reducing NAD+ or NADP+ to produce relevant hydrazides and acetate, the latter of which the yeast assimilated. The deduced amino acid sequence revealed that Hdh belongs to the aldehyde dehydrogenase (Aldh) superfamily. Kinetic and mutagenesis studies showed that Hdh formed a ternary complex with the substrates and that conserved Cys is essential for the activity. The mechanism of Hdh is similar to that of Aldh, except that it catalyzed oxidative hydrolysis of hydrazones that requires adding a water molecule to the reaction catalyzed by conventional Aldh. Surprisingly, both Hdh and Aldh from baker's yeast (Ald4p) catalyzed the Hdh reaction as well as aldehyde oxidation. Our findings are unique in that we discovered a biological mechanism for hydrazone utilization and a novel function of proteins in the Aldh family that act on C=N compounds. PMID:18096698

  13. Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT-20 cells.

    PubMed Central

    McFerran, B. W.; MacEwan, D. J.; Guild, S. B.

    1995-01-01

    1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)-mediated enhancement of calcium- and guanine nucleotide-evoked adrenocorticotrophin (ACTH) secretion. 2. A profile of the PKC isozymes present in AtT-20 cells was obtained by Western blotting analysis and it was found that AtT-20 cells express the alpha, beta, epsilon and zeta isoforms of PKC. 3. PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically-permeabilized cells. Phorbol 12-myristate 13-acetate (PMA, EC50 = 1 +/- 0.05 nM, which activates all isozymes of PKC, except the zeta isozyme), thymeleatoxin (TMX, EC50 = 10 +/- 0.5 nM, which activates the alpha, beta and gamma isozymes) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA, EC50 = 3 +/- 0.5 nM, a beta 1-selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration-dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA-stimulated ACTH secretion was only 60-70% of that obtained in response to PMA or TMX. 4. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 100 nM to 10 microM. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 PMID:7670732

  14. Sorbitol dehydrogenase is a zinc enzyme.

    PubMed Central

    Jeffery, J; Chesters, J; Mills, C; Sadler, P J; Jörnvall, H

    1984-01-01

    Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins. PMID:6370679

  15. Saturation transfer difference NMR studies on substrates and inhibitors of succinic semialdehyde dehydrogenases

    SciTech Connect

    Jaeger, Martin Rothacker, Boris; Ilg, Thomas

    2008-08-01

    Saturation transfer difference (STD) NMR experiments on Escherichia coli and Drosophila melanogaster succinic semialdehyde dehydrogenase (SSADH, EC1.2.1.24) suggest that only the aldehyde forms and not the gem-diol forms of the specific substrate succinic semialdehyde (SSA), of selected aldehyde substrates, and of the inhibitor 3-tolualdehyde bind to these enzymes. Site-directed mutagenesis of the active site cysteine311 to alanine in D. melanogaster SSADH leads to an inactive product binding both SSA aldehyde and gem-diol. Thus, the residue cysteine311 is crucial for their discrimination. STD experiments on SSADH and NAD{sup +}/NADP{sup +} indicate differential affinity in agreement with the respective cosubstrate properties. Epitope mapping by STD points to a strong interaction of the NAD{sup +}/NADP{sup +} adenine H2 proton with SSADH. Adenine H8, nicotinamide H2, H4, and H6 also show STD signals. Saturation transfer to the ribose moieties is limited to the anomeric protons of E. coli SSADH suggesting that the NAD{sup +}/NADP{sup +} adenine and nicotinamide, but not the ribose moieties are important for the binding of the coenzymes.

  16. Protein kinase C in the wood frog, Rana sylvatica: reassessing the tissue-specific regulation of PKC isozymes during freezing

    PubMed Central

    Storey, Kenneth B.

    2014-01-01

    The wood frog, Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites—in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study

  17. Gibberellic acid controls specific acid-phosphatase isozymes in aleurone cells and protoplasts of Avena fatua L.

    PubMed

    Hooley, R

    1984-06-01

    In the presence of gibberellic acid (GA3) aleurone layers and isolated aleurone protoplasts of Avena fatua accumulate specific isozymes of acid phosphatase (EC 3.1.3.2). Some of these may be involved in mobilizing aleurone-grain phosphate reserves during germination. The hormone also controls secretion of other specific molecular forms of the enzyme that probably assist in endosperm hydrolysis. The accumulation and secretion of putative cell-wall-associated isozymes are stimulated by the action of GA3 in isolated protoplasts. This effect however, is apparently over-ridden in the intact tissue, possibly by a cell-wall-based feedback mechanism.

  18. Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

    PubMed Central

    Miyake, Masayuki; Fushinobu, Shinya

    2016-01-01

    Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system. PMID:26808202

  19. Inhibitory effect of Aristolochia fruit on Cytochrome P450 isozymes in vitro and in vivo.

    PubMed

    Zhu, Shuzhen; Gao, Jingwen; Shi, Zhan; QLi, George; Chen, Zuanguang; Yao, Meicun

    2015-05-01

    The mature fruits of Aristolochia debilis, known in China by the name, "Madouling" has been popularly prescribed in Asia, particularly in China, to treat a range of conditions including gynaecological problems, arthritis and wound healing. This study was aimed to evaluate the potential effect of Madouling on the cytochrome P450 (CYP) isozymes in vitro in microsomal fractions and in vivo in rats. The influence of Madouling on CYPs activity was first explored by an in vitro method of estimating levels of four respective metabolites in rat liver microsomes. The results were re-examined in vivo in rats by using a cocktail approach involving the probe drugs theophylline, tolbutamide, chlorzoxazone and dapsone. Pharmacokinetics of the four substrates was used to analyze the activities of the targeting isozymes. In vitro study revealed that Madouling decreased the activity of CYP1A2, 3A1 and 2E1. However, no significant influence on CYP2C6 was found. These results coincided with those of in vivo study to a great degree except that in vivo estimation the herb didn't inhibit CYP1A2 significantly. From the data obtained, Madouling is suggested as a candidate for clinically significant CYP interactions. Drug co-administrated with Madouling may need dose adjustment.

  20. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis.

    PubMed

    Maisonneuve, Sylvie; Bessoule, Jean-Jacques; Lessire, René; Delseny, Michel; Roscoe, Thomas J

    2010-02-01

    In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.

  1. Applications of β-gal-III isozyme from Bacillus coagulans RCS3, in lactose hydrolysis.

    PubMed

    Batra, Navneet; Singh, Jagtar; Joshi, Amit; Bhatia, Sonu

    2011-12-01

    Bacillus coagulans RCS3 isolated from hot water springs secreted five isozymes i.e. β-gal I-V of β-galactosidase. β-gal III isozyme was purified using DEAE cellulose and Sephadex G 100 column chromatography. Its molecular weight characterization showed a single band at 315kD in Native PAGE, while two subunits of 50.1 and 53.7 kD in SDS PAGE. β-Gal III had pH optima in the range of 6-7 and temperature optima at 65°C. It preferred nitro-aryl-β-d-galactoside as substrate having K(m) of 4.16 mM with ONPG. More than 85% and 80% hydrolysis of lactose (1-5%, w/v) was recorded within 48 h of incubation at 55°C and 50°C respectively and pH range of 6-7. About 78-86% hydrolysis of lactose in various brands of standardized milk was recorded at incubation temperature of 50°C. These results marked the applications of β-gal III in processing of milk/whey industry. PMID:21855568

  2. Distinct physiological roles for the two L-asparaginase isozymes of Escherichia coli

    SciTech Connect

    Srikhanta, Yogitha N.; Atack, John M.; Beacham, Ifor R.; Jennings, Michael P.

    2013-07-05

    Highlights: •Escherichia coli contains two L-asparaginase isozymes with distinct localization, kinetics and regulation. •Mutant strains were used to examine the roles of these enzymes in L-asparagine utilization. •We report that L-asparaginase II permits growth on asparagine and glycerol under anaerobic conditions. •We propose that this enzyme is the first step in a co-regulated pathway leading to fumarate. •The pathway is regulated by anaerobiosis and cAMP and provides a terminal elector acceptor. -- Abstract: Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.

  3. Role of PKC isozymes in low-power light-stimulated proliferation of cultured skin cells

    NASA Astrophysics Data System (ADS)

    Grossman, Nili; Kleitman, Vered; Meller, Julia; Kaufmann, Roland; Akgun, Nermin; Ruck, Angelika; Livneh, Etta; Lubart, Rachel

    2000-11-01

    Exposure of cultured skin cells to low power visible light leads to a transiently stimulated proliferation. Facilitation of this response requires the presence of active PKC, elevation of intracellular calcium, and involves reactive oxygen species. In the present study, the role of PKC(alpha) and PCK(eta) was examined using paired murine fibroblasts, differing in the level of these isozymes expression. The ability of the cells to respond to low power UVA light or HeNe laser by stimulated proliferation was correlated with an active state or overexpression of PKC(alpha) , but not PKC(eta) . A parallel response was obtained in cells that were loaded with A1PcS4 before photosensitization. Whenever this latter treatment caused a light-stimulated inhibition, it was accompanied by the intracellular calcium and photosensitizer dynamics typical of the effect of PDT on rate epithelial cells. Accordingly, added antioxidants that suppressed light-stimulated proliferation also suppressed this light-stimulated inhibition. The model systems employed in this study are the first to demonstrate the specific effect of PKC isozymes on light-stimulated proliferation, in relation to oxidative stress, and indicate their dual role in light-tissue interaction.

  4. Soybean roots retain the seed urease isozyme synthesized during embryo development

    SciTech Connect

    Torisky, R.S.; Polacco, J.C. )

    1990-05-01

    Roots of young soybean plants contain two urease isozymes which are separable by hydroxyapatite chromatography. These two urease species (HAP1 and HAP2) differ in: (1) native gel electrophoretic mobility, (2) pH optima, and (3) recognition by a monoclonal antibody specific for the embryo-specific urease. By these parameters HAP1 is similar to the abundant embryo-specific urease isozyme while HAP2 resembles the ubiquitous urease, found in all soybean tissues previously examined (embryo, seed coat, cultured cells). Roots of mutant soybean plants lacking the seed urease contain no HAP1 urease activity, whereas roots of mutants lacking the ubiquitous urease contain no HAP2 urease activity. However, adventitious roots generated from cuttings of any urease genotype lack HAP1 urease activity. Furthermore, ({sup 35}S) methionine labelling shows no {und de novo} synthesis of the HAP1 urease in the root, and total root HAP1 urease activity decreases sharply following germination. We conclude: (1) HAP1 is a remnant of the seed urease accumulated in the embryonic root axis during seed development, and (2) HAP2 is ubiquitous urease synthesized de novo in the root.

  5. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

    PubMed Central

    Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity

  6. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

    PubMed

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong; Hassan, Maizom

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards

  7. Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

    SciTech Connect

    Hondred, D. ); Hanson, A.D. Univ. de Montreal, Quebec )

    1990-09-01

    In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

  8. Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella.

    PubMed Central

    Israel, D W; Gronostajski, R M; Yeung, A T; Schmidt, R R

    1977-01-01

    Earlier studies indicated that the gene of an ammonium-inducible glutamate dehydrogenase (GDH) was inducible throughout the cell cycle and was expressible shortly after replication early in the S-phase in synchronous Chlorella cells growing at a rate of 13% per h in the absence of inducer. In the present study, synchronous cells cultured at the same growth rate in the continuous presence of inducer accumulated this enzyme in a linear manner, with a positive rate change observed late instead of early in the S-phase. At a growth rate of 26% per h, the positive rate change appeared to be displaced to 1.5 h before the S-phase in the next cell cycle. With 2'-deoxyadenosine, an in vivo inhibitor of deoxyribonucleic acid (DNA) synthesis, the magnitude of the positive rate change was shown to be proportional to the relative increase in DNA in the previous cell cycle. Collectively, these data support the idea that expression of newly replicated genes of this enzyme can be delayed into the subsequent cell cycle in cells in the continuous presence of inducer. Studies with cycloheximide indicated that the inducible GDH and another GDH isozyme were stable in fully induced cells in the absence of protein synthesis. However, after ammonium was removed from the culture medium, the activity of the inducible GDH decreased rapidly in vivo, with a half-time of 5 to 10 min at 38.5 degrees C, whereas the rate of accumulation of the other GDH isozyme did not change. Addition of cycloheximide, at the time of inducer removal, prevented this loss in activity of the inducible GDH. The inability to rescue the activity of the inducible GDH, by readdition of ammonium during the deinduction period, indicates that this enzyme probably underwent irreversible inactivation and/or proteolytic degradation. PMID:45486

  9. Contribution of ozone to airborne aldehyde formation in Paris homes.

    PubMed

    Rancière, Fanny; Dassonville, Claire; Roda, Célina; Laurent, Anne-Marie; Le Moullec, Yvon; Momas, Isabelle

    2011-09-15

    Indoor aldehydes may result from ozone-initiated chemistry, mainly documented by experimental studies. As part of an environmental investigation included in the PARIS birth cohort, the aim of this study was to examine ozone contribution to airborne aldehyde formation in Paris homes. Formaldehyde, acetaldehyde and hexaldehyde levels, as well as styrene, nitrogen dioxide and nicotine concentrations, comfort parameters and carbon dioxide levels, were measured twice during the first year of life of the babies. Ambient ozone concentrations were collected from the closest background station of the regional air monitoring network. Traffic-related nitrogen oxide concentrations in front of the dwellings were estimated by an air pollution dispersion model. Home characteristics and families' way of life were described by questionnaires. Stepwise multiple linear regression models were used to link aldehyde levels with ambient ozone concentrations and a few aldehyde precursors involved in oxidation reactions, adjusting for other indoor aldehyde sources, comfort parameters and traffic-related nitrogen oxides. A 4 and 11% increase in formaldehyde and hexaldehyde levels was pointed out when 8-hour ozone concentrations increased by 20 μg/m(3). The influence of potential precursors such as indoor styrene level and frequent use of air fresheners, containing unsaturated volatile organic compounds as terpenes, was also found. Thus, our results suggest that ambient ozone can significantly impact indoor air quality, especially with regard to formaldehyde and hexaldehyde levels.

  10. Maternal aldehyde elimination during pregnancy preserves the fetal genome.

    PubMed

    Oberbeck, Nina; Langevin, Frédéric; King, Gareth; de Wind, Niels; Crossan, Gerry P; Patel, Ketan J

    2014-09-18

    Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2(-/-)Fanca(-/-) embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2. PMID:25155611

  11. Polyvinyl alcohol cross-linked with two aldehydes

    NASA Technical Reports Server (NTRS)

    Sheibley, D. W.; Rieker, L. L.; Hsu, L. C.; Manzo, M. A. (Inventor)

    1982-01-01

    A film forming polyvinyl alcohol resin is admixed, in aqueous solution, with a dialdehyde crosslinking agent which is capable of crosslinking the polyvinyl alcohol resin and a water soluble acid aldehyde containing a reactive aldehyde group capable of reacting with hydroxyl groups in the polyvinyl alcohol resin and an ionizable acid hydrogen atom. The dialdehyde is present in an amount sufficient to react with from 1 to 20% by weight of the theoretical amount required to react with all of the hydroxyl groups of the polyvinyl alcohol. The amount of acid aldehyde is from 1 to 50% by weight, same basis, and is sufficient to reduce the pH of the aqueous admixture to 5 or less. The admixture is then formed into a desired physical shape, such as by casting a sheet or film, and the shaped material is then heated to simultaneously dry and crosslink the article.

  12. Molecular cloning and biochemical characterization of two cinnamyl alcohol dehydrogenases from a liverwort Plagiochasma appendiculatum.

    PubMed

    Sun, Yi; Wu, Yifeng; Zhao, Yu; Han, Xiaojuan; Lou, Hongxiang; Cheng, Aixia

    2013-09-01

    Cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195) is a key enzyme in lignin biosynthesis. It catalyzes cinnamyl aldehydes as substrates to form corresponding alcohols, the last step in monolignol biosynthesis. Almost all CAD members of land plants could be divided into three classes according to the phylogenetic analysis, together with gene structure and function. In the present investigation, two cDNAs encoding CADs were obtained from a Chinese liverwort Plagiochasma appendiculatum thallus library and were designated as PaCAD1 and PaCAD2. Phylogenetic analysis showed that PaCAD1 and PaCAD2 belonged to Class II. Full length cDNAs were heterologously expressed in E. coli and the recombinant PaCAD proteins displayed high activity levels using p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl and sinapyl aldehydes as substrates to form corresponding alcohols. The enzyme kinetics results showed that PaCAD1 and PaCAD2 used coniferyl aldehyde as the favourite substrate and showed high catalytic efficiency towards p-coumaryl aldehyde but lowest catalytic efficiency towards 5-hydroxyconiferaldehyde. In accord with the higher lignin content in the thallus than in the callus, the expression level of PaCAD2 was also higher in thallus than in the callus. The expression of PaCAD1 and PaCAD2 was induced by Methyl jasmonic acid (MeJA) treatment. This suggested that these two PaCADs played twin roles in lignin biosynthesis and the defencedefence of abiotic stress in P. appendiculatum. This is the first time that the CADs in liverworts have been functionally characterized.

  13. RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1.

    PubMed

    Mezzar, Serena; de Schryver, Evelyn; Van Veldhoven, Paul P

    2014-03-01

    Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates.

  14. Characterization of a new liver- and kidney-specific pfkfb3 isozyme that is downregulated by cell proliferation and dedifferentiation

    SciTech Connect

    Duran, Joan; Gomez, Marta; Navarro-Sabate, Aurea; Riera-Sans, Lluis; Obach, Merce; Manzano, Anna; Perales, Jose C.; Bartrons, Ramon

    2008-03-21

    The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P{sub 2}), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1-4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cultured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiquitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism.

  15. Direct β-Alkylation of Aldehydes via Photoredox Organocatalysis

    PubMed Central

    2015-01-01

    Direct β-alkylation of saturated aldehydes has been accomplished by synergistically combining photoredox catalysis and organocatalysis. Photon-induced enamine oxidation provides an activated β-enaminyl radical intermediate, which readily combines with a wide range of Michael acceptors to produce β-alkyl aldehydes in a highly efficient manner. Furthermore, this redox-neutral, atom-economical C–H functionalization protocol can be achieved both inter- and intramolecularly. Mechanistic studies by various spectroscopic methods suggest that a reductive quenching pathway is operable. PMID:24754456

  16. Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

    NASA Astrophysics Data System (ADS)

    Kobayashi, Yasuhito; Motohashi, Yutaka; Miyazaki, Yoshifumi; Yatagai, Mitsuyoshi; Takano, Takehito

    1991-12-01

    Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.

  17. Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships.

    PubMed

    Ko, Wun-Chang; Shih, Chwen-Ming; Lai, Ya-Hsin; Chen, Jun-Hao; Huang, Hui-Lin

    2004-11-15

    The structure-activity relationships of flavonoids with regard to their inhibitory effects on phosphodiesterase (PDE) isozymes are little known. The activities of PDE1-5 were measured by a two-step procedure using cAMP with [(3)H]-cAMP or cGMP with [(3)H]-cGMP as substrates. In the present results, PDE1, 5, 2, and 4 isozymes were partially purified from guinea pig lungs in that order, and PDE3 was from the heart. The IC(50) values of PDE1-5 were greater than those reported previously for the reference drugs, vinpocetin, EHNA, milrinone, Ro 20-1724, and zaprinast, by 5-, 5-, 7-, 5-, and 3-fold, respectively. As shown in Table 2, luteolin revealed non-selective inhibition of PDE1-5 with IC(50) values in a range of 10-20 microM, as did genistein except with a low potency on PDE5. Daidzein, an inactive analogue of genistein in tyrosine kinase inhibition, showed selective inhibition of PDE3 with an IC(50) value of around 30 microM, as did eriodictyol with an IC(50) value of around 50 microM. Hesperetin and prunetin exhibited more-selective inhibition of PDE4 with IC(50) values of around 30 and 60 microM, respectively. Luteolin-7-glucoside exhibited dual inhibition of PDE2/PDE4 with an IC(50) value of around 40 microM. Diosmetin more-selectively inhibited PDE2 (IC(50) of 4.8 microM) than PDE1, PDE4, or PDE5. However, biochanin A more-selectively inhibited PDE4 (IC(50) of 8.5 microM) than PDE1 or PDE2. Apigenin inhibited PDE1-3 with IC(50) values of around 10-25 microM. Myricetin inhibited PDE1-4 with IC(50) values of around 10-40 microM. The same was true for quercetin, but we rather consider that it more-selectively inhibited PDE3 and PDE4 (IC(50) of < 10 microM). In conclusion, it is possible to synthesize useful drugs through elucidating the structure-activity relationships of flavonoids with respect to inhibition of PDE isozymes at concentrations used in this in vitro study.

  18. Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships.

    PubMed

    Ko, Wun-Chang; Shih, Chwen-Ming; Lai, Ya-Hsin; Chen, Jun-Hao; Huang, Hui-Lin

    2004-11-15

    The structure-activity relationships of flavonoids with regard to their inhibitory effects on phosphodiesterase (PDE) isozymes are little known. The activities of PDE1-5 were measured by a two-step procedure using cAMP with [(3)H]-cAMP or cGMP with [(3)H]-cGMP as substrates. In the present results, PDE1, 5, 2, and 4 isozymes were partially purified from guinea pig lungs in that order, and PDE3 was from the heart. The IC(50) values of PDE1-5 were greater than those reported previously for the reference drugs, vinpocetin, EHNA, milrinone, Ro 20-1724, and zaprinast, by 5-, 5-, 7-, 5-, and 3-fold, respectively. As shown in Table 2, luteolin revealed non-selective inhibition of PDE1-5 with IC(50) values in a range of 10-20 microM, as did genistein except with a low potency on PDE5. Daidzein, an inactive analogue of genistein in tyrosine kinase inhibition, showed selective inhibition of PDE3 with an IC(50) value of around 30 microM, as did eriodictyol with an IC(50) value of around 50 microM. Hesperetin and prunetin exhibited more-selective inhibition of PDE4 with IC(50) values of around 30 and 60 microM, respectively. Luteolin-7-glucoside exhibited dual inhibition of PDE2/PDE4 with an IC(50) value of around 40 microM. Diosmetin more-selectively inhibited PDE2 (IC(50) of 4.8 microM) than PDE1, PDE4, or PDE5. However, biochanin A more-selectively inhibited PDE4 (IC(50) of 8.5 microM) than PDE1 or PDE2. Apigenin inhibited PDE1-3 with IC(50) values of around 10-25 microM. Myricetin inhibited PDE1-4 with IC(50) values of around 10-40 microM. The same was true for quercetin, but we rather consider that it more-selectively inhibited PDE3 and PDE4 (IC(50) of < 10 microM). In conclusion, it is possible to synthesize useful drugs through elucidating the structure-activity relationships of flavonoids with respect to inhibition of PDE isozymes at concentrations used in this in vitro study. PMID:15476679

  19. Tunable Ether Production via Coupling of Aldehydes or Aldehyde/Alcohol over Hydrogen-Modified Gold Catalysts at Low Temperatures.

    PubMed

    Pan, Ming; Brush, Adrian J; Dong, Guangbin; Mullins, C Buddie

    2012-09-01

    Ethers are an important group of organic compounds that are primarily prepared via homogeneous catalysis, which can lead to operational and environmental issues. Here we demonstrate the production of ethers via heterogeneous catalysis over H adatom-covered gold at temperatures lower than 250 K. Symmetrical ethers can be formed via a self-coupling reaction of corresponding aldehydes; for example, homocoupling of acetaldehyde and propionaldehyde yields diethyl ether and di-n-propyl ether, respectively. In addition, coupling reactions between alcohols and aldehydes, with different carbon chain lengths, are observed via the production of the corresponding unsymmetrical ethers. A reaction mechanism is proposed, suggesting that an alcohol-like intermediate via partial hydrogenation of aldehydes on the surface plays a key role in these reactions. These surface chemical reactions suggest possible heterogeneous routes to low-temperature production of ethers. PMID:26292142

  20. Influence of rare earth elements on metabolism and related enzyme activity and isozyme expression in Tetrastigma hemsleya