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Sample records for acid o-methyltransferase gene

  1. Phylogenetic, Molecular, and Biochemical Characterization of Caffeic Acid o-Methyltransferase Gene Family in Brachypodium distachyon

    PubMed Central

    Wu, Xianting; Wu, Jiajie; Luo, Yangfan; Bragg, Jennifer; Anderson, Olin; Vogel, John; Gu, Yong Q.

    2013-01-01

    Caffeic acid o-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of the new grass model Brachypodium distachyon identified four COMT gene homologs, designated as BdCOMT1, BdCOMT2, BdCOMT3, and BdCOMT4. Phylogenetic analysis suggested that they belong to the COMT gene family, whereas syntenic analysis through comparisons with rice and sorghum revealed that BdCOMT4 on Chromosome 3 is the orthologous copy of the COMT genes well characterized in other grass species. The other three COMT genes are unique to Brachypodium since orthologous copies are not found in the collinear regions of rice and sorghum genomes. Expression studies indicated that all four Brachypodium COMT genes are transcribed but with distinct patterns of tissue specificity. Full-length cDNAs were cloned in frame into the pQE-T7 expression vector for the purification of recombinant Brachypodium COMT proteins. Biochemical characterization of enzyme activity and substrate specificity showed that BdCOMT4 has significant effect on a broad range of substrates with the highest preference for caffeic acid. The other three COMTs had low or no effect on these substrates, suggesting that a diversified evolution occurred on these duplicate genes that not only impacted their pattern of expression, but also altered their biochemical properties. PMID:23431288

  2. Down-regulation of the Caffeic acid O-methyltransferase Gene in Switchgrass Reveals a Novel Monolignol Analog

    SciTech Connect

    Tschaplinski, Timothy J; Standaert, Robert F; Engle, Nancy L; Martin, Madhavi Z; Sangha, Amandeep K; Parks, Jerry M; Smith, Jeremy C; Samuel, Reichel; Pu, Yunqiao; Ragauskas, A J; Hamilton, Choo Yieng; Fu, Chunxiang; Wang, Zeng-Yu; Davison, Brian H; Dixon, Richard A; Mielenz, Jonathan R

    2012-01-01

    Down-regulation of the caffeic acid 3-O-methyltransferase (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography-mass spectrometry-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors, confirming the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. Although there was no indication that iso-sinapyl alcohol was integrated into the cell wall, diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth.

  3. Stress Responses in Alfalfa (XXI. Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures).

    PubMed Central

    Ni, W.; Sewalt, VJH.; Korth, K. L.; Blount, J. W.; Ballance, G. M.; Dixon, R. A.

    1996-01-01

    Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.). However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity. Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts. Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions. In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities. Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis. PMID:12226420

  4. Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

    PubMed Central

    2012-01-01

    Background Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. Results GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. Conclusions Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl

  5. Functional Analyses of Caffeic Acid O-Methyltransferase and Cinnamoyl-CoA-Reductase Genes from Perennial Ryegrass (Lolium perenne)[W

    PubMed Central

    Tu, Yi; Rochfort, Simone; Liu, Zhiqian; Ran, Yidong; Griffith, Megan; Badenhorst, Pieter; Louie, Gordon V.; Bowman, Marianne E.; Smith, Kevin F.; Noel, Joseph P.; Mouradov, Aidyn; Spangenberg, German

    2010-01-01

    Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference–mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production. PMID:20952635

  6. Acid detergent lignin, lodging resistance index, and expression of the caffeic acid O-methyltransferase gene in brown midrib-12 sudangrass.

    PubMed

    Li, Yuan; Liu, Guibo; Li, Jun; You, Yongliang; Zhao, Haiming; Liang, Huan; Mao, Peisheng

    2015-09-01

    Understanding the relationship between acid detergent lignin (ADL) and lodging resistance index (LRI) is essential for breeding new varieties of brown midrib (bmr) sudangrass (Sorghum sudanense (Piper) Stapf.). In this study, bmr-12 near isogenic lines and their wild-types obtained by back cross breeding were used to compare relevant forage yield and quality traits, and to analyze expression of the caffeic acid O-methyltransferase (COMT) gene using quantitative real time-PCR. The research showed that the mean ADL content of bmr-12 mutants (20.94 g kg(-1)) was significantly (P < 0.05) lower than measured in N-12 lines (43.45 g kg(-1)), whereas the LRI of bmr-12 mutants (0.29) was significantly (P < 0.05) higher than in N-12 lines (0.22). There was no significant correlation between the two indexes in bmr-12 materials (r = -0.44, P > 0.05). Sequence comparison of the COMT gene revealed two point mutations present in bmr-12 but not in the wild-type, the second mutation changed amino acid 129 of the protein from Gln (CAG) to a stop codon (UAG). The relative expression level of COMT gene was significantly reduced, which likely led to the decreased ADL content observed in the bmr-12 mutant.

  7. Cloning and Phylogenetic Analysis of Brassica napus L. Caffeic Acid O-Methyltransferase 1 Gene Family and Its Expression Pattern under Drought Stress

    PubMed Central

    Lu, Kun; Yuan, Jianglian; Huang, Jieheng; Du, Hai; Li, Jiana

    2016-01-01

    For many plants, regulating lignin content and composition to improve lodging resistance is a crucial issue. Caffeic acid O-methyltransferase (COMT) is a lignin monomer-specific enzyme that controls S subunit synthesis in plant vascular cell walls. Here, we identified 12 BnCOMT1 gene homologues, namely BnCOMT1-1 to BnCOMT1-12. Ten of 12 genes were composed of four highly conserved exons and three weakly conserved introns. The length of intron I, in particular, showed enormous diversification. Intron I of homologous BnCOMT1 genes showed high identity with counterpart genes in Brassica rapa and Brassica oleracea, and intron I from positional close genes in the same chromosome were relatively highly conserved. A phylogenetic analysis suggested that COMT genes experience considerable diversification and conservation in Brassicaceae species, and some COMT1 genes are unique in the Brassica genus. Our expression studies indicated that BnCOMT1 genes were differentially expressed in different tissues, with BnCOMT1-4, BnCOMT1-5, BnCOMT1-8, and BnCOMT1-10 exhibiting stem specificity. These four BnCOMT1 genes were expressed at all developmental periods (the bud, early flowering, late flowering and mature stages) and their expression level peaked in the early flowering stage in the stem. Drought stress augmented and accelerated lignin accumulation in high-lignin plants but delayed it in low-lignin plants. The expression levels of BnCOMT1s were generally reduced in water deficit condition. The desynchrony of the accumulation processes of total lignin and BnCOMT1s transcripts in most growth stages indicated that BnCOMT1s could be responsible for the synthesis of a specific subunit of lignin or that they participate in other pathways such as the melatonin biosynthesis pathway. PMID:27832102

  8. Plant isoflavone and isoflavanone O-methyltransferase genes

    DOEpatents

    Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

    2014-08-19

    The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

  9. Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

  10. Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa

    PubMed Central

    Guo, Dianjing; Chen, Fang; Inoue, Kentaro; Blount, Jack W.; Dixon, Richard A.

    2001-01-01

    Transgenic alfalfa plants were generated harboring caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) cDNA sequences under control of the bean phenylalanine ammonia-lyase PAL2 promoter. Strong downregulation of COMT resulted in decreased lignin content, a reduction in total guaiacyl (G) lignin units, a near total loss of syringyl (S) units in monomeric and dimeric lignin degradation products, and appearance of low levels of 5-hydroxy guaiacyl units and a novel dimer. No soluble monolignol precursors accumulated. In contrast, strong downregulation of CCOMT led to reduced lignin levels, a reduction in G units without reduction in S units, and increases in β-5 linked dimers of G units. Accumulation of soluble caffeic acid β-d-glucoside occurred only in CCOMT downregulated plants. The results suggest that CCOMT does not significantly contribute to the 3-O-methylation step in S lignin biosynthesis in alfalfa and that there is redundancy with respect to the 3-O-methylation reaction of G lignin biosynthesis. COMT is unlikely to catalyze the in vivo methylation of caffeic acid during lignin biosynthesis. PMID:11158530

  11. Phylogenetic, molecular, and biochemical characterization of caffeic aicd O-methyltransferase (COMT) gene family in Brachypodium distachyon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Caffeic acid O-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of new grass model Brachypodium distachyon identified four COMT gene homologues, designated as BdCOMT1, BdCOMT2, BdCOMT3, and ...

  12. Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

    PubMed Central

    Inoue, Kentaro; Sewalt, Vincent J.H.; Murray Ballance, G.; Ni, Weiting; Stürzer, Cornelia; Dixon, Richard A.

    1998-01-01

    The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content. PMID:9662519

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is a significant recalcitrant in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired...

  14. A Continuous, Quantitative Fluorescent Assay for Plant Caffeic acid O-Methyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant caffeic acid O-methyltransferases (COMTs) use s-adenosylmethionine (ado-met), as a methyl donor to transmethylate their preferred (phenolic) substrates in-vivo, and will generally utilize a range of phenolic compounds in-vitro. Collazo et al. (2005; Analytical Biochemistry 342: 86-92) have pu...

  15. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

    PubMed

    Byeon, Yeong; Back, Kyoungwhan

    2016-08-01

    Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

  16. The catechol-O-methyltransferase gene (COMT) and cognitive function from childhood through adolescence.

    PubMed

    Gaysina, Darya; Xu, Man K; Barnett, Jennifer H; Croudace, Tim J; Wong, Andrew; Richards, Marcus; Jones, Peter B

    2013-02-01

    Genetic variation in the catechol-O-methyltransferase gene (COMT) can influence cognitive function, and this effect may depend on developmental stage. Using a large representative British birth cohort, we investigated the effect of COMT on cognitive function (verbal and non-verbal) at ages 8 and 15 years taking into account the possible modifying effect of pubertal stage. Five functional COMT polymorphisms, rs6269, rs4818, rs4680, rs737865 and rs165599 were analysed. Associations between COMT polymorphisms and cognition were tested using regression and latent variable structural equation modelling (SEM). Before correction for multiple testing, COMT rs737865 showed association with reading comprehension, verbal ability and global cognition at age 15 years in pubescent boys only. Although there was some evidence for age- and sex-specific effects of the COMT rs737865 none remained significant after correction for multiple testing. Further studies are necessary in order to make firmer conclusions.

  17. Genetic influences on insight problem solving: the role of catechol-O-methyltransferase (COMT) gene polymorphisms

    PubMed Central

    Jiang, Weili; Shang, Siyuan; Su, Yanjie

    2015-01-01

    People may experience an “aha” moment, when suddenly realizing a solution of a puzzling problem. This experience is called insight problem solving. Several findings suggest that catecholamine-related genes may contribute to insight problem solving, among which the catechol-O-methyltransferase (COMT) gene is the most promising candidate. The current study examined 753 healthy individuals to determine the associations between 7 candidate single nucleotide polymorphisms on the COMT gene and insight problem-solving performance, while considering gender differences. The results showed that individuals carrying A allele of rs4680 or T allele of rs4633 scored significantly higher on insight problem-solving tasks, and the COMT gene rs5993883 combined with gender interacted with correct solutions of insight problems, specifically showing that this gene only influenced insight problem-solving performance in males. This study presents the first investigation of the genetic impact on insight problem solving and provides evidence that highlights the role that the COMT gene plays in insight problem solving. PMID:26528222

  18. Genetic influences on insight problem solving: the role of catechol-O-methyltransferase (COMT) gene polymorphisms.

    PubMed

    Jiang, Weili; Shang, Siyuan; Su, Yanjie

    2015-01-01

    People may experience an "aha" moment, when suddenly realizing a solution of a puzzling problem. This experience is called insight problem solving. Several findings suggest that catecholamine-related genes may contribute to insight problem solving, among which the catechol-O-methyltransferase (COMT) gene is the most promising candidate. The current study examined 753 healthy individuals to determine the associations between 7 candidate single nucleotide polymorphisms on the COMT gene and insight problem-solving performance, while considering gender differences. The results showed that individuals carrying A allele of rs4680 or T allele of rs4633 scored significantly higher on insight problem-solving tasks, and the COMT gene rs5993883 combined with gender interacted with correct solutions of insight problems, specifically showing that this gene only influenced insight problem-solving performance in males. This study presents the first investigation of the genetic impact on insight problem solving and provides evidence that highlights the role that the COMT gene plays in insight problem solving.

  19. Is catechol-o-methyltransferase gene polymorphism a risk factor in the development of premenstrual syndrome?

    PubMed Central

    Deveci, Esma Ozturk; Selek, Salih; Camuzcuoglu, Aysun; Hilali, Nese Gul; Camuzcuoglu, Hakan; Erdal, Mehmet Emin; Vural, Mehmet

    2014-01-01

    Objective The objective of this study was to investigate whether there was a correlation between catechol-o-methyltransferase (COMT) gene polymorphism, which is believed to play a role in the etiology of psychotic disorders, and premenstrual syndrome (PMS). Methods Fifty-three women with regular menstrual cycles, aged between 18 and 46 years and diagnosed with PMS according to the American Congress of Obstetrics and Gynecology criteria were included in this study as the study group, and 53 healthy women having no health problems were selected as the controls. Venous blood was collected from all patients included in the study and kept at -18℃ prior to analysis. Results There was no significant difference between the groups in terms of demographic features such as age, body mass index, number of pregnancies, parity, and number of children. No statistically significant difference was observed in terms of COMT gene polymorphism (p=0.61) between women in the PMS and the control groups. However, a significant difference was found between arthralgia, which is an indicator of PMS, and low-enzyme activity COMT gene (Met/Met) polymorphism (p=0.04). Conclusion These results suggested that there was no significant relationship between PMS and COMT gene polymorphism. Since we could not find a direct correlation between the COMT gene polymorphism and PMS, further studies including alternative neurotransmitter pathways are needed to find an effective treatment for this disease. PMID:25045629

  20. Cloning and functional characterization of a caffeic acid O-methyltransferase from Trigonella foenum-graecum L.

    PubMed

    Qin, Jian-Chun; Zhang, Ya-Mei; Lang, Chen-Yong; Yao, Yan-Hua; Pan, Hong-Yu; Li, Xiang

    2012-02-01

    A cDNA encoding an O-methyltransferase (namely FGCOMT1) was identified from the medicinal plant Trigonella foenum-graecum L. The FGCOMT1 enzyme is a functional caffeic acid O-methyltransferase (COMT) and is localized in the cytosol. Kinetic analysis indicated that FGCOMT1 protein exhibited the highest catalyzing efficiency towards 5-hydroxy ferulic acid and caffeic acid as substrates, but did not possess the abilities to methylate either quercetin or tricetin in vitro. Furthermore, transformation of Arabidopsis loss-of-function Atomt1 mutant with a FGCOMT1 cDNA partially complements accumulation of sinapoyl derivatives but did not function to produce the major methylated flavonol isorhamnetin in seeds. The results from this study indicated that FGCOMT1 is a COMT with substrate preference to monomeric lignin precursors but is not involved in the flavonoid methylation in T. foenum-graecum L.

  1. Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene.

    PubMed

    Rastogi, Smita; Dwivedi, Upendra Nath

    2006-01-01

    In the present study, a 0.47 kb OMT gene construct from aspen, encoding for an enzyme O-methyltransferase (OMT, EC 2.1.1.6), in antisense orientation was used to down-regulate lignin biosynthesis in Leucaena leucocephala. The plants were transformed with Agrobacterium tumefaciens strain harboring the antisense gene, and the transformation was confirmed by PCR amplification of the npt II gene. The integration of a heterologous antisense OMT gene construct in transformed plants led to a maximum of 60% reduction in OMT activity relative to control. The evaluation of total lignin content by the Klason method revealed a maximum of 28% reduction. Histochemical analyses of stem sections depicted a reduction in lignin content and normal xylem development. The results also suggested a probable increase in aldehyde levels and a decrease in syringyl units. Lignin down-regulation was accompanied by an increase in methanol soluble phenolics to an extent that had no impact on wood discoloration, and the plants displayed a normal phenotype. Concomitantly, an increase of up to 9% in cellulose content was also observed. Upon alkali extraction, modified lignin was more extractable as evident from reduced Klason lignin in saponified residue and increased alkali soluble phenolics. The results together suggested that the extent of down-regulation of OMT activity achieved may lead to quality amelioration of Leucaena with respect to its applicability in pulp and paper manufacture as well as nutritive and easily digestible forage production.

  2. Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia coli.

    PubMed Central

    Madduri, K; Torti, F; Colombo, A L; Hutchinson, C R

    1993-01-01

    Sequence analysis of a portion of the Streptomyces peucetius daunorubicin biosynthetic gene cluster revealed a complete open reading frame (dnrK) that showed DNA and protein sequence homology to several O-methyltransferases. Expression of dnrK in Streptomyces lividans and Escherichia coli was done to show that this gene codes for carminomycin 4-O-methyltransferase. The deduced carminomycin 4-O-methyltransferase protein shows a conserved nucleotide binding site for its S-adenosyl-L-methionine cofactor. Images PMID:8509343

  3. Catechol-O-Methyltransferase Gene Polymorphisms in Specific Obsessive-Compulsive Disorder Patients' Subgroups.

    PubMed

    Melo-Felippe, Fernanda Brito; de Salles Andrade, Juliana Braga; Giori, Isabele Gomes; Vieira-Fonseca, Tamiris; Fontenelle, Leonardo Franklin; Kohlrausch, Fabiana Barzotti

    2016-01-01

    Pharmacological data and animal models support the hypothesis that the dopaminergic (DA) system is implicated in obsessive-compulsive disorder (OCD). Therefore, this case-control study assessed whether genetics variations in catechol-O-methyltransferase gene (COMT) could influence susceptibility to OCD and OCD features in a Brazilian sample. A sample of 199 patients with OCD and 200 healthy individuals was genotyped for -287A > G (rs2075507) and Val158Met (rs4680) single nucleotide polymorphisms (SNPs) by TaqMan(®) or restriction mapping. We observed a statistically significant predominance of the Met low-activity allele in the male patient group as compared to the male healthy control group. The -287A > G polymorphism's genotypes and alleles were significantly overrepresented among male individuals with ordering and female subjects with washing symptoms. We also found female hoarders to exhibit a significant higher frequency of the low activity Met/Met genotype of Val158Met polymorphism compared to female patients who did not express this dimension. Our data suggest an influence of COMT polymorphisms on OCD and OCD patients' features, such as gender, and ordering, washing, and hoarding symptom dimensions. Further studies to confirm the clinical importance of COMT SNPs in OCD are warranted.

  4. Influence of catechol-O-methyltransferase (COMT) gene polymorphisms in pain sensibility of Brazilian fibromialgia patients.

    PubMed

    Barbosa, Flávia Regina; Matsuda, Josie Budag; Mazucato, Mendelson; de Castro França, Suzelei; Zingaretti, Sônia Marli; da Silva, Lucienir Maria; Martinez-Rossi, Nilce Maria; Júnior, Milton Faria; Marins, Mozart; Fachin, Ana Lúcia

    2012-02-01

    Fibromyalgia syndrome (FS) is a rheumatic syndrome affecting to 2-3% of individuals of productive age, mainly women. Neuroendocrine and genetic factors may play a significant role in development of the disease which is characterized by diffuse chronic pain and presence of tender points. Several studies have suggested an association between FS, especially pain sensitivity, and polymorphism of the catechol-O-methyltransferase (COMT) gene. The aim of the present study was to characterize the SNPs rs4680 and rs4818 of the COMT gene and assess its influence in pain sensitivity of patients with fibromyalgia screened by the Fibromyalgia Impact Questionnaire (FIQ). DNA was extracted from peripheral blood of 112 patients with fibromyalgia and 110 healthy individuals and was used as template in PCR for amplification of a 185-bp fragment of the COMT gene. The amplified fragment was sequenced for analyses of the SNPs rs4680 and rs4818. The frequency of mutant genotype AA of SNP rs6860 was 77.67% in patients with FS and 28.18% for the control group. For the SNP rs4818, the frequency of mutant genotype CC was 73.21 and 39.09% for patients with FS and controls, respectively. Moreover, the FIQ score was higher in patients with the homozygous mutant genotype for SNPs rs4680 (87.92 points) and rs4818 (86.14 points). These results suggest that SNPs rs4680 and rs4818 of the COMT gene may be associated with fibromyalgia and pain sensitivity in FS Brazilian patients.

  5. Catechol-O-methyltransferase gene haplotypes in Mexican and Spanish patients with fibromyalgia

    PubMed Central

    Vargas-Alarcón, Gilberto; Fragoso, José-Manuel; Cruz-Robles, David; Vargas, Angélica; Vargas, Alfonso; Lao-Villadóniga, José-Ignacio; García-Fructuoso, Ferrán; Ramos-Kuri, Manuel; Hernández, Fernando; Springall, Rashidi; Bojalil, Rafael; Vallejo, Maite; Martínez-Lavín, Manuel

    2007-01-01

    Autonomic dysfunction is frequent in patients with fibromyalgia (FM). Heart rate variability analyses have demonstrated signs of ongoing sympathetic hyperactivity. Catecholamines are sympathetic neurotransmitters. Catechol-O-methyltransferase (COMT), an enzyme, is the major catecholamine-clearing pathway. There are several single-nucleotide polymorphisms (SNPs) in the COMT gene associated with the different catecholamine-clearing abilities of the COMT enzyme. These SNPs are in linkage disequilibrium and segregate as 'haplotypes'. Healthy females with a particular COMT gene haplotype (ACCG) producing a defective enzyme are more sensitive to painful stimuli. The objective of our study was to define whether women with FM, from two different countries (Mexico and Spain), have the COMT gene haplotypes that have been previously associated with greater sensitivity to pain. All the individuals in the study were female. Fifty-seven Mexican patients and 78 Spanish patients were compared with their respective healthy control groups. All participants filled out the Fibromyalgia Impact Questionnaire (FIQ). Six COMT SNPs (rs2097903, rs6269, rs4633, rs4818, rs4680, and rs165599) were genotyped from peripheral blood DNA. In Spanish patients, there was a significant association between three SNPs (rs6269, rs4818, and rs4680) and the presence of FM when compared with healthy controls. Moreover, in Spanish patients with the 'high pain sensitivity' haplotype (ACCG), the disease, as assessed by the FIQ, was more severe. By contrast, Mexican patients displayed only a weak association between rs6269 and rs165599, and some FIQ subscales. In our group of Spanish patients, there was an association between FM and the COMT haplotype previously associated with high pain sensitivity. This association was not observed in Mexican patients. Studies with a larger sample size are needed in order to verify or amend these preliminary results. PMID:17961261

  6. The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis.

    PubMed

    Dekkers, Katherine L; You, Bang-Jau; Gowda, Vivek S; Liao, Hui-Ling; Lee, Miin-Huey; Bau, Huey-Jiunn; Ueng, Peter P; Chung, Kuang-Ren

    2007-05-01

    Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Deltactb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Deltactb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Deltactb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Deltactb3 disruptants drastically reduced lesion formation.

  7. Analysis of catechol-O-methyltransferase gene mutation and identification of new pathogenic gene for paroxysmal kinesigenic dyskinesia.

    PubMed

    Gu, Chengzhi; Li, Jia; Zhu, Lianhai; Lu, Zhenhui; Huang, Huaiyu

    2016-03-01

    We aimed to analyze the mutation site and frequency of catechol-O-methyltransferase (COMT) gene, to explore the relationship between COMT genotype and phenotype, and to find new pathogenic genes for paroxysmal kinesigenic dyskinesia (PKD). PKD patients who were treated from December 2011 to January 2014 were selected and subjected to genetic testing in the exon region of COMT. Two patients and one intrafamilial healthy control were subjected to exome sequencing using whole exome capture in combination with high-throughput sequencing to find candidate pathogenic gene sites. The results were verified by Sanger sequencing. A total of 11 familial PKD patients from 4 families and 9 sporadic patients without family history were included. Pathogenic c.634dupC(p.P220fsX7) mutation of COMT gene was found in 7 familial PKD patients and3 sporadic patients. Mutated COMT gene carriers suffered from PKD earlier (average age of onset: 11.61 ± 2.33 vs 16.21 ± 2.58, P = 0.001) with symmetric symptoms in most cases, while the mutation-negative group only showed unilateral symptoms (P = 0.001). The mutation-positive group also had more daily attacks (P = 0.038). Carbamazepine worked for all mutation-positive patients (10/10, 100%), but only for a part of mutation-negative patients (3/10, 30.0%). About 90000 single nucleotide polymorphisms and 2000 insertion-deletion polymorphisms were detected in each of the three samples. c.737C → T(p.T246 M) mutation of POC1B gene was a new pathogenic site for a selected family. COMT gene mutation, which was the pathogenesis of most familial PKD patients and a part of sporadic patients, predicted the response to carbamazepine. POC1B may be a novel pathogenic gene for PKD.

  8. Determination of the structure and catalytic mechanism of Sorghum bicolor caffeic acid O-methyltransferase and the structural impact of three brown midrib12 mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With S-adenosylmethionine (SAM) acting as the methyl donor, caffeic acid O-methyltransferase from Sorghum bicolor (SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde, to form sinapaldehyde. In order to determine the mechanism of SbCOMT and understand the red...

  9. Sugarcane DIRIGENT and O-methyltransferase promoters confer stem-regulated gene expression in diverse monocots.

    PubMed

    Damaj, Mona B; Kumpatla, Siva P; Emani, Chandrakanth; Beremand, Phillip D; Reddy, Avutu S; Rathore, Keerti S; Buenrostro-Nava, Marco T; Curtis, Ian S; Thomas, Terry L; Mirkov, T Erik

    2010-05-01

    Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Omicron-Methyltransferase (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the beta-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both Pro( DIR16 ):GUS and Pro( OMT ):GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. Pro( DIR16 ) and Pro( OMT ) will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

  10. Catechol-O-methyltransferase (COMT) gene modulates private self-consciousness and self-flexibility.

    PubMed

    Wang, Bei; Ru, Wenzhao; Yang, Xing; Yang, Lu; Fang, Pengpeng; Zhu, Xu; Shen, Guomin; Gao, Xiaocai; Gong, Pingyuan

    2016-08-01

    Dopamine levels in the brain influence human consciousness. Inspired by the role of Catechol-O-methyltransferase (COMT) in inactivating dopamine in the brain, we investigated to what extent COMT could modulate individual's self-consciousness dispositions and self-consistency by genotyping the COMT Val158Met (rs4680) polymorphism and measuring self-consciousness and self-consistency and congruence in a college student population. The results indicated that COMT Val158Met polymorphism significantly modulated the private self-consciousness. The individuals with Val/Val genotype, corresponding to lower dopamine levels in the brain, were more likely to be aware of their feelings and beliefs. The results also indicated that this polymorphism modulated one's self-flexibility. The individuals with Val/Val genotype showed higher levels of stereotype in self-concept compared with those with Met/Met genotype. These findings suggest that COMT is a predictor of the individual differences in self-consciousness and self-flexibility.

  11. Association of catechol-O-methyltransferase gene polymorphisms with schizophrenia and negative symptoms in a Chinese population

    PubMed Central

    Li, Wen Jun; Kou, Chang Gui; Yu, Yaqin; Sun, Shilong; Zhang, Xuan; Kosten, Thomas R; Zhang, Xiang Yang

    2014-01-01

    The gene encoding Catechol O-methyltransferase (COMT), a dopamine catabolic enzyme, has been associated inconsistently with schizophrenia in spite of consistent evidence for dopaminergic dysfunction in the prefrontal cortex (PFC) of schizophrenia. Since one contribution to this inconsistency might be genetic heterogeneity, this study investigated whether the COMT gene was associated with the development and symptoms of schizophrenia in relatively genetically homogeneous Chinese schizophrenic patients. We analyzed two polymorphisms (rs740603 and rs4818) of the COMT gene in a case–control study of 604 Han Chinese (284 patients and 320 controls). The patients’ psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS). We found no significant differences in the rs740603 and rs4818 genotype and allele distributions between the patient and control groups. Quantitative trait analysis by the UNPHASED program showed that the rs740603 and rs740603(G)-rs4818(G) haplotypes were associated with negative symptoms in the schizophrenic patients, particularly among female patients. Thus, the COMT gene polymorphisms may not contribute to the susceptibility to schizophrenia, but may contribute to the negative symptoms of schizophrenia among Han Chinese. PMID:22354729

  12. Down-Regulation of Caffeic Acid O-Methyltransferase in Maize Revisited Using a Transgenic Approach1

    PubMed Central

    Piquemal, Joel; Chamayou, Simon; Nadaud, Isabelle; Beckert, Michel; Barrière, Yves; Mila, Isabelle; Lapierre, Catherine; Rigau, Joan; Puigdomenech, Pere; Jauneau, Alain; Digonnet, Catherine; Boudet, Alain-Michel; Goffner, Deborah; Pichon, Magalie

    2002-01-01

    Transgenic maize (Zea mays) plants were generated with a construct harboring a maize caffeic acid O-methyltransferase (COMT) cDNA in the antisense (AS) orientation under the control of the maize Adh1 (alcohol dehydrogenase) promoter. Adh1-driven β-glucuronidase expression was localized in vascular tissues and lignifying sclerenchyma, indicating its suitability in transgenic experiments aimed at modifying lignin content and composition. One line of AS plants, COMT-AS, displayed a significant reduction in COMT activity (15%–30% residual activity) and barely detectable amounts of COMT protein as determined by western-blot analysis. In this line, transgenes were shown to be stably integrated in the genome and transmitted to the progeny. Biochemical analysis of COMT-AS showed: (a) a strong decrease in Klason lignin content at the flowering stage, (b) a decrease in syringyl units, (c) a lower p-coumaric acid content, and (d) the occurrence of unusual 5-OH guaiacyl units. These results are reminiscent of some characteristics already observed for the maize bm3 (brown-midrib3) mutant, as well as for COMT down-regulated dicots. However, as compared with bm3, COMT down-regulation in the COMT-AS line is less severe in that it is restricted to sclerenchyma cells. To our knowledge, this is the first time that an AS strategy has been applied to modify lignin biosynthesis in a grass species. PMID:12481050

  13. Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality

    PubMed Central

    Ho-Yue-Kuang, Séverine; Alvarado, Camille; Antelme, Sébastien; Bouchet, Brigitte; Cézard, Laurent; Le Bris, Philippe; Legée, Frédéric; Maia-Grondard, Alessandra; Yoshinaga, Arata; Saulnier, Luc; Guillon, Fabienne; Sibout, Richard; Lapierre, Catherine; Chateigner-Boutin, Anne-Laure

    2016-01-01

    Cereal crop by-products are a promising source of renewable raw material for the production of biofuel from lignocellulose. However, their enzymatic conversion to fermentable sugars is detrimentally affected by lignins. Here the characterization of the Brachypodium Bd5139 mutant provided with a single nucleotide mutation in the caffeic acid O-methyltransferase BdCOMT6 gene is reported. This BdCOMT6-deficient mutant displayed a moderately altered lignification in mature stems. The lignin-related BdCOMT6 gene was also found to be expressed in grains, and the alterations of Bd5139 grain lignins were found to mirror nicely those evidenced in stem lignins. The Bd5139 grains displayed similar size and composition to the control. Complementation experiments carried out by introducing the mutated gene into the AtCOMT1-deficient Arabidopsis mutant demonstrated that the mutated BdCOMT6 protein was still functional. Such a moderate down-regulation of lignin-related COMT enzyme reduced the straw recalcitrance to saccharification, without compromising the vegetative or reproductive development of the plant. PMID:26433202

  14. Association of codon 108/158 catechol-O-methyltransferase gene polymorphism with the psychiatric manifestations of velo-cardio-facial syndrome

    SciTech Connect

    Lachman, H.M.; Papolos, D.F.; Veit, S.

    1996-09-20

    Velo-cardio-facial-syndrome (VCFS) is a common congenital disorder associated with typical facial appearance, cleft palate, cardiac defects, and learning disabilities. The majority of patients have an interstitial deletion on chromosome 22q11. In addition to physical abnormalities, a variety of psychiatric illnesses have been reported in patients with VCFS, including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. The psychiatric manifestations of VCFS could be due to haploinsufficiency of a gene(s) within 22q11. One candidate that has been mapped to this region is catechol-O-methyltransferase (COMT). We recently identified a polymorphism in the COMT gene that leads to a valine{r_arrow}methionine substitution at amino acid 158 of the membrane-bound form of the enzyme. Homozygosity for COMT158{sup met} leads to a 3- to 4-fold reduction in enzymatic activity, compared with homozygotes for COMT158{sup met}. We now report that in a population of patients with VCFS, there is an apparent association between the low-activity allele, COMT158{sup met}, and the development of bipolar spectrum disorder, and in particular, a rapid-cycling form. 33 refs., 3 tabs.

  15. Sequencing around 5-Hydroxyconiferyl Alcohol-Derived Units in Caffeic Acid O-Methyltransferase-Deficient Poplar Lignins1[OA

    PubMed Central

    Lu, Fachuang; Marita, Jane M.; Lapierre, Catherine; Jouanin, Lise; Morreel, Kris; Boerjan, Wout; Ralph, John

    2010-01-01

    Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its β-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure. PMID:20427467

  16. Interaction of catechol O-methyltransferase and serotonin transporter genes modulates effective connectivity in a facial emotion-processing circuitry.

    PubMed

    Surguladze, S A; Radua, J; El-Hage, W; Gohier, B; Sato, J R; Kronhaus, D M; Proitsi, P; Powell, J; Phillips, M L

    2012-01-17

    Imaging genetic studies showed exaggerated blood oxygenation level-dependent response in limbic structures in carriers of low activity alleles of serotonin transporter-linked promoter region (5-HTTLPR) as well as catechol O-methyltransferase (COMT) genes. This was suggested to underlie the vulnerability to mood disorders. To better understand the mechanisms of vulnerability, it is important to investigate the genetic modulation of frontal-limbic connectivity that underlies emotional regulation and control. In this study, we have examined the interaction of 5-HTTLPR and COMT genetic markers on effective connectivity within neural circuitry for emotional facial expressions. A total of 91 healthy Caucasian adults underwent functional magnetic resonance imaging experiments with a task presenting dynamic emotional facial expressions of fear, sadness, happiness and anger. The effective connectivity within the facial processing circuitry was assessed with Granger causality method. We have demonstrated that in fear processing condition, an interaction between 5-HTTLPR (S) and COMT (met) low activity alleles was associated with reduced reciprocal connectivity within the circuitry including bilateral fusiform/inferior occipital regions, right superior temporal gyrus/superior temporal sulcus, bilateral inferior/middle prefrontal cortex and right amygdala. We suggest that the epistatic effect of reduced effective connectivity may underlie an inefficient emotion regulation that places these individuals at greater risk for depressive disorders.

  17. The O-methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit.

    PubMed

    Yauk, Yar-Khing; Chagné, David; Tomes, Sumathi; Matich, Adam J; Wang, Mindy Y; Chen, Xiuyin; Maddumage, Ratnasiri; Hunt, Martin B; Rowan, Daryl D; Atkinson, Ross G

    2015-06-01

    Phenylpropenes, such as eugenol and trans-anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non-producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co-located with seven candidate genes for phenylpropene O-methyltransferases (MdoOMT1-7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over-expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.

  18. Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations1[W

    PubMed Central

    Green, Abigail R.; Lewis, Kevin M.; Barr, John T.; Jones, Jeffrey P.; Lu, Fachuang; Ralph, John; Vermerris, Wilfred; Sattler, Scott E.; Kang, ChulHee

    2014-01-01

    Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion. PMID:24948836

  19. The role of the catechol-O-methyltransferase (COMT) gene in personality and related psychopathological disorders.

    PubMed

    Montag, Christian; Jurkiewicz, Magdalena; Reuter, Martin

    2012-05-01

    This review provides a short overview of the most significant biologically oriented theories of human personality. Personality concepts of Eysenck, Gray and McNaughton, Cloninger and Panksepp will be introduced and the focal evidence for the heritability of personality will be summarized. In this context, a synopsis of a large number of COMT genetic association studies (with a focus on the COMT Val158Met polymorphism) in the framework of the introduced biologically oriented personality theories will be given. In line with the theory of a continuum model between healthy anxious behavior and related psychopathological behavior, the role of the COMT gene in anxiety disorders will be discussed. A final outlook considers new research strategies such as genetic imaging and epigenetics for a better understanding of human personality.

  20. The Role of the Catechol-O-Methyltransferase (COMT) Gene in Personality and Related Psychopathological Disorders

    PubMed Central

    Montag, Christian; Jurkiewicz, Magdalena; Reuter, Martin

    2015-01-01

    This review provides a short overview of the most significant biologically oriented theories of human personality. Personality concepts of Eysenck, Gray and McNaughton, Cloninger and Panksepp will be introduced and the focal evidence for the heritability of personality will be summarized. In this context, a synopsis of a large number of COMT genetic association studies (with a focus on the COMT Val158Met polymorphism) in the framework of the introduced biologically oriented personality theories will be given. In line with the theory of a continuum model between healthy anxious behavior and related psychopathological behavior, the role of the COMT gene in anxiety disorders will be discussed. A final outlook considers new research strategies such as genetic imaging and epigenetics for a better understanding of human personality. PMID:22483293

  1. Genetic polymorphisms of estrogen receptor alpha and catechol-O-methyltransferase genes in Turkish patients with familial prostate carcinoma

    PubMed Central

    Pazarbasi, Ayfer; Yilmaz, M. Bertan; Alptekin, Davut; Luleyap, Umit; Tansug, Zuhtu; Ozpak, Lutfiye; Izmirli, Muzeyyen; Onatoglu-Arikan, Dilge; Kocaturk-Sel, Sabriye; Erkoc, Mehmet Ali; Turgut, Ozgur; Bereketoglu, Ceyhun; Tunc, Erdal; Akbal, Eylul

    2013-01-01

    OBJECTIVES: Estrogen is one of the most crucial hormones participating in the proliferation and carcinogenesis of the prostate glands. Genetic polymorphisms in the estrogen metabolism pathway might be involved in the risk of prostate carcinoma development. We evaluated the association between genetic polymorphisms in estrogen receptor alpha (ESR1) and catechol-O-methyltransferase (COMT) genes and the risk of developing familial prostate carcinoma. MATERIALS AND METHODS: In this study, 34 cases with prostate carcinoma whose first-degree relatives had prostate carcinoma and 30 healthy age-matched male controls were enrolled. The genotypes of ESR1 and COMT genes were analyzed employing polymerase chain reaction-restriction fragment length polymorphism method. 34 cases with prostate carcinoma, whose first degree relatives had prostate carcinoma and 14 age-matched male controls were enrolled to analyze the genotype of these two genes. RESULTS: Among control patients, the ESR1 PvuII genotypes of C/C, C/T and T/T were observed in 37%, 26% and 37%, respectively, whereas the C/C, C/T and T/T genotypes were observed in 18%, 41% and 41% of case patients, respectively. Among controls, the ESR1 PvuII allele frequencies of C and T were equally observed, whereas the C and T allele frequencies were observed in 38% and 62% of patients, respectively. Among ESR1 PvuII genotypes there were not any significant difference in terms of genotype (P = 0.199) and allele (P = 0.181) frequencies. Among controls, the ESR1 XbaI genotypes of G/G, G/A and A/A were observed in 33%, 37% and 33%, respectively, whereas the G/G, G/A and A/A genotypes were observed in 12%, 47% and 41% of patients, respectively. Among controls, the ESR1 XbaI allele frequencies of A and G were observed equally, respectively, whereas the A and G frequencies were observed in 65% and 35% of patients, respectively. Among ESR1 Χ baI, there was not any significant difference in terms of genotype (P = 0.111) and allele (P = 0

  2. Systematic analysis of O-methyltransferase gene family and identification of potential members involved in the formation of O-methylated flavonoids in Citrus.

    PubMed

    Liu, Xiaogang; Luo, Yan; Wu, Hongkun; Xi, Wanpeng; Yu, Jie; Zhang, Qiuyun; Zhou, Zhiqin

    2016-01-10

    The O-methylation of various secondary metabolites is mainly catalyzed by S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase (OMT) proteins that are encoded by the O-methyltransferase gene family. Citrus fruits are a rich source of O-methylated flavonoids that have a broad spectrum of biological activities, including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. However, little is known about this gene family and its members that are involved in the O-methylation of flavonoids and their regulation in Citrus. In this study, 58 OMT genes were identified from the entire Citrus sinensis genome and compared with those from 3 other representative dicot plants. A comprehensive analysis was performed, including functional/substrate predictions, identification of chromosomal locations, phylogenetic relationships, gene structures, and conserved motifs. Distribution mapping revealed that the 58 OMT genes were unevenly distributed on the 9 citrus chromosomes. Phylogenetic analysis of 164 OMT proteins from C.sinensis, Arabidopsis thaliana, Populus trichocarpa, and Vitis vinifera showed that these proteins were categorized into group I (COMT subfamily) and group II (CCoAOMT subfamily), which were further divided into 10 and 2 subgroups, respectively. Finally, digital gene expression and quantitative real-time polymerase chain reaction analyses revealed that citrus OMT genes had distinct temporal and spatial expression patterns in different tissues and developmental stages. Interestingly, 18 and 11 of the 27 genes predicted to be involved in O-methylation of flavonoids had higher expression in the peel and pulp during fruit development, respectively. The citrus OMT gene family identified in this study might help in the selection of appropriate candidate genes and facilitate functional studies in Citrus.

  3. The Role of the Catechol-o-methyltransferase (COMT) Gene Val158Met in Aggressive Behavior, A Review of Genetic Studies

    PubMed Central

    Qayyum, Arqam; Zai, Clement C.; Hirata, Yuko; Tiwari, Arun K.; Cheema, Sheraz; Nowrouzi, Behdin; Beitchman, Joseph H.; Kennedy, James L.

    2015-01-01

    Aggressive behaviors have become a major public health problem, and early-onset aggression can lead to outcomes such as substance abuse, antisocial personality disorder among other issues. In recent years, there has been an increase in research in the molecular and genetic underpinnings of aggressive behavior, and one of the candidate genes codes for the catechol-O-methyltransferase (COMT). COMT is involved in catabolizing catecholamines such as dopamine. These neurotransmitters appear to be involved in regulating mood which can contribute to aggression. The most common gene variant studied in the COMT gene is the Valine (Val) to Methionine (Met) substitution at codon 158. We will be reviewing the current literature on this gene variant in aggressive behavior. PMID:26630958

  4. Association between cerebrospinal fluid dopamine concentrations and catechol-O-methyltransferase gene polymorphisms in forensic autopsy cases of methamphetamine abusers.

    PubMed

    Matsusue, Aya; Ishikawa, Takaki; Michiue, Tomomi; Waters, Brian; Hara, Kenji; Kashiwagi, Masayuki; Takayama, Mio; Ikematsu, Natsuki; Kubo, Shin-Ichi

    2017-01-01

    Methamphetamine (MA) is an illicit psychostimulant that stimulates the release of catecholamines from sympathetic nerve terminals and is widely abused worldwide. Since catechol-O-methyltransferase (COMT) metabolizes catecholamines and mediates adrenergic, noradrenergic, and dopaminergic signaling responses, we investigated the effects of the COMT polymorphisms rs4633 and rs4680 on cerebrospinal fluid (CSF) catecholamine concentrations in autopsies of subjects who died of drug intoxication. 28 MA abusers and 22 fatal psychotropic drug intoxication cases were evaluated. No correlations were identified between rs4633 or rs4680 polymorphisms and CSF concentrations of adrenaline (Adr), noradrenaline (Nad), or dopamine (DA) in fatal psychotropic cases. However, among MA abusers, DA concentrations in the CSF were significantly higher in those with the T allele (CT and TT) of rs4633 than in CC genotype carriers (p=0.004). Moreover, among MA abusers, DA concentrations were significantly higher in those with the A allele (GA and AA) of rs4680 than in GG genotype carriers (p=0.017). In subsequent haplotype analyses of MA abusers, a strong correlation was identified between two COMT haplotypes and CSF DA concentrations (p=0.002). However, the CSF concentrations of Adr and Nad were not associated with COMT genotypes or haplotypes. The present results indicate that rs4633 and rs4680 polymorphisms influence CSF DA concentrations and MA toxicity in MA abusers.

  5. Function Analysis of Caffeoyl-CoA O-Methyltransferase for Biosynthesis of Lignin and Phenolic Acid in Salvia miltiorrhiza.

    PubMed

    Wang, Zhengjun; Ge, Qian; Chen, Chen; Jin, Xinxin; Cao, Xiaoyan; Wang, Zhezhi

    2017-02-01

    In this study, we cloned a full-length cDNA and the genomic DNA sequence of SmCCoAOMT (GenBank ID JQ007585) from Salvia miltiorrhiza. The 744-bp open-reading frame encodes a protein of 247 amino acids that shares 95 % similarity with one in Vitis vinifera. Real-time quantitative PCR analysis revealed that SmCCoAOMT is most highly expressed in the stems and can be induced by methyl jasmonate (MeJA) and XC-1 treatment. To evaluate its function in vivo, we generated RNA interference transgenic plants through Agrobacterium tumefaciens-mediated gene transfer. Compared with untransformed control plants, the transgenics had significantly less lignin and the expression of lignin-biosynthetic genes SmCCR and SmCOMT was depressed. In 90-day-old roots from plants of transgenic line M5, accumulations of rosmarinic acid and salvianolic acid B (Sal B) were greatly reduced by 0.89- and 0.69-fold, respectively. This low-Sal B phenotype was stable in the roots, with the level of accumulation being approximately 43.58 mg g(-1) dry weight, which was 52 % of the amount measured in the untransformed control. Our results suggest that SmCCoAOMT is involved in lignin biosynthesis and affects the accumulation of phenolic acids. This study also provides potential guidance for using lignin-related genes to genetically engineer Salvia miltiorrhiza.

  6. Association of aggressive behavior in Korean male schizophrenic patients with polymorphisms in the serotonin transporter promoter and catecholamine-O-methyltransferase genes.

    PubMed

    Han, Doug Hyun; Park, Doo Byung; Na, Chul; Kee, Baik Seok; Lee, Young Sik

    2004-11-30

    The incidence of aggressive behavior in patients with schizophrenia is higher than in the general population. Among particular gene polymorphisms posited to be involved in psychiatric disorders, the catecholamine-O-methyltransferase (COMT) and serotonin transporter (5-HTTPR) genes have been the focus of recent research on aggression. In this study, we hypothesized that both the COMT and the 5-HTTPR genotypes may be dependent on and related to aggression in Korean patients with schizophrenia. The subjects were 168 unrelated male schizophrenic patients diagnosed according to DSM-IV. Among two psychiatric hospital staff and medical university students, 158 unrelated male subjects with no lifetime history of psychiatric disorders were recruited to establish the COMT and 5-HTTPR genotype distribution in the general population. All episodes of aggression from the last discharge to readmission were rated. The Total Overt Aggression Scale (OAS) score (sum of the scores of all episodes of aggression), highest OAS score (highest individual episode score, 0-16), OAS category, and OAS category score (mean score within each category) were recorded. There were statistically significant effects of COMT genotype on the mean OAS 4 (physical aggression against other people) score and the highest OAS score. The most predictive was the OAS 4 score. There was a statistically significant effect of 5-HTTPR genotype on mean total score. Thus, the COMT gene is associated with the severity of aggression and with physical aggression against other people, whereas the 5-HTTPR gene is associated with the summary score of all episodes of aggression.

  7. Structure-Function Analyses of a Caffeic Acid O-Methyltransferase from Perennial Ryegrass Reveal the Molecular Basis for Substrate Preference[W][OA

    PubMed Central

    Louie, Gordon V.; Bowman, Marianne E.; Tu, Yi; Mouradov, Aidyn; Spangenberg, German; Noel, Joseph P.

    2010-01-01

    Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-l-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT’s catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde. PMID:21177481

  8. Abiotic stresses differentially affect the expression of O-methyltransferase genes related to methoxypyrazine biosynthesis in seeded and parthenocarpic fruits of Vitis vinifera (L.).

    PubMed

    Vallarino, José G; Gainza-Cortés, Felipe; Verdugo-Alegría, Claudio; González, Enrique; Moreno, Yerko M

    2014-07-01

    MPs (3-alkyl-2-methoxypyrazines) are grape-derived aroma compounds that are associated with detrimental herbaceous flavours in some wines. It is well known that several viticultural and environmental parameters can modulate MP concentrations in grapes, although comprehensive molecular studies have not been conducted in this field. Although the biosynthesis pathway of MPs has not been fully elucidated, four Vitis vinifera O-methyltransferase genes (VvOMT1-4) have been related to be involved in MP biosynthesis. We assessed whether different abiotic stresses induction have an impact on MP levels in grapes and wines from seeded and parthenocarpic fruits. Our results show that the timing of VvOMT3 expression is associated with the period of MPs accumulation in seeded fruits during both abiotic stresses, whereas no association was found in parthenocarpic fruits. These results are discussed in the context of how different viticultural practices can modulate VvOMT gene expression, which has a direct impact on MPs levels in wines.

  9. Association of the functional polymorphism in the catechol-O-methyltransferase gene with schizophrenia in the three ethnic groups of the Malaysian population.

    PubMed

    Wan, Ching-Lee; Zainal, Nor Zuraida; Lian, Lay-Hoong; Mohamed, Zahurin

    2011-08-30

    The catechol-O-methyltransferase (COMT) gene is a candidate gene for schizophrenia as its encoded enzyme is involved in the metabolic inactivation of dopamine and noradrenaline. Several molecular genetic studies thus far have demonstrated that the COMT functional polymorphism of Val158Met is susceptible with schizophrenia. Hence, the present study aims to determine this genetic association of this SNP in the three major ethnic groups of the Malaysian population. A total of 317 patients (79 Malays, 154 Chinese and 84 Indians) meeting DSM-IV criteria for schizophrenia and 417 healthy subjects (160 Malays, 164 Chinese and 93 Indians) were recruited. A PCR-RFLP method was used to determine the genotypes and alleles present. We found a significant association of genotypes within the total pooled samples, as well as in the female subgroup, with a higher frequency of heterozygotes in schizophrenia subjects. However, there were no significant differences in allele and genotype frequency between the schizophrenic patients and normal controls in all three ethnic groups. Our current findings suggest that the Val158Met polymorphism has a weak association with schizophrenia in the Malaysian population and does not play a major role in conferring susceptibility to the schizophrenia in any of the three major local ethnicities.

  10. Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Lee, Kyungjin; Lee, Hye-Jung; Back, Kyoungwhan

    2014-11-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis.

  11. Catechol-O-methyltransferase gene variants may associate with negative symptom response and plasma concentrations of prolactin in schizophrenia after amisulpride treatment.

    PubMed

    Chen, Chun-Yen; Yeh, Yi-Wei; Kuo, Shin-Chang; Ho, Pei-Shen; Liang, Chih-Sung; Yen, Che-Hung; Lu, Ru-Band; Huang, San-Yuan

    2016-03-01

    Catechol-O-methyltransferase (COMT) enzyme is involved in the pathogenesis of psychotic symptoms and may be associated with a therapeutic response to antipsychotic drugs. The aim of this study was to examine the relationship between COMT variants, plasma prolactin level, and the therapeutic effectiveness of amisulpride treatment in patients with schizophrenia. A 12-week naturalistic study of amisulpride treatment was carried out in 185 Han Chinese patients with schizophrenia. The patients were screened for 14 single-nucleotide polymorphisms of the COMT gene. The Positive and Negative Syndrome Scale (PANSS) was used to assess the improvement of psychopathological symptoms from the baseline to the end point in each subject. For better presentation of time-course changes in response status, a mixed model for repeated-measures (MMRM) analysis of symptom improvement during the 12-week treatment period was conducted. The change in plasma prolactin level after amisulpride treatment was also examined (n=51). No significant differences in the genotype frequencies of the COMT variants investigated were observed between responders and non-responders. Moreover, an MMRM analysis of psychopathological symptom improvement during the 12-week treatment course showed that it depended significantly on COMT variants (rs4680, rs4633, and rs6267), particularly regarding changes in negative symptoms. The increase in plasma prolactin levels observed was influenced by the COMT rs4680 variant and was positively correlated with a reduction in PANSS negative scores. Our results suggest that variation of the COMT gene is associated with treatment response regarding negative symptoms and prolactin changes after amisulpride treatment in patients with schizophrenia.

  12. Effect of Catechol-O-methyltransferase-gene (COMT) Variants on Experimental and Acute Postoperative Pain in 1,000 Women undergoing Surgery for Breast Cancer

    PubMed Central

    Kambur, Oleg; Kaunisto, Mari A.; Tikkanen, Emmi; Leal, Suzanne M.; Ripatti, Samuli; Kalso, Eija A.

    2016-01-01

    Background Catechol-O-methyltransferase (COMT) metabolizes catecholamines in different tissues. Polymorphisms in COMT gene can attenuate COMT activity and increase sensitivity to pain. Human studies exploring the effect of COMT polymorphisms on pain sensitivity have mostly included small, heterogeneous samples and have ignored several important single nucleotide polymorphisms (SNPs). This study examines the effect of COMT polymorphisms on experimental and postoperative pain phenotypes in a large ethnically homogeneous female patient cohort. Methods Intensity of cold (+2–4°C) and heat (+48°C) pain and tolerance to cold pain were assessed in 1,000 patients scheduled for breast cancer surgery. Acute postoperative pain and oxycodone requirements were recorded. Twenty-two COMT SNPs were genotyped and their association with six pain phenotypes analyzed with linear regression. Results There was no association between any of the tested pain phenotypes and SNP rs4680. The strongest association signals were seen between rs165774 and heat pain intensity as well as rs887200 and cold pain intensity. In both cases, minor allele carriers reported less pain. Neither of these results remained significant after strict multiple testing corrections. When analyzed further, the effect of rs887200 was, however, shown to be significant and consistent throughout the cold pressure test. No evidence of association between the SNPs and postoperative oxycodone consumption was found. Conclusions SNPs rs887200 and rs165774 located in the untranslated regions of the gene had the strongest effects on pain sensitivity. Their effect on pain is described here for the first time. These results should be confirmed in further studies and the potential functional mechanisms of the variants studied. PMID:24343288

  13. How to consistently link extraversion and intelligence to the catechol-O-methyltransferase (COMT) gene: on defining and measuring psychological phenotypes in neurogenetic research.

    PubMed

    Wacker, Jan; Mueller, Erik M; Hennig, Jürgen; Stemmler, Gerhard

    2012-02-01

    The evidence for associations between genetic polymorphisms and complex behavioral/psychological phenotypes (traits) has thus far been weak and inconsistent. Using the well-studied Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene as an example, we demonstrate that using theoretical models to guide phenotype definition and measuring the phenotypes of interest with a high degree of specificity reveals strong gene-behavior associations that are consistent with prior work and that would have otherwise gone unnoticed. Only after statistically controlling for irrelevant portions of phenotype variance did we observe strong (Cohen's d = 0.33-0.70) and significant associations between COMT Val158Met and both cognitive and affective traits in a healthy male sample (N = 201) in Study 1: Carriers of the Met allele scored higher in fluid intelligence (reasoning) but lower in both crystallized intelligence (general knowledge) and the agency facet of extraversion. In Study 2, we conceptually replicated the association of COMT Val158Met with the agency facet of extraversion after partialing irrelevant phenotype variance in a female sample (N = 565). Finally, through reanalysis of a large published data set we showed that Met allele carriers also scored higher in indicators of fluid intelligence after partialing verbal fluency. Because the Met allele codes for a less efficient variant of the enzyme COMT, resulting in higher levels of extrasynaptic prefrontal dopamine, these observations provide further support for a role for dopamine in both intelligence and extraversion. More importantly, the present findings have important implications for the definition of psychological phenotypes in neurogenetic research.

  14. TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol.

    PubMed

    Jung, Je Hyeong; Altpeter, Fredy

    2016-09-01

    Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production. Reduction of lignin content significantly improves the conversion of lignocellulosic biomass into ethanol. Conventional mutagenesis is not expected to confer reduction in lignin content in sugarcane due to its high polyploidy (x = 10-13) and functional redundancy among homo(eo)logs. Here we deploy transcription activator-like effector nuclease (TALEN) to induce mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane. Capillary electrophoresis (CE) was validated by pyrosequencing as reliable and inexpensive high throughput method for identification and quantitative characterization of TALEN mediated mutations. Targeted COMT mutations were identified by CE in up to 74 % of the lines. In different events 8-99 % of the wild type COMT were converted to mutant COMT as revealed by pyrosequencing. Mutation frequencies among mutant lines were positively correlated to lignin reduction. Events with a mutation frequency of 99 % displayed a 29-32 % reduction of the lignin content compared to non-transgenic controls along with significantly reduced S subunit content and elevated hemicellulose content. CE analysis displayed similar peak patterns between primary COMT mutants and their vegetative progenies suggesting that TALEN mediated mutations were faithfully transmitted to vegetative progenies. This is the first report on genome editing in sugarcane. The findings demonstrate that targeted mutagenesis can improve cell wall characteristics for production of lignocellulosic ethanol in crops with highly complex genomes.

  15. Genotype status of the dopamine-related catechol-O-methyltransferase (COMT) gene corresponds with desirability of “unhealthy” foods

    PubMed Central

    Wallace, Deanna L.; Aarts, Esther; Uquillas, Federico d’Oleire; Dang, Linh C.; Greer, Stephanie M.; Jagust, William J.; D’Esposito, Mark

    2015-01-01

    The role of dopamine is extensively documented in weight regulation and food intake in both animal models and humans. Yet the role of dopamine has not been well studied in individual differences for food desirability. Genotype status of the dopamine-related catechol-O-methyltransferase (COMT) gene has been shown to influence dopamine levels, with greater COMT enzymatic activity in val/val individuals corresponding to greater degradation of dopamine. Decreased dopamine has been associated with poorer cognitive control and diminished goal-directed behavior in various behavioral paradigms. Additionally, dopaminergic-rich regions such as the frontal cortex and dorsal striatum have been shown to be important for supporting food-related decision-making. However, the role of dopamine, as assessed by COMT genotype status, in food desirability has not been fully explored. Therefore, we utilized an individual’s COMT genotype status (n=61) and investigated food desirability based on self-rated “healthy” and “unhealthy” food perceptions. Here we found val/val individuals (n=19) have greater desirability for self-rated “unhealthy” food items, but not self-rated “healthy” food items, as compared to val/met (n=24) and met/met (n=18) individuals (p<0.005). Utilizing an objective health measure for the food items, we also found val/val and val/met individuals have greater desirability for objectively defined “unhealthy” food items, as compared to met/met individuals (p<0.01). This work further substantiates a role of dopamine in food-related behaviors and more specifically in relationship to food desirability for “unhealthy” food items. PMID:25963102

  16. Characterization and functional analysis of eugenol O-methyltransferase gene reveal metabolite shifts, chemotype specific differential expression and developmental regulation in Ocimum tenuiflorum L.

    PubMed

    Renu, Indu Kumari; Haque, Inamul; Kumar, Manish; Poddar, Raju; Bandopadhyay, Rajib; Rai, Amit; Mukhopadhyay, Kunal

    2014-03-01

    Eugenol-O-methyltransferase (EOMT) catalyzes the conversion of eugenol to methyleugenol in one of the final steps of phenylpropanoid pathway. There are no comprehensive reports on comparative EOMT gene expression and developmental stage specific accumulation of phenylpropenes in Ocimum tenuiflorum. Seven chemotypes, rich in eugenol and methyleugenol, were selected by assessment of volatile metabolites through multivariate data analysis. Isoeugenol accumulated in higher levels during juvenile stage (36.86 ng g(-1)), but reduced sharply during preflowering (8.04 ng g(-1)), flowering (2.29 ng g(-1)) and postflowering stages (0.17 ng g(-1)), whereas methyleugenol content gradually increased from juvenile (12.25 ng g(-1)) up to preflowering (16.35 ng g(-1)) and then decreased at flowering (7.13 ng g(-1)) and post flowering (5.95 ng g(-1)) from fresh tissue. Extreme variations of free intracellular and alkali hydrolysable cell wall released phenylpropanoid compounds were observed at different developmental stages. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90 bp intron. The translated proteins had eight catalytic domains, the major two being dimerisation superfamily and methyltransferase_2 superfamily. A validated 3D structure of EOMT protein was also determined. The chemotype Ot7 had a reduced reading frame that lacked both dimerisation domains and one of the two protein-kinase-phosphorylation sites; this was also reflected in reduced accumulation of methyleugenol compared to other chemotypes. EOMT transcripts showed enhanced expression in juvenile stage that increased further during preflowering but decreased at flowering and further at postflowering. The expression patterns may possibly be compared and correlated to the amounts of eugenol/isoeugenol and methyleugenol in different developmental stages of all chemotypes.

  17. Catechol-O-methyltransferase Val158Met genotype and the clinical responses to duloxetine treatment or plasma levels of 3-methoxy-4-hydroxyphenylglycol and homovanillic acid in Japanese patients with major depressive disorder

    PubMed Central

    Atake, Kiyokazu; Yoshimura, Reiji; Hori, Hikaru; Katsuki, Asuka; Nakamura, Jun

    2015-01-01

    Purpose This study investigated the relationships among the plasma levels of catecholamine metabolites, the clinical response to duloxetine treatment, and Val158Met polymorphism of the catechol-O-methyltransferase (COMT) gene. Subjects and methods Sixty-four patients and 30 healthy control subjects were recruited. Major depressive episodes were diagnosed using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision criteria. The severity of depression was evaluated using the 17-item Hamilton Rating Scale for Depression (HAMD17). Patients whose HAMD17 scores were 15 or greater were enrolled in the study. Blood sampling and clinical evaluation were performed at week 0 and week 8. The levels of plasma catecholamine metabolites were measured using high-performance liquid chromatography with electrochemical detection. Genotyping was performed using direct sequencing. Results Thirty of 45 patients (67%) responded to duloxetine treatment during the 8 weeks of treatment. The baseline plasma levels of 3-methoxy-4-hydroxyphenylglycol (MHPG), but not homovanillic acid (HVA), were lower in patients with major depressive disorder (MDD) who had the Val/Val genotype than in patients who were Met-carriers. Patients with MDD and the Val/Val genotype, but not Met carriers, had increased plasma levels of MHPG after 8 weeks of duloxetine treatment. The baseline plasma MHPG levels in healthy control subjects with the Val/Val genotype were significantly higher than those in patients with MDD. Among the subjects in the MDD group with the Val/Val genotype, the plasma MHPG levels increased to the same degree as in the healthy control subjects with the Val/Val genotype after 8 weeks of duloxetine treatment. Conclusion The relationship among the COMT Val158Met polymorphism, plasma levels of catecholamine metabolites, and responses to duloxetine is complex. Nevertheless, our results suggest that patients with MDD and the

  18. Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

    SciTech Connect

    Bhuiya, M.W.; Liu, C.

    2010-01-01

    Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily 'plastic' amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

  19. Catechol O-methyltransferase and monoamine oxidase A genotypes, and plasma catecholamine metabolites in bipolar and schizophrenic patients.

    PubMed

    Zumárraga, Mercedes; Dávila, Ricardo; Basterreche, Nieves; Arrue, Aurora; Goienetxea, Biotza; Zamalloa, María I; Erkoreka, Leire; Bustamante, Sonia; Inchausti, Lucía; González-Torres, Miguel A; Guimón, José

    2010-01-01

    Metabolites of dopamine and norepinephrine measured in the plasma have long been associated with symptomatic severity and response to treatment in schizophrenic, bipolar and other psychiatric patients. Plasma concentrations of catecholamine metabolites are genetically regulated. The genes encoding enzymes that are involved in the synthesis and degradation of these monoamines are candidate targets for this genetic regulation. We have studied the relationship between the Val158Met polymorphism in catechol O-methyltransferase gene, variable tandem repeat polymorphisms in the monoamine oxidase A gene promoter, and plasma concentrations of 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid and homovanillic acid in healthy control subjects as well as in untreated schizophrenic and bipolar patients. We found that the Val158Met substitution in catechol O-methyltransferase gene influences the plasma concentrations of homovanillic and 3,4-dihydroxyphenylacetic acids. Although higher concentrations of plasma homovanillic acid were found in the high-activity ValVal genotype, this mutation did not affect the plasma concentration of 3-methoxy-4-hydroxyphenylglycol. 3,4-dihydroxyphenylacetic acid concentrations were higher in the low-activity MetMet genotype. Interestingly, plasma values 3-methoxy-4-hydroxyphenylglycol were greater in schizophrenic patients and in bipolar patients than in healthy controls. Our results are compatible with the previously reported effect of the Val158Met polymorphism on catechol O-methyltransferase enzymatic activity. Thus, our results suggest that this polymorphism, alone or associated with other polymorphisms, could have an important role in the genetic control of monoamine concentration and its metabolites.

  20. Age-Dependent Effects of Catechol-O-Methyltransferase (COMT) Gene Val158Met Polymorphism on Language Function in Developing Children.

    PubMed

    Sugiura, Lisa; Toyota, Tomoko; Matsuba-Kurita, Hiroko; Iwayama, Yoshimi; Mazuka, Reiko; Yoshikawa, Takeo; Hagiwara, Hiroko

    2016-11-30

    The genetic basis controlling language development remains elusive. Previous studies of the catechol-O-methyltransferase (COMT) Val(158)Met genotype and cognition have focused on prefrontally guided executive functions involving dopamine. However, COMT may further influence posterior cortical regions implicated in language perception. We investigated whether COMT influences language ability and cortical language processing involving the posterior language regions in 246 children aged 6-10 years. We assessed language ability using a language test and cortical responses recorded during language processing using a word repetition task and functional near-infrared spectroscopy. The COMT genotype had significant effects on language performance and processing. Importantly, Met carriers outperformed Val homozygotes in language ability during the early elementary school years (6-8 years), whereas Val homozygotes exhibited significant language development during the later elementary school years. Both genotype groups exhibited equal language performance at approximately 10 years of age. Val homozygotes exhibited significantly less cortical activation compared with Met carriers during word processing, particularly at older ages. These findings regarding dopamine transmission efficacy may be explained by a hypothetical inverted U-shaped curve. Our findings indicate that the effects of the COMT genotype on language ability and cortical language processing may change in a narrow age window of 6-10 years.

  1. Monoamine Oxidase A (MAOA) and Catechol-O-Methyltransferase (COMT) Gene Polymorphisms Interact with Maternal Parenting in Association with Adolescent Reactive Aggression but not Proactive Aggression: Evidence of Differential Susceptibility.

    PubMed

    Zhang, Wenxin; Cao, Cong; Wang, Meiping; Ji, Linqin; Cao, Yanmiao

    2016-04-01

    To date, whether and how gene-environment (G × E) interactions operate differently across distinct subtypes of aggression remains untested. More recently, in contrast with the diathesis-stress hypothesis, an alternative hypothesis of differential susceptibility proposes that individuals could be differentially susceptible to environments depending on their genotypes in a "for better and for worse" manner. The current study examined interactions between monoamine oxidase A (MAOA) T941G and catechol-O-methyltransferase (COMT) Val158Met polymorphisms with maternal parenting on two types of aggression: reactive and proactive. Moreover, whether these potential G × E interactions would be consistent with the diathesis-stress versus the differential susceptibility hypothesis was tested. Within the sample of 1399 Chinese Han adolescents (47.2 % girls, M age = 12.32 years, SD = 0.50), MAOA and COMT genes both interacted with positive parenting in their associations with reactive but not proactive aggression. Adolescents with T alleles/TT homozygotes of MAOA gene or Met alleles of COMT gene exhibited more reactive aggression when exposed to low positive parenting, but less reactive aggression when exposed to high positive parenting. These findings provide the first evidence for distinct G × E interaction effects on reactive versus proactive aggression and lend further support for the differential susceptibility hypothesis.

  2. Microdialysis with radiometric monitoring of L-[β-11C]DOPA to assess dopaminergic metabolism: effect of inhibitors of L-amino acid decarboxylase, monoamine oxidase, and catechol-O-methyltransferase on rat striatal dialysate.

    PubMed

    Okada, Maki; Nakao, Ryuji; Hosoi, Rie; Zhang, Ming-Rong; Fukumura, Toshimitsu; Suzuki, Kazutoshi; Inoue, Osamu

    2011-01-01

    The catecholamine, dopamine (DA), is synthesized from 3,4-dihydroxy-L-phenylalanine (L-DOPA) by aromatic L-amino acid decarboxylase (AADC). Dopamine metabolism is regulated by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). To measure dopaminergic metabolism, we used microdialysis with radiometric detection to monitor L-[β-(11)C]DOPA metabolites in the extracellular space of the rat striatum. We also evaluated the effects of AADC, MAO, and COMT inhibitors on metabolite profiles. The major early species measured after administration of L-[β-(11)C]DOPA were [(11)C]3,4-dihydroxyphenylacetic acid ([(11)C]DOPAC) and [(11)C]homovanillic acid ([(11)C]HVA) in a 1:1 ratio, which shifted toward [(11)C]HVA with time. An AADC inhibitor increased the uptake of L-[β-(11)C]DOPA and L-3-O-methyl-[(11)C]DOPA and delayed the accumulation of [(11)C]DOPAC and [(11)C]HVA. The MAO and COMT inhibitors increased the production of [(11)C]3-methoxytyramine and [(11)C]DOPAC, respectively. These results reflect the L-DOPA metabolic pathway, suggesting that this method may be useful for assessing dopaminergic metabolism.

  3. Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama)

    PubMed Central

    Van Ekert, Evelien; Shatters, Robert G.; Rougé, Pierre; Powell, Charles A.; Smagghe, Guy; Borovsky, Dov

    2015-01-01

    The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus ‘Liberibacter’ asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10−3 and 0.217 × 10−3 s−1, respectively) as compared to JH acid I (JHA I) (cis/trans/cis; 2Z, 6E, 10cis), JHA III (2E, 6E, 10cis), and JHA I (trans/cis/cis; 2E, 2Z, 10cis) (kcat of 0.081 × 10−3, 0.013 × 10−3, and 0.003 × 10−3 s−1, respectively). This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), not a jmtD, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). DcFA-o-MT does not require Ca2+, Mg2+ or Zn2+, however, Zn2+ (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA. PMID:25893162

  4. Association of Single Nucleotide Polymorphisms in Catechol-O-Methyltransferase and Serine-Threonine Protein Kinase Genes in the Pakistani Schizophrenic Population: A Study with Special Emphasis on Cannabis and Smokeless Tobacco.

    PubMed

    Nawaz, Rukhsana; Siddiqui, Sonia

    2015-01-01

    Schizophrenia is a neuropsychiatric disorder in which abnormalities in the prefrontal cortex lead to impaired synthesis of dopamine. It is associated with hallucination, psychosis and hearing impairments. Many susceptible genes have been identified in schizophrenia such as catechol-O-methyltransferase (COMT) and serine/threonine kinase (AKT1). Single nucleotide polymorphisms (SNPs) in these genes have not been identified in Pakistan. Therefore, we investigated the allelic and genotypic frequencies in COMT and AKT1 genes in the Pakistani population. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to identify SNPs in the genes. The present study shows that COMT Val and COMT Met allelic frequencies for the controls were p=0.52, q=0.48 and for the schizophrenic cases they were p=0.34, q=0.66 respectively. The distribution of polymorphism in COMT Val158Met genotype by Hardy-Weinberg equilibrium (HWE) was P=0.61 for controls and P=0.005 for cases. The data reveal that SNP rs1130214 T allele mutation was found neither in patients nor in controls in the 5' untranslated region (UTR). This proves that no association of AKT1 and positive association of COMT with schizophrenia exist in the population of Pakistan. Moreover, a study based on a single family showed COMT Met allele inheritance in schizophrenic offspring. This suggested that COMT allele alteration influences susceptibility to at least some forms of psychosis in the Pakistani population. Interestingly, according to our socio-economical survey, COMT genotype has no association with cannabis but it is strongly associated with tobacco. The Pakistani population with Val158Met SNP showed more susceptibility towards developing schizophrenia. This study highlights the genetic differences between Pakistani and other Caucasian populations.

  5. Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

    PubMed Central

    2012-01-01

    Background Silene latifolia and its pollinator, the noctuid moth Hadena bicruris, represent an open nursery pollination system wherein floral volatiles, especially veratrole (1, 2-dimethoxybenzene), lilac aldehydes, and phenylacetaldehyde are of key importance for floral signaling. Despite the important role of floral scent in ensuring reproductive success in S. latifolia, the molecular basis of scent biosynthesis in this species has not yet been investigated. Results We isolated two full-length cDNAs from S. latifolia that show similarity to rose orcinol O-methyltransferase. Biochemical analysis showed that both S. latifolia guaiacol O-methyltransferase1 (SlGOMT1) &S. latifolia guaiacol O-methyltransferase2 (SlGOMT2) encode proteins that catalyze the methylation of guaiacol to form veratrole. A large Km value difference between SlGOMT1 (~10 μM) and SlGOMT2 (~501 μM) resulted that SlGOMT1 is 31-fold more catalytically efficient than SlGOMT2. qRT-PCR expression analysis showed that the SlGOMT genes are specifically expressed in flowers and male S. latifolia flowers had 3- to 4-folds higher level of GOMT gene transcripts than female flower tissues. Two related cDNAs, S. dioica O-methyltransferase1 (SdOMT1) and S. dioica O-methyltransferase2 (SdOMT2), were also obtained from the sister species Silene dioica, but the proteins they encode did not methylate guaiacol, consistent with the lack of veratrole emission in the flowers of this species. Our evolutionary analysis uncovered that SlGOMT1 and SlGOMT2 genes evolved under positive selection, whereas SdOMT1 and SdOMT2 genes show no evidence for selection. Conclusions Altogether, we report the identification and functional characterization of the gene, SlGOMT1 that efficiently catalyzes veratrole formation, whereas another copy of this gene with only one amino acid difference, SlGOMT2 was found to be less efficient for veratrole synthesis in S. latifolia. PMID:22937972

  6. Structural characterization of the mitomycin 7-O-methyltransferase

    SciTech Connect

    Singh, Shanteri; Chang, Aram; Goff, Randal D.; Bingman, Craig A.; Grüschow, Sabine; Sherman, David H.; Phillips, Jr., George N.; Thorson, Jon S.

    2014-10-02

    Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.

  7. Functional characterization of a plastidal cation-dependent O-methyltransferase from the liverwort Plagiochasma appendiculatum.

    PubMed

    Xu, Rui-Xue; Zhao, Yu; Gao, Shuai; Zhang, Yu-Ying; Li, Dan-Dan; Lou, Hong-Xiang; Cheng, Ai-Xia

    2015-10-01

    Caffeoyl CoA O-methyltransferases (CCoAOMTs), known to be involved in phenylpropanoid metabolism and lignin synthesis, have been characterized from several higher plant species, which also harbor CCoAOMT-like enzymes responsible for methylation of a variety of flavonoids, anthocyanins, coumarins and phenylpropanoids. Here, a gene encoding a CCoAOMT (PaOMT1) was isolated from a sequenced cDNA library of the liverwort species Plagiochasma appendiculatum, a species belonging to the Family Aytoniaceae. The full-length cDNA sequence of PaOMT1 contains 909 bp, and is predicted to encode a protein with 302 amino acids. The gene products were 40-50% identical to CCoAOMT sequences of other plants. Experiments based on recombinant PaOMT1 showed that the enzyme was able to methylate phenylpropanoids, flavonoids and coumarins, with a preference for the flavonoid quercetin (19). Although the substrate selectivity and biochemical feature of PaOMT1 is similar to CCoAOMT-like enzymes, the sequence alignment results indicated PaOMT1 is closer to true CCoAOMT enzymes. A phylogenetic analysis indicated that PaOMT1 is intermediate between true CCoAOMTs and CCoAOMT-like enzymes. The transient expression of a PaOMT1-GFP fusion in tobacco demonstrated that PaOMT1 is directed to the plastids. PaOMT1 may represent an ancestral form of higher plant true CCoAOMT and CCoAOMT-like enzymes. This is the first time an O-methyltransferase was characterized in liverworts.

  8. Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis.

    PubMed

    Byeon, Yeong; Lee, Hye-Jung; Lee, Hyoung Yool; Back, Kyoungwhan

    2016-01-01

    The N-acetylserotonin O-methyltransferase (ASMT) gene encodes the enzyme that catalyzes the conversion of N-acetylserotonin to melatonin as the last step in melatonin biosynthesis. The first plant ASMT gene to be cloned was from rice. An orthologous gene encoding a protein with ASMT activity and only 39.7% amino acid sequence identity to the rice ASMT protein was recently isolated from apple (Malus zumi). The low homology of the apple ASMT sequence prompted us to screen the Arabidopsis genome for a homologous ASMT gene. The At4g35160 gene exhibited the highest sequence identity (31%) to the rice ASMT gene, followed by the At1g76790 gene with 29% sequence identity. We purified recombinant proteins expressed from the two Arabidopsis genes. The At4g35160 recombinant protein exhibited ASMT enzyme activity, but the At1g76790 recombinant protein did not; thus, we designated At4g35160 as an Arabidopsis thaliana ASMT (AtASMT) gene. The AtASMT protein catalyzed the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine with Vmax values of 0.11 and 0.29 pkat/mg protein, respectively. However, AtASMT exhibited no caffeic acid O-methyltransferase activity, suggesting that its function was highly specific to melatonin synthesis. AtASMT transcripts were induced by cadmium treatment in Arabidopsis followed by increased melatonin synthesis. Similar to other ASMT proteins, AtASMT was localized in the cytoplasm and its ectopic overexpression in rice resulted in increased ASMT enzyme activity and melatonin production, indicating the involvement of AtASMT in melatonin synthesis.

  9. Perinatal Risk Factors Interacting with Catechol O-Methyltransferase and the Serotonin Transporter Gene Predict ASD Symptoms in Children with ADHD

    ERIC Educational Resources Information Center

    Nijmeijer, Judith S.; Hartman, Catharina A.; Rommelse, Nanda N. J.; Altink, Marieke E.; Buschgens, Cathelijne J. M.; Fliers, Ellen A.; Franke, Barbara; Minderaa, Ruud B.; Ormel, Johan; Sergeant, Joseph A.; Verhulst, Frank C.; Buitelaar, Jan K.; Hoekstra, Pieter J.

    2010-01-01

    Background: Symptoms of autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD) often co-occur. Given the previously found familiality of ASD symptoms in children with ADHD, addressing these symptoms may be useful for genetic association studies, especially for candidate gene findings that have not been consistently…

  10. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    PubMed

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods.

  11. Methylation mediated by an anthocyanin, O-methyltransferase, is involved in purple flower coloration in Paeonia

    PubMed Central

    Du, Hui; Wu, Jie; Ji, Kui-Xian; Zeng, Qing-Yin; Bhuiya, Mohammad-Wadud; Su, Shang; Shu, Qing-Yan; Ren, Hong-Xu; Liu, Zheng-An; Wang, Liang-Sheng

    2015-01-01

    Anthocyanins are major pigments in plants. Methylation plays a role in the diversity and stability of anthocyanins. However, the contribution of anthocyanin methylation to flower coloration is still unclear. We identified two homologous anthocyanin O-methyltransferase (AOMT) genes from purple-flowered (PsAOMT) and red-flowered (PtAOMT) Paeonia plants, and we performed functional analyses of the two genes in vitro and in vivo. The critical amino acids for AOMT catalytic activity were studied by site-directed mutagenesis. We showed that the recombinant proteins, PsAOMT and PtAOMT, had identical substrate preferences towards anthocyanins. The methylation activity of PsAOMT was 60 times higher than that of PtAOMT in vitro. Interestingly, this vast difference in catalytic activity appeared to result from a single amino acid residue substitution at position 87 (arginine to leucine). There were significant differences between the 35S::PsAOMT transgenic tobacco and control flowers in relation to their chromatic parameters, which further confirmed the function of PsAOMT in vivo. The expression levels of the two homologous AOMT genes were consistent with anthocyanin accumulation in petals. We conclude that AOMTs are responsible for the methylation of cyanidin glycosides in Paeonia plants and play an important role in purple coloration in Paeonia spp. PMID:26208646

  12. Monolignol 4-O-methyltransferases and uses thereof

    DOEpatents

    Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

    2014-11-18

    Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

  13. Cloning and Characterization of a Norbelladine 4′-O-Methyltransferase Involved in the Biosynthesis of the Alzheimer’s Drug Galanthamine in Narcissus sp. aff. pseudonarcissus

    PubMed Central

    Kilgore, Matthew B.; Augustin, Megan M.; Starks, Courtney M.; O’Neil-Johnson, Mark; May, Gregory D.; Crow, John A.; Kutchan, Toni M.

    2014-01-01

    Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway. PMID:25061748

  14. Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation.

    PubMed

    Thanh Mai Pham, Le; Kim, Yong Hwan

    2016-01-01

    Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5'-adenosyl)-L-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones.

  15. Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii.

    PubMed

    Weng, Jing-Ke; Akiyama, Takuya; Ralph, John; Chapple, Clint

    2011-07-01

    Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts.

  16. Independent Recruitment of an O-Methyltransferase for Syringyl Lignin Biosynthesis in Selaginella moellendorffii[W

    PubMed Central

    Weng, Jing-Ke; Akiyama, Takuya; Ralph, John; Chapple, Clint

    2011-01-01

    Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts. PMID:21742988

  17. Two distinct O-methyltransferases in aflatoxin biosynthesis.

    PubMed Central

    Yabe, K; Ando, Y; Hashimoto, J; Hamasaki, T

    1989-01-01

    The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium. Images PMID:2802602

  18. Hypnotizability and Catechol-O-Methyltransferase (COMT) polymorphysms in Italians

    PubMed Central

    Presciuttini, Silvano; Gialluisi, Alessandro; Barbuti, Serena; Curcio, Michele; Scatena, Fabrizio; Carli, Giancarlo; Santarcangelo, Enrica L.

    2014-01-01

    Higher brain dopamine content depending on lower activity of Catechol-O-Methyltransferase (COMT) in subjects with high hypnotizability scores (highs) has been considered responsible for their attentional characteristics. However, the results of the previous genetic studies on association between hypnotizability and the COMT single nucleotide polymorphism (SNP) rs4680 (Val158Met) were inconsistent. Here, we used a selective genotyping approach to re-evaluate the association between hypnotizability and COMT in the context of a two-SNP haplotype analysis, considering not only the Val158Met polymorphism, but also the closely located rs4818 SNP. An Italian sample of 53 highs, 49 low hypnotizable subjects (lows), and 57 controls, were genotyped for a segment of 805 bp of the COMT gene, including Val158Met and the closely located rs4818 SNP. Our selective genotyping approach had 97.1% power to detect the previously reported strongest association at the significance level of 5%. We found no evidence of association at the SNP, haplotype, and diplotype levels. Thus, our results challenge the dopamine-based theory of hypnosis and indirectly support recent neuropsychological and neurophysiological findings reporting the lack of any association between hypnotizability and focused attention abilities. PMID:24431998

  19. Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn

    PubMed Central

    Zhao, Yucheng; Wang, Nana; Zeng, Zhixiong; Xu, Sheng; Huang, Chuanlong; Wang, Wei; Liu, Tingting; Luo, Jun; Kong, Lingyi

    2016-01-01

    Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin, and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006) as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA). Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226, and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum. PMID:27252733

  20. Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn.

    PubMed

    Zhao, Yucheng; Wang, Nana; Zeng, Zhixiong; Xu, Sheng; Huang, Chuanlong; Wang, Wei; Liu, Tingting; Luo, Jun; Kong, Lingyi

    2016-01-01

    Coumarins are main active components of Peucedanum praeruptorum Dunn. Among them, methoxylated coumarin compound, such as bergapten, xanthotoxin, and isopimpinellin, has high officinal value and plays an important role in medicinal field. However, major issues associated with the biosynthesis mechanism of coumarins remain unsolved and no corresponding enzyme has been cloned from P. praeruptorum. In this study, a local BLASTN program was conducted to find the candidate genes from P. praeruptorum transcriptome database using the nucleotide sequence of Ammi majus bergaptol O-methyltransferase (AmBMT, GenBank accession No: AY443006) as a template. As a result, a 1335 bp full-length of cDNA sequence which contains an open reading frame of 1080 bp encoding a BMT polypeptide of 359 amino acids was obtained. The recombinant protein was functionally expressed in Escherichia coli and displayed an observed activity to bergaptol. In vitro experiments show that the protein has narrow substrate specificity for bergaptol. Expression profile indicated that the cloned gene had a higher expression level in roots and can be induced by methyl jasmonate (MeJA). Subcellular localization analysis showed that the BMT protein was located in cytoplasm in planta. Homology modeling and docking based site-directed mutagenesis have been employed to investigate the amino acid residues in BMT required for substrate binding and catalysis. Conservative amino acid substitutions at residue H264 affected BMT catalysis, whereas substitutions at residues F171, M175, D226, and L312 affected substrate binding. The systemic study summarized here will enlarge our knowledge on OMTs and provide useful information in investigating the coumarins biosynthesis mechanism in P. praeruptorum.

  1. Synthesis and optimization of N-heterocyclic pyridinones as catechol-O-methyltransferase (COMT) inhibitors.

    PubMed

    Zhao, Zhijian; Harrison, Scott T; Schubert, Jeffrey W; Sanders, John M; Polsky-Fisher, Stacey; Zhang, Nanyan Rena; McLoughlin, Debra; Gibson, Christopher R; Robinson, Ronald G; Sachs, Nancy A; Kandebo, Monika; Yao, Lihang; Smith, Sean M; Hutson, Pete H; Wolkenberg, Scott E; Barrow, James C

    2016-06-15

    A series of N-heterocyclic pyridinone catechol-O-methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and clogP, were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

  2. Regioselectivity of catechol O-methyltransferase confers enhancement of catalytic activity

    NASA Astrophysics Data System (ADS)

    Tsao, Douglas; Liu, Shubin; Dokholyan, Nikolay V.

    2011-04-01

    Catechol O-methyltransferase (COMT) metabolizes catechol moieties by methylating a single hydroxyl group at the meta- or para- hydroxyl position. Hydrophobic amino acids near the active site of COMT influence the regioselectivity of this reaction. Our sequence analysis highlights their importance by showing that these residues are highly conserved throughout evolution. Reaction barriers calculated in the gas phase reveal a lower barrier during methylation at the meta- position, suggesting that the observed meta-regioselectivity of COMT can be attributed to the substrate itself, and that COMT has evolved residues to orient the substrate in a manner that increases the rate of catalysis.

  3. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    SciTech Connect

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. )

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  4. Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same

    DOEpatents

    Liu, Chang-Jun; Cai, Yuanheng

    2017-01-31

    Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.

  5. Structure and Mechanism of the Rebeccamycin Sugar 4'-O-Methyltransferase RebM

    SciTech Connect

    Singh, Shanteri; McCoy, Jason G.; Zhang, Changsheng; Bingman, Craig A.; Phillips, Jr., George N.; Thorson, Jon S.

    2008-12-12

    The 2.65-{angstrom} crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His{sup 140/141} and Asp{sup 166}) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His{sup 140} and Asp{sup 166} to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.

  6. Catechol-O-Methyltransferase "Val[superscript 158]Met" Genotype, Parenting Practices and Adolescent Alcohol Use: Testing the Differential Susceptibility Hypothesis

    ERIC Educational Resources Information Center

    Laucht, Manfred; Blomeyer, Dorothea; Buchmann, Arlette F.; Treutlein, Jens; Schmidt, Martin H.; Esser, Gunter; Jennen-Steinmetz, Christine; Rietschel, Marcella; Zimmermann, Ulrich S.; Banaschewski, Tobias

    2012-01-01

    Background: Recently, first evidence has been reported for a gene-parenting interaction (G x E) with regard to adolescent alcohol use. The present investigation set out to extend this research using the catechol-O-methyltransferase ("COMT") "Val[superscript 158]Met" polymorphism as a genetic susceptibility factor. Moreover, the current study…

  7. An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

    PubMed

    Oliva Chávez, Adela S; Fairman, James W; Felsheim, Roderick F; Nelson, Curtis M; Herron, Michael J; Higgins, LeeAnn; Burkhardt, Nicole Y; Oliver, Jonathan D; Markowski, Todd W; Kurtti, Timothy J; Edwards, Thomas E; Munderloh, Ulrike G

    2015-01-01

    Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.

  8. An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum

    PubMed Central

    Oliva Chávez, Adela S.; Fairman, James W.; Felsheim, Roderick F.; Nelson, Curtis M.; Herron, Michael J.; Higgins, LeeAnn; Burkhardt, Nicole Y.; Oliver, Jonathan D.; Markowski, Todd W.; Kurtti, Timothy J.; Edwards, Thomas E.; Munderloh, Ulrike G.

    2015-01-01

    Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations

  9. Functional characterization of a novel benzylisoquinoline O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L).

    PubMed

    Pienkny, Silke; Brandt, Wolfgang; Schmidt, Jürgen; Kramell, Robert; Ziegler, Jörg

    2009-10-01

    The benzylisoquinoline alkaloids are a highly diverse group of about 2500 compounds which accumulate in a species-specific manner. Despite the numerous compounds which could be identified, the biosynthetic pathways and the participating enzymes or cDNAs could be characterized only for a few selected members, whereas the biosynthesis of the majority of the compounds is still largely unknown. In an attempt to characterize additional biosynthetic steps at the molecular level, integration of alkaloid and transcript profiling across Papaver species was performed. This analysis showed high expression of an expressed sequence tag (EST) of unknown function only in Papaver somniferum varieties. After full-length cloning of the open reading frame and sequence analysis, this EST could be classified as a member of the class II type O-methyltransferase protein family. It was related to O-methyltransferases from benzylisoquinoline biosynthesis, and the amino acid sequence showed 68% identical residues to norcoclaurine 6-O-methyltransferase. However, rather than methylating norcoclaurine, the recombinant protein methylated norreticuline at position seven with a K(m) of 44 mum using S-adenosyl-l-methionine as a cofactor. Of all substrates tested, only norreticuline was converted. Even minor changes in the benzylisoquinoline backbone were not tolerated by the enzyme. Accordingly, the enzyme was named norreticuline 7-O-methyltransferase (N7OMT). This enzyme represents a novel O-methyltransferase in benzylisoquinoline metabolism. Expression analysis showed slightly increased expression of N7OMT in P. somniferum varieties containing papaverine, suggesting its involvement in the partially unknown biosynthesis of this pharmaceutically important compound.

  10. Characterization of three O-methyltransferases involved in noscapine biosynthesis in opium poppy.

    PubMed

    Dang, Thu-Thuy T; Facchini, Peter J

    2012-06-01

    Noscapine is a benzylisoquinoline alkaloid produced in opium poppy (Papaver somniferum) and other members of the Papaveraceae. It has been used as a cough suppressant and more recently was shown to possess anticancer activity. However, the biosynthesis of noscapine in opium poppy has not been established. A proposed pathway leading from (S)-reticuline to noscapine includes (S)-scoulerine, (S)-canadine, and (S)-N-methylcanadine as intermediates. Stem cDNA libraries and latex extracts of eight opium poppy cultivars displaying different alkaloid profiles were subjected to massively parallel pyrosequencing and liquid chromatography-tandem mass spectrometry, respectively. Comparative transcript and metabolite profiling revealed the occurrence of three cDNAs encoding O-methyltransferases designated as SOMT1, SOMT2, and SOMT3 that correlated with the accumulation of noscapine in the eight cultivars. SOMT transcripts were detected in all opium poppy organs but were most abundant in aerial organs, where noscapine primarily accumulates. SOMT2 and SOMT3 showed strict substrate specificity and regiospecificity as 9-O-methyltransferases targeting (S)-scoulerine. In contrast, SOMT1 was able to sequentially 9- and 2-O-methylate (S)-scoulerine, yielding (S)-tetrahydropalmatine. SOMT1 also sequentially 3'- and 7-O-methylated both (S)-norreticuline and (S)-reticuline with relatively high substrate affinity, yielding (S)-tetrahydropapaverine and (S)-laudanosine, respectively. The metabolic functions of SOMT1, SOMT2, and SOMT3 were investigated in planta using virus-induced gene silencing. Reduction of SOMT1 or SOMT2 transcript levels resulted in a significant decrease in noscapine accumulation. Reduced SOMT1 transcript levels also caused a decrease in papaverine accumulation, confirming the selective roles for these enzymes in the biosynthesis of both alkaloids in opium poppy.

  11. Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy1[W

    PubMed Central

    Dang, Thu-Thuy T.; Facchini, Peter J.

    2012-01-01

    Noscapine is a benzylisoquinoline alkaloid produced in opium poppy (Papaver somniferum) and other members of the Papaveraceae. It has been used as a cough suppressant and more recently was shown to possess anticancer activity. However, the biosynthesis of noscapine in opium poppy has not been established. A proposed pathway leading from (S)-reticuline to noscapine includes (S)-scoulerine, (S)-canadine, and (S)-N-methylcanadine as intermediates. Stem cDNA libraries and latex extracts of eight opium poppy cultivars displaying different alkaloid profiles were subjected to massively parallel pyrosequencing and liquid chromatography-tandem mass spectrometry, respectively. Comparative transcript and metabolite profiling revealed the occurrence of three cDNAs encoding O-methyltransferases designated as SOMT1, SOMT2, and SOMT3 that correlated with the accumulation of noscapine in the eight cultivars. SOMT transcripts were detected in all opium poppy organs but were most abundant in aerial organs, where noscapine primarily accumulates. SOMT2 and SOMT3 showed strict substrate specificity and regiospecificity as 9-O-methyltransferases targeting (S)-scoulerine. In contrast, SOMT1 was able to sequentially 9- and 2-O-methylate (S)-scoulerine, yielding (S)-tetrahydropalmatine. SOMT1 also sequentially 3′- and 7-O-methylated both (S)-norreticuline and (S)-reticuline with relatively high substrate affinity, yielding (S)-tetrahydropapaverine and (S)-laudanosine, respectively. The metabolic functions of SOMT1, SOMT2, and SOMT3 were investigated in planta using virus-induced gene silencing. Reduction of SOMT1 or SOMT2 transcript levels resulted in a significant decrease in noscapine accumulation. Reduced SOMT1 transcript levels also caused a decrease in papaverine accumulation, confirming the selective roles for these enzymes in the biosynthesis of both alkaloids in opium poppy. PMID:22535422

  12. Low catechol-O-methyltransferase activity in a Saami population.

    PubMed

    Klemetsdal, B; Straume, B; Giverhaug, T; Aarbakke, J

    1994-01-01

    Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of catechol hormones, neurotransmitters and certain drugs. It is subject to genetic polymorphism and ethnic differences. High red blood cell (RBC) COMT activity has been correlated with a poor response to levodopa treatment in Parkinson's disease. RBC COMT was determined in a Norwegian population (n = 213) of whom 115 were Saami (Laaps). The Saami had 16.5% lower RBC COMT activity compared to a non-Saami population sample from the northern part of Norway (n = 50), 13.9 vs. 16.4 units/ml RBC (U) (P = 0.04). This is the first report of any population with lower RBC COMT activity than a Caucasian population. A wide range of RBC COMT activities was found in the entire population examined (1.3-38.3 U).

  13. Design of electrochemical biosensor systems for the detection of specific DNA sequences in PCR-amplified nucleic acids related to the catechol-O-methyltransferase Val108/158Met polymorphism based on intrinsic guanine signal.

    PubMed

    Ozkan-Ariksoysal, Dilsat; Tezcanli, Burcin; Kosova, Buket; Ozsoz, Mehmet

    2008-02-01

    Psychiatric disorders are common and complex diseases that show polygenic and multifactorial heredity. A single nucleotide polymorphism (Val108/158Met) in the catechol-O-methyl transferase (COMT) gene is related to many psychiatric disorders such as schizophrenia, alcoholism, bipolar disorder, and obsessive-compulsive disorder. Schizophrenia is a complex disorder and a single nucleotide polymorphism (Val108/158Met) at the COMT gene is related to schizophrenia susceptibility. A novel hybridization-based disposable electrochemical DNA biosensor for the detection of a common functional polymorphism in the COMT gene from polymerase chain reaction (PCR) amplicons has been described without using an external label. This developed technology combined with a disposable carbon graphite electrode and differential pulse voltammetry was performed by using short synthetic oligonucleotides and PCR amplicons in length 203 bp to measure the change of guanine oxidation signal obtained at approximately +1.0 V after DNA hybridization between probe and target (synthetic target or denatured PCR samples). COMT-specific oligonucleotides were immobilized onto the carbon surface with a simple adsorption method in two different modes: (a) Guanine-containing targets were attached or (b) inosine-substituted probes were attached onto an electrode. By controlling the surface coverage of the target DNA, the hybridization event between the probes and their synthetic targets or specific PCR products was optimized. The wild-type or polymorphic allele-specific probes/targets were also interacted with an equal amount of noncomplementary and one-base mismatch-containing DNAs in order to measure the sensor selectivity. The decrease or appearance in the intrinsic guanine signal simplified the detection procedure and shortened the assay time because protocol eliminates the label-binding step. The nonspecific binding effects were minimized by using sodium dodecyl sulfate with different washing methods

  14. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

  15. Mapping the conformational space accessible to catechol-O-methyltransferase

    PubMed Central

    Ehler, Andreas; Benz, Jörg; Schlatter, Daniel; Rudolph, Markus G.

    2014-01-01

    Methylation catalysed by catechol-O-methyltransferase (COMT) is the main pathway of catechol neurotransmitter deactivation in the prefrontal cortex. Low levels of this class of neurotransmitters are held to be causative of diseases such as schizophrenia, depression and Parkinson’s disease. Inhibition of COMT may increase neurotransmitter levels, thus offering a route for treatment. Structure-based drug design hitherto seems to be based on the closed enzyme conformation. Here, a set of apo, semi-holo, holo and Michaelis form crystal structures are described that define the conformational space available to COMT and that include likely intermediates along the catalytic pathway. Domain swaps and sizeable loop movements around the active site testify to the flexibility of this enzyme, rendering COMT a difficult drug target. The low affinity of the co-substrate S-adenosylmethionine and the large conformational changes involved during catalysis highlight significant energetic investment to achieve the closed conformation. Since each conformation of COMT is a bona fide target for inhibitors, other states than the closed conformation may be promising to address. Crystallographic data for an alternative avenue of COMT inhibition, i.e. locking of the apo state by an inhibitor, are presented. The set of COMT structures may prove to be useful for the development of novel classes of inhibitors. PMID:25084335

  16. Cloning and expression of a novel catechol-O-methyltransferase in common marmosets.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-02-04

    Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of endogenous catechol amines and estrogens and exogenous catechol-type of drugs. A Parkinson's disease model of common marmoset (Callithrix jacchus) has been widely used in preclinical studies to evaluate inhibitory potential of new drug candidates on marmoset COMT. Despite COMT inhibitors could potentiate the pharmacological action of levodopa on Parkinson's disease in animal models, marmoset COMT cDNA has not yet been identified and characterized. In this study, a cDNA highly homologous to human COMT was cloned from marmoset livers. This cDNA encoded 268 amino acids containing a transmembrane region and critical amino acid residues for catalytic function. The amino acid sequences of marmoset COMT shared high sequence identity (90%) with human COMT. COMT mRNA was expressed in all five tissues tested, including brain, lung, liver, kidney and small intestine, and was more abundant in marmoset liver and kidney. Membrane-bound COMT was immunochemically detected in livers and kidneys, whereas soluble COMT was detected in livers, similar to humans. These results indicated that the molecular characteristics of marmoset COMT were generally similar to the human ortholog.

  17. Cloning and expression of a novel catechol-O-methyltransferase in common marmosets

    PubMed Central

    UEHARA, Shotaro; UNO, Yasuhiro; INOUE, Takashi; SASAKI, Erika; YAMAZAKI, Hiroshi

    2016-01-01

    Catechol-O-methyltransferase (COMT) catalyzes the O-methylation of endogenous catechol amines and estrogens and exogenous catechol-type of drugs. A Parkinson’s disease model of common marmoset (Callithrix jacchus) has been widely used in preclinical studies to evaluate inhibitory potential of new drug candidates on marmoset COMT. Despite COMT inhibitors could potentiate the pharmacological action of levodopa on Parkinson’s disease in animal models, marmoset COMT cDNA has not yet been identified and characterized. In this study, a cDNA highly homologous to human COMT was cloned from marmoset livers. This cDNA encoded 268 amino acids containing a transmembrane region and critical amino acid residues for catalytic function. The amino acid sequences of marmoset COMT shared high sequence identity (90%) with human COMT. COMT mRNA was expressed in all five tissues tested, including brain, lung, liver, kidney and small intestine, and was more abundant in marmoset liver and kidney. Membrane-bound COMT was immunochemically detected in livers and kidneys, whereas soluble COMT was detected in livers, similar to humans. These results indicated that the molecular characteristics of marmoset COMT were generally similar to the human ortholog. PMID:27890888

  18. Analysis of Oxidative Stress Status, Catalase and Catechol-O-Methyltransferase Polymorphisms in Egyptian Vitiligo Patients

    PubMed Central

    Mehaney, Dina A.; Darwish, Hebatallah A.; Hegazy, Rehab A.; Nooh, Mohammed M.; Tawdy, Amira M.; Gawdat, Heba I.; El-Sawalhi, Maha M.

    2014-01-01

    Vitiligo is the most common depigmentation disorder of the skin. Oxidative stress is implicated as one of the probable events involved in vitiligo pathogenesis possibly contributing to melanocyte destruction. Evidence indicates that certain genes including those involved in oxidative stress and melanin synthesis are crucial for development of vitiligo. This study evaluates the oxidative stress status, the role of catalase (CAT) and catechol-O-Methyltransferase (COMT) gene polymorphisms in the etiology of generalized vitiligo in Egyptians. Total antioxidant capacity (TAC) and malondialdehyde (MDA) levels as well as CAT exon 9 T/C and COMT 158 G/A polymorphisms were determined in 89 patients and 90 age and sex-matched controls. Our results showed significantly lower TAC along with higher MDA levels in vitiligo patients compared with controls. Meanwhile, genotype and allele distributions of CAT and COMT polymorphisms in cases were not significantly different from those of controls. Moreover, we found no association between both polymorphisms and vitiligo susceptibility. In conclusion, the enhanced oxidative stress with the lack of association between CAT and COMT polymorphisms and susceptibility to vitiligo in our patients suggest that mutations in other genes related to the oxidative pathway might contribute to the etiology of generalized vitiligo in Egyptian population. PMID:24915010

  19. Catechol-o-methyltransferase genotype and childhood trauma may interact to impact schizotypal personality traits.

    PubMed

    Savitz, Jonathan; van der Merwe, Lize; Newman, Timothy K; Stein, Dan J; Ramesar, Raj

    2010-05-01

    We attempt to identify gene by childhood abuse interactions which predispose to the development of schizotypal traits in a familial bipolar disorder (BD) sample. Self-report measures of schizotypal personality traits (Schizotypal Personality Scale) and childhood maltreatment (Childhood Trauma Questionnaire) were administered to 222 participants from 44 families with BD. Variants of catechol-o-methyltransferase (COMT) and four other dopamine pathway-related genes: DRD4, DRD2,MAOA, and SLC6A3, were typed. BD type I (BD I) subjects scored significantly higher than their unaffected relatives on the Schizotypal Personality Scale. The val allele of the Val158 Met polymorphism of the COMT gene was associated with increased schizotypal personality trait scores in individuals exposed to higher levels of self-reported childhood trauma (p < 0.05). There was no direct effect of the val158met polymorphism on schizotypal personality traits. Further, no passive correlation between COMT genotype and childhood trauma was found. We raise the possibility that genetically-driven variation in COMT may interact with childhood trauma to contribute to the risk of developing schizotypal personality traits.

  20. Water Deficits Affect Caffeate O-Methyltransferase, Lignification, and Related Enzymes in Maize Leaves. A Proteomic Investigation1[w

    PubMed Central

    Vincent, Delphine; Lapierre, Catherine; Pollet, Brigitte; Cornic, Gabriel; Negroni, Luc; Zivy, Michel

    2005-01-01

    Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-l-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants. PMID:15728345

  1. The Flexible Mind Is Associated with the Catechol-O-Methyltransferase (COMT) Val[superscript 158]Met Polymorphism: Evidence for a Role of Dopamine in the Control of Task-Switching

    ERIC Educational Resources Information Center

    Colzato, Lorenza S.; Waszak, Florian; Nieuwenhuis, Sander; Posthuma, Danielle; Hommel, Bernhard

    2010-01-01

    Genetic variability related to the catechol-O-methyltransferase (COMT) gene Val[superscript 128]Met polymorphism) has received increasing attention as a possible modulator of cognitive control functions. Recent evidence suggests that the Val[superscript 128]Met genotype may differentially affect cognitive stability and flexibility, in such a way…

  2. Genetic variation in catechol-O-methyltransferase modifies effects of clonidine treatment in chronic fatigue syndrome

    PubMed Central

    Hall, Kathryn T.; Kossowsky, Joe; Oberlander, Tim F.; Kaptchuk, Ted J.; Saul, J. Philip; Wyller, Vegard Bruun; Fagermoen, Even; Sulheim, Dag; Gjerstad, Johannes; Winger, Anette; Mukamal, Kenneth J.

    2016-01-01

    Clonidine, an α2-adrenergic receptor agonist, decreases circulating norepinephrine and epinephrine, attenuating sympathetic activity. Although catechol-O-methyltransferase (COMT) metabolizes catecholamines, main effectors of sympathetic function, COMT genetic variation effects on clonidine treatment are unknown. Chronic fatigue syndrome (CFS) is hypothesized to result in part from dysregulated sympathetic function. A candidate gene analysis of COMT rs4680 effects on clinical outcomes in the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial (NorCAPITAL), a randomized double-blinded clonidine versus placebo trial, was conducted (N=104). Patients homozygous for rs4680 high-activity allele randomized to clonidine took 2,500 fewer steps compared to placebo (pinteraction=0.04). There were no differences between clonidine and placebo amongst patients with COMT low-activity alleles. Similar gene-drug interactions were observed for sleep (pint=0.003) and quality of life (pint=0.018). Detrimental effects of clonidine in the subset of CFS patients homozygous for COMT high-activity allele warrant investigation of potential clonidine-COMT interaction effects in other conditions. PMID:27457818

  3. Genetic variation in catechol-O-methyltransferase modifies effects of clonidine treatment in chronic fatigue syndrome.

    PubMed

    Hall, K T; Kossowsky, J; Oberlander, T F; Kaptchuk, T J; Saul, J P; Wyller, V B; Fagermoen, E; Sulheim, D; Gjerstad, J; Winger, A; Mukamal, K J

    2016-10-01

    Clonidine, an α2-adrenergic receptor agonist, decreases circulating norepinephrine and epinephrine, attenuating sympathetic activity. Although catechol-O-methyltransferase (COMT) metabolizes catecholamines, main effectors of sympathetic function, COMT genetic variation effects on clonidine treatment are unknown. Chronic fatigue syndrome (CFS) is hypothesized to result in part from dysregulated sympathetic function. A candidate gene analysis of COMT rs4680 effects on clinical outcomes in the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial (NorCAPITAL), a randomized double-blinded clonidine versus placebo trial, was conducted (N=104). Patients homozygous for rs4680 high-activity allele randomized to clonidine took 2500 fewer steps compared with placebo (Pinteraction=0.04). There were no differences between clonidine and placebo among patients with COMT low-activity alleles. Similar gene-drug interactions were observed for sleep (Pinteraction=0.003) and quality of life (Pinteraction=0.018). Detrimental effects of clonidine in the subset of CFS patients homozygous for COMT high-activity allele warrant investigation of potential clonidine-COMT interaction effects in other conditions.

  4. Role of Petal-Specific Orcinol O-Methyltransferases in the Evolution of Rose Scent1

    PubMed Central

    Scalliet, Gabriel; Lionnet, Claire; Le Bechec, Mickaël; Dutron, Laurence; Magnard, Jean-Louis; Baudino, Sylvie; Bergougnoux, Véronique; Jullien, Frédéric; Chambrier, Pierre; Vergne, Philippe; Dumas, Christian; Cock, J. Mark; Hugueney, Philippe

    2006-01-01

    Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominanty in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses. PMID:16361520

  5. Catechol-O-methyltransferase association with hemoglobin A1c

    PubMed Central

    Hall, Kathryn T.; Jablonski, Kathleen A.; Chen, Ling; Harden, Maegan; Tolkin, Benjamin R.; Kaptchuk, Ted J.; Bray, George A.; Ridker, Paul M.; Florez, Jose C.; Chasman, Daniel I.

    2016-01-01

    Aims Catecholamines have metabolic effects on blood pressure, insulin sensitivity and blood glucose. Genetic variation in catechol-O-methyltransferase (COMT), an enzyme that degrades catecholamines, is associated with cardiometabolic risk factors and incident cardiovascular disease (CVD). Here we examined COMT effects on glycemic function and type 2 diabetes. Methods We tested whether COMT polymorphisms were associated with baseline HbA1c in the Women’s Genome Health Study (WGHS), and Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC), and with susceptibility to type 2 diabetes in WGHS, DIAbetes Genetics Replication And Meta-analysis consortium (DIAGRAM), and the Diabetes Prevention Program (DPP). Given evidence that COMT modifies some drug responses, we examined association with type 2 diabetes and randomized metformin and aspirin treatment. Results COMT rs4680 high-activity G-allele was associated with lower HbA1c in WGHS (β = −0.032% [0.012], p = 0.008) and borderline significant in MAGIC (β = −0.006% [0.003], p = 0.07). Combined COMT per val allele effects on type 2 diabetes were significant (OR = 0.98 [0.96–0.998], p = 0.03) in fixed-effects analyses across WGHS, DIAGRAM, and DPP. Similar results were obtained for 2 other COMT SNPs rs4818 and rs4633. In the DPP, the rs4680 val allele was borderline associated with lower diabetes incidence among participants randomized to metformin (HR = 0.81 [0.65–1.00], p = 0.05). Conclusions COMT rs4680 high-activity G-allele was associated with lower HbA1c and modest protection from type 2 diabetes. The directionality of COMT associations was concordant with those previously observed for cardiometabolic risk factors and CVD. PMID:27282867

  6. Essential Role of Caffeoyl Coenzyme A O-Methyltransferase in Lignin Biosynthesis in Woody Poplar Plants

    PubMed Central

    Zhong, Ruiqin; Morrison, W. Herbert; Himmelsbach, David S.; Poole, Farris L.; Ye, Zheng-Hua

    2000-01-01

    Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) has recently been shown to participate in lignin biosynthesis in herbacious tobacco plants. Here, we demonstrate that CCoAOMT is essential in lignin biosynthesis in woody poplar (Populus tremula × Populus alba) plants. In poplar stems, CCoAOMT was found to be expressed in all lignifying cells including vessel elements and fibers as well as in xylem ray parenchyma cells. Repression of CCoAOMT expression by the antisense approach in transgenic poplar plants caused a significant decrease in total lignin content as detected by both Klason lignin assay and Fourier-transform infrared spectroscopy. The reduction in lignin content was the result of a decrease in both guaiacyl and syringyl lignins as determined by in-source pyrolysis mass spectrometry. Fourier-transform infrared spectroscopy indicated that the reduction in lignin content resulted in a less condensed and less cross-linked lignin structure in wood. Repression of CCoAOMT expression also led to coloration of wood and an elevation of wall-bound p-hydroxybenzoic acid. Taken together, these results indicate that CCoAOMT plays a dominant role in the methylation of the 3-hydroxyl group of caffeoyl CoA, and the CCoAOMT-mediated methylation reaction is essential to channel substrates for 5-methoxylation of hydroxycinnamates. They also suggest that antisense repression of CCoAOMT is an efficient means for genetic engineering of trees with low lignin content. PMID:11027707

  7. Catechol-O-methyltransferase promoter hypomethylation is associated with the risk of coronary heart disease.

    PubMed

    Zhong, Jinyan; Chen, Xiaoying; Wu, Nan; Shen, Caijie; Cui, Hanbin; Du, Weiping; Zhang, Zhaoxia; Feng, Mingjun; Liu, Junsong; Lin, Shaoyi; Zhang, Lulu; Wang, Jian; Chen, Xiaomin; Duan, Shiwei

    2016-11-01

    Catechol-O-methyltransferase (COMT) gene variation is known to be associated with the risk of acute coronary events. The purpose of the present study was to investigate the contribution of COMT promoter methylation towards the risk of coronary heart disease (CHD). COMT methylation was evaluated in 48 CHD cases and 48 well-matched non-CHD controls using bisulfite pyrosequencing technology. The results demonstrated that CHD cases had a significantly lower level of methylation at COMT CpG3 sites compared with the controls (33.77±5.71 vs. 36.42±5.00%; P=0.018). Further analysis, according to gender, showed that CpG3 methylation was associated with CHD in males (P=0.038) but not in females (P=0.253), suggesting that there is a gender disparity in the association between COMT methylation and CHD. In conclusion, it was determined that COMT CpG3 hypomethylation is associated with an increased risk of CHD in males.

  8. Catechol-O-methyltransferase promoter hypomethylation is associated with the risk of coronary heart disease

    PubMed Central

    Zhong, Jinyan; Chen, Xiaoying; Wu, Nan; Shen, Caijie; Cui, Hanbin; Du, Weiping; Zhang, Zhaoxia; Feng, Mingjun; Liu, Junsong; Lin, Shaoyi; Zhang, Lulu; Wang, Jian; Chen, Xiaomin; Duan, Shiwei

    2016-01-01

    Catechol-O-methyltransferase (COMT) gene variation is known to be associated with the risk of acute coronary events. The purpose of the present study was to investigate the contribution of COMT promoter methylation towards the risk of coronary heart disease (CHD). COMT methylation was evaluated in 48 CHD cases and 48 well-matched non-CHD controls using bisulfite pyrosequencing technology. The results demonstrated that CHD cases had a significantly lower level of methylation at COMT CpG3 sites compared with the controls (33.77±5.71 vs. 36.42±5.00%; P=0.018). Further analysis, according to gender, showed that CpG3 methylation was associated with CHD in males (P=0.038) but not in females (P=0.253), suggesting that there is a gender disparity in the association between COMT methylation and CHD. In conclusion, it was determined that COMT CpG3 hypomethylation is associated with an increased risk of CHD in males. PMID:27882177

  9. Characterization and structure of DhpI, a phosphonate O-methyltransferase involved in dehydrophos biosynthesis

    SciTech Connect

    Lee, Jin-Hee; Bae, Brian; Kuemin, Michael; Circello, Benjamin T.; Metcalf, William W.; Nair, Satish K.; van der Donk, Wilfred A.

    2012-03-15

    Phosphonate natural products possess a range of biological activities as a consequence of their ability to mimic phosphate esters or tetrahedral intermediates formed in enzymatic reactions involved in carboxyl group metabolism. The dianionic form of these compounds at pH 7 poses a drawback with respect to their ability to mimic carboxylates and tetrahedral intermediates. Microorganisms producing phosphonates have evolved two solutions to overcome this hurdle: biosynthesis of monoanionic phosphinates containing two P-C bonds or esterification of the phosphonate group. The latter solution was first discovered for the antibiotic dehydrophos that contains a methyl ester of a phosphonodehydroalanine group. We report here the expression, purification, substrate scope, and structure of the O-methyltransferase from the dehydrophos biosynthetic gene cluster. The enzyme utilizes S-adenosylmethionine to methylate a variety of phosphonates including 1-hydroxyethylphosphonate, 1,2-dihydroxyethylphosphonate, and acetyl-1-aminoethylphosphonate. Kinetic analysis showed that the best substrates are tripeptides containing as C-terminal residue a phosphonate analog of alanine suggesting the enzyme acts late in the biosynthesis of dehydrophos. These conclusions are corroborated by the X-ray structure that reveals an active site that can accommodate a tripeptide substrate. Furthermore, the structural studies demonstrate a conformational change brought about by substrate or product binding. Interestingly, the enzyme has low substrate specificity and was used to methylate the clinical antibiotic fosfomycin and the antimalaria clinical candidate fosmidomycin, showing its promise for applications in bioengineering.

  10. Catechol-O-Methyltransferase Val158Met Polymorphism Is Associated with Somatosensory Amplification and Nocebo Responses

    PubMed Central

    Benson, Sven; Engler, Harald; Engler, Andrea; Hinney, Anke; Rief, Winfried; Witzke, Oliver; Schedlowski, Manfred

    2014-01-01

    A large number of unwanted adverse events and symptoms reported by patients in clinical trials are not caused by the drug provided, since most of adverse events also occur in corresponding placebo groups. These nocebo effects also play a major role in drug discontinuation in clinical practice, negatively affecting treatment efficacy as well as patient adherence and compliance. Experimental and clinical data document a large interindividual variability in nocebo responses, however, data on psychological, biological or genetic predictors of nocebo responses are lacking. Thus, with an established paradigm of behaviorally conditioned immunosuppressive effects we analyzed possible genetic predictors for nocebo responses. We focused on the genetic polymorphisms in the catechol-O-methyltransferase (COMT) gene (Val158Met) and analyzed drug specific and general side effects before and after immunosuppressive medication and subsequent placebo intake in 62 healthy male subjects. Significantly more drug-specific as well as general side effects were reported from homozygous carriers of the Val158 variant during medication as well as placebo treatment compared to the other genotype groups. Val158/Val158 carriers also had significantly higher scores in the somatosensory amplification scale (SSAS) and the BMQ (beliefs about medicine questionnaire). Together these data demonstrate potential genetic and psychological variables predicting nocebo responses after drug and placebo intake, which might be utilized to minimize nocebo effects in clinical trials and medical practice. PMID:25222607

  11. An O-methyltransferase modifies accumulation of methylated anthocyanins in seedlings of tomato.

    PubMed

    Gomez Roldan, Maria Victoria; Outchkourov, Nikolay; van Houwelingen, Adèle; Lammers, Michiel; Romero de la Fuente, Irene; Ziklo, Noa; Aharoni, Asaph; Hall, Robert D; Beekwilder, Jules

    2014-11-01

    Anthocyanins contribute to the appearance of fruit by conferring to them a red, blue or purple colour. In a food context, they have also been suggested to promote consumer health. In purple tomato tissues, such as hypocotyls, stems and purple fruits, various anthocyanins accumulate. These molecules have characteristic patterns of modification, including hydroxylations, methylations, glycosylations and acylations. The genetic basis for many of these modifications has not been fully elucidated, and nor has their role in the functioning of anthocyanins. In this paper, AnthOMT, an O-methyltransferase (OMT) mediating the methylation of anthocyanins, has been identified and functionally characterized using a combined metabolomics and transcriptomics approach. Gene candidates were selected from the draft tomato genome, and their expression was subsequently monitored in a tomato seedling system comprising three tissues and involving several time points. In addition, we also followed gene expression in wild-type red and purple transgenic tomato fruits expressing Rosea1 and Delila transcription factors. Of the 57 candidates identified, only a single OMT gene showed patterns strongly correlating with both accumulation of anthocyanins and expression of anthocyanin biosynthesis genes. This candidate (AnthOMT) was compared to a closely related caffeoyl CoA OMT by recombinant expression in Escherichia coli, and then tested for substrate specificity. AnthOMT showed a strong affinity for glycosylated anthocyanins, while other flavonoid glycosides and aglycones were much less preferred. Gene silencing experiments with AnthOMT resulted in reduced levels of the predominant methylated anthocyanins. This confirms the role of this enzyme in the diversification of tomato anthocyanins.

  12. Association Between the Catechol-O-Methyltransferase Val158Met Polymorphism and Self-Perceived Social Acceptance in Adolescent Girls

    PubMed Central

    Dearing, Karen F.; Joormann, Jutta; Gotlib, Ian H.

    2009-01-01

    Abstract Low perceived social acceptance is a significant risk factor for emotional difficulties in children. No studies, however, have examined genetic factors that may underlie individual differences in perceived social acceptance. In the present study we examined the relation between polymorphisms on the catechol-O-methyltransferase (COMT) Val158Met and serotonin transporter promoter (5-HTTLPR) genes and perceived social acceptance in 103 adolescent girls. Only the COMT polymorphism was related to perceived social acceptance: Val-allele carriers reported greater perceived social acceptance than did homozygous Met-allele carriers. In a subsample of these participants, homozygous Val-allele carriers reported greater maintenance of positive emotions during stress. This, in turn, predicted social acceptance, suggesting that COMT exerts its effects on social functioning through emotion regulation. These data are the first to show an association between COMT and social functioning in children. Future research might profitably examine emotion regulation as a mediator between COMT and social acceptance. PMID:19702491

  13. Association of Catechol-O-Methyltransferase (COMT) Polymorphism and Academic Achievement in a Chinese Cohort

    ERIC Educational Resources Information Center

    Yeh, Ting-Kuang; Chang, Chun-Yen; Hu, Chung-Yi; Yeh, Ting-Chi; Lin, Ming-Yeh

    2009-01-01

    Catechol-O-methyltransferase (COMT) is a methylation enzyme that catalyzes the degradation pathway and inactivation of dopamine. It is accepted widely as being involved in the modulation of dopaminergic physiology and prefrontal cortex (PFC) function. The COMT Val158Met polymorphism is associated with variation in COMT activity. COMT 158Met allele…

  14. Catechol-O-methyltransferase: a method for autoradiographic visualization of isozymes in cellogel

    SciTech Connect

    Brahe, C.; Crosti, N.; Meera Khan, P.; Serra, A.

    1984-02-01

    An electrophoretic procedure for separating the molecular forms of catechol-O-methyltransferase in cellulose acetate gel is described; the zones of enzyme activity were revealed by autoradiography. The electrophoretic patterns of the enzyme in several tissues and cell lines derived from four different species are presented.

  15. Molecular docking studies for the identification of novel melatoninergic inhibitors for acetylserotonin-O-methyltransferase using different docking routines

    PubMed Central

    2013-01-01

    Background N-Acetylserotonin O-methyltransferase (ASMT) is an enzyme which by converting nor-melatonin to melatonin catalyzes the final reaction in melatonin biosynthesis in tryptophan metabolism pathway. High Expression of ASMT gene is evident in PPTs. The presence of abnormally high levels of ASMT in pineal gland could serve as an indication of the existence of pineal parenchymal tumors (PPTs) in the brain (J Neuropathol Exp Neurol 65: 675–684, 2006). Different levels of melatonin are used as a trait marker for prescribing the mood disorders e.g. Seasonal affective disorder, bipolar disorder, or major depressive disorder. In addition, melatonin levels can also be used to calculate the severity of a patient’s illness at a given point in time. Methods Seventy three melatoninergic inhibitors were docked with acetylserotonin-O-methyltransferase in order to identify the potent inhibitor against the enzyme. The chemical nature of the protein and ligands greatly influence the performance of docking routines. Keeping this fact in view, critical evaluation of the performance of four different commonly used docking routines: AutoDock/Vina, GOLD, FlexX and FRED were performed. An evaluation criterion was based on the binding affinities/docking scores and experimental bioactivities. Results and conclusion Results indicated that both hydrogen bonding and hydrophobic interactions contributed significantly for its ligand binding and the compound selected as potent inhibitor is having minimum binding affinity, maximum GoldScore and minimum FlexX energy. The correlation value of r2 = 0. 66 may be useful in the selection of correct docked complexes based on the energy without having prior knowledge of the active site. This may lead to further understanding of structures, their reliability and Biomolecular activity especially in connection with bipolar disorders. PMID:24156411

  16. Catechol-O-Methyltransferase and 3,4-(±)-Methylenedioxymethamphetamine Toxicity

    PubMed Central

    Herndon, Joseph M.; Cholanians, Aram B.; Lizarraga, Lucina E.; Lau, Serrine S.; Monks, Terrence J.

    2014-01-01

    Metabolism of 3,4-(±)-methylenedioxymethamphetamine (MDMA) is necessary to elicit its neurotoxic effects. Perturbations in phase I and phase II hepatic enzymes can alter the neurotoxic profile of systemically administered MDMA. In particular, catechol-O-methyltransferase (COMT) plays a critical role in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites. Thus, cytochrome P450 mediated demethylenation of MDMA, or its N-demethylated metabolite, 3,4-(±)-methylenedioxyamphetamine, give rise to the catechols, N-methyl-α-methyldopamine and α-methyldopamine, respectively. Methylation of these catechols by COMT limits their oxidation and conjugation to glutathione, a process that ultimately gives rise to neurotoxic metabolites. We therefore determined the effects of modulating COMT, a critical enzyme involved in determining the fraction of MDMA that is converted to potentially neurotoxic metabolites, on MDMA-induced toxicity. Pharmacological inhibition of COMT in the rat potentiated MDMA-induced serotonin deficits and exacerbated the acute MDMA-induced hyperthermic response. Using a genetic mouse model of COMT deficiency, in which mice lack a functional COMT gene, such mice displayed greater reductions in dopamine concentrations relative to their wild-type (WT) counterparts. Neither WT nor COMT deficient mice were susceptible to MDMA-induced decreases in serotonin concentrations. Interestingly, mice devoid of COMT were far more susceptible to the acute hyperthermic effects of MDMA, exhibiting greater increases in body temperature that ultimately resulted in death. Our findings support the view that COMT plays a pivotal role in determining the toxic response to MDMA. PMID:24591155

  17. A Novel Catechol-O-Methyltransferase Variant Associated with Human Disc Degeneration

    PubMed Central

    Gruber, Helen E.; Sha, Wei; Brouwer, Cory R.; Steuerwald, Nury; Hoelscher, Gretchen L.; Hanley, Edward N. Jr.

    2014-01-01

    Background: Disc degeneration and its associated low back pain are a major health care concern causing disability with a prominent role in this country's medical, social and economic structure. Low back pain is devastating and influences the quality of life for millions. Low back pain lifetime prevalence approximates 80% with an estimated direct cost burden of $86 billion per year. Back pain patients incur higher costs, greater health care utilization, and greater work loss than patients without back pain. Methods: Research was performed following approval of our Institutional Review Board. DNA was isolated, processed and amplified using routine techniques. Amplified DNA was hybridized to Affymetrix Genome-Wide Human SNP Arrays. Quality control and genotyping analysis were performed using Affymetrix Genotyping Console. The Birdseed v2 algorithm was used for genotyping analysis. 2589 SNPs were selected a priori to enter statistical analysis using lotistic regression in SAS. Results: Our objective was to search for novel single nucleotide polymorphisms (SNPs) associated with disc degeneration. Four SNPs were found to have a significant relationship to disc degeneration; three are novel. Rs165656, a new SNP found to be associated with disc degeneration, was in catechol-O-methyltransferase (COMT), a gene with well-recognized pain involvement, especially in female subjects (p=0.01). Analysis confirmed the previously association between COMT SNP rs4633 and disc degeneration. We also report two novel disc degeneration-related SNPs (rs2095019 and rs470859) located in intergenic regions upstream to thrombospondin 2. Conclusions: Findings contribute to the challenging field of disc degeneration and pain, and are important in light of the high clinical relevance of low back pain and the need for improved understanding of its fundamental basis. PMID:24904231

  18. Differential regulation of catechol-O-methyltransferase expression in a mouse model of aggression

    PubMed Central

    Che, Shaoli; Hashim, Audrey; Zavadil, Jiri; Cancro, Robert; Lee, Sang H.; Petkova, Eva; Sershen, Henry W.; Volavka, Jan

    2011-01-01

    This study was designed to understand molecular and cellular mechanisms underlying aggressive behaviors in mice exposed to repeated interactions in their homecage with conspecifics. A resident–intruder procedure was employed whereby two males were allowed to interact for 10 min trials, and aggressive and/or submissive behaviors (e.g., degree of attacking, biting, chasing, grooming, rearing, or upright posture) were assessed. Following 10 days of behavioral trials, brains were removed and dissected into specific regions including the cerebellum, frontal cortex, hippocampus, midbrain, pons, and striatum. Gene expression analysis was performed using real-time quantitative polymerase-chain reaction (qPCR) for catechol-O-methyltransferase (COMT) and tyrosine hydroxylase (TH). Compared to naive control mice, significant up regulation of COMT expression of residents was observed in the cerebellum, frontal cortex, hippocampus, midbrain, and striatum; in all of these brain regions the COMT expression of residents was also significantly higher than that of intruders. The intruders also had a significant down regulation (compared to naive control mice) within the hippocampus, indicating a selective decrease in COMT expression in the hippocampus of submissive subjects. Immunoblot analysis confirmed COMT up regulation in the midbrain and hippocampus of residents and down regulation in intruders. qPCR analysis of TH expression indicated significant up regulation in the midbrain of residents and concomitant down regulation in intruders. These findings implicate regionally- and behaviorally-specific regulation of COMT and TH expression in aggressive and submissive behaviors. Additional molecular and cellular characterization of COMT, TH, and other potential targets is warranted within this animal model of aggression. PMID:21512897

  19. Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex

    SciTech Connect

    Rodrigues, M. L.; Bonifácio, M. J.; Soares-da-Silva, P.; Carrondo, M. A.; Archer, M.

    2005-01-01

    Catechol-O-methyltransferase has been co-crystallized with a novel inhibitor, which has potential therapeutic application in the Parkinson’s disease therapy. Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson’s disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°.

  20. Cell-Specific and Conditional Expression of Caffeoyl-Coenzyme A-3-O-Methyltransferase in Poplar1

    PubMed Central

    Chen, Cuiying; Meyermans, Hugo; Burggraeve, Bart; De Rycke, Riet M.; Inoue, Kentaro; De Vleesschauwer, Vera; Steenackers, Marijke; Van Montagu, Marc C.; Engler, Gilbert J.; Boerjan, Wout A.

    2000-01-01

    Caffeoyl coenzyme A-3-O-methyltransferase (CCoAOMT) plays an important role in lignin biosynthesis and is encoded by two genes in poplar (Populus trichocarpa). Here, we describe the expression pattern conferred by the two CCoAOMT promoters when fused to the gus-coding sequence in transgenic poplar (Populus tremula × Populus alba). Both genes were expressed similarly in xylem and differentially in phloem. In xylem, expression was preferentially observed in vessels and contact rays, whereas expression was barely detectable in storage rays and fibers, suggesting different routes to monolignol biosynthesis in the different xylem types. Furthermore, after wounding, fungal infection, and bending, the expression of both genes was induced concomitantly with de novo lignin deposition. Importantly, upon bending and leaning of the stem, the cell-specific expression pattern was lost, and both genes were expressed in all cell types of the xylem. CCoAOMT promoter activity correlated well with the presence of the CCoAOMT protein, as shown by immunolocalization. These expression data may explain, at least in part, the heterogeneity in lignin composition that is observed between cell types and upon different environmental conditions. PMID:10889235

  1. Complex multilocus effects of catechol-O-methyltransferase haplotypes predict pain and pain interference 6 weeks after motor vehicle collision

    PubMed Central

    Bortsov, Andrey V.; Diatchenko, Luda; McLean, Samuel A.

    2013-01-01

    Catechol-O-methyltransferase, encoded by COMT gene, is the primary enzyme that metabolizes catecholamines. COMT haplotypes have been associated with vulnerability to persistent non-traumatic pain. In this prospective observational study, we investigated the influence of COMT on persistent pain and pain interference with life functions after motor vehicle collision (MVC) in 859 European American adults for whom overall pain (0–10 numeric rating scale) and pain interference (Brief Pain Inventory) were assessed at week 6 after MVC. Ten single nucleotide polymorphisms (SNPs) spanning the COMT gene were successfully genotyped, nine were present in three haploblocks: block 1 (rs2020917, rs737865, rs1544325), block 2 (rs4633, rs4818, rs4680, rs165774) and block 3 (rs174697, rs165599). After adjustment for multiple comparisons, haplotype TCG from block 1 predicted decreased pain interference (p =.004). The pain-protective effect of the low pain sensitivity (LPS, CGGG) haplotype from block 2 was only observed if at least one TCG haplotype was present in block 1 (haplotype × haplotype interaction p=.002 and <.0001 for pain and pain interference, respectively). Haplotype AG from block 3 was associated with pain and interference in males only (sex × haplotype interaction p=.005 and .0005, respectively). These results suggest that genetic variants in the distal promoter are important contributors to the development of persistent pain after MVC, directly and via the interaction with haplotypes in the coding region of the gene. PMID:23963787

  2. Perceived stress during pregnancy and the catechol-O-methyltransferase (COMT) rs165599 polymorphism impacts on childhood IQ.

    PubMed

    Lamb, Yvette N; Thompson, John M D; Murphy, Rinki; Wall, Clare; Kirk, Ian J; Morgan, Angharad R; Ferguson, Lynnette R; Mitchell, Edwin A; Waldie, Karen E

    2014-09-01

    Maternal stress during pregnancy has been associated with a range of adverse outcomes in offspring and the catechol-O-methyltransferase (COMT) gene has been linked to differential susceptibility to the consequences of antenatal stress. This study examined two functional polymorphisms of the COMT gene (rs4680 and rs165599) in relation to maternal perceived stress and childhood cognitive performance. Data from the longitudinal Auckland Birthweight Collaborative (ABC) study was used. Maternal perceived stress over the prior month was measured at birth, 3.5 and 7years. Full-Scale IQ (FSIQ) was measured at ages 7 and 11. At age 11, a total of 546 DNA samples were collected from the child participants. Data were subjected to a series of split-plot ANCOVAs with birthweight for gestational age and maternal school leaving age as covariates. There were direct effects of maternal stress during the last month of pregnancy on offspring FSIQ at ages 7 and 11years. A significant interaction revealed that children exposed to high maternal antenatal stress had significantly lower FSIQ scores at both 7 and 11years of age than those exposed to low stress, only when they were carriers of the rs165599 G allele. At each age, this difference was of approximately 5 IQ points. The G allele of the rs165599 polymorphism may confer genetic susceptibility to negative cognitive outcomes arising from exposure to antenatal stress. This finding highlights the need to consider gene-environment interactions when investigating the outcomes of antenatal stress exposure.

  3. Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

    PubMed

    Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

    2010-09-20

    KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer.

  4. Structural and functional characterization of CalS11, a TDP-rhamnose 3′-O-methyltransferase involved in calicheamicin biosynthesis

    PubMed Central

    Singh, Shanteri; Chang, Aram; Helmich, Kate E.; Bingman, Craig A.; Wrobel, Russel L.; Beebe, Emily T.; Makino, Shin-Ichi; Aceti, David J.; Dyer, Kevin; Hura, Greg L.; Sunkara, Manjula; Morris, Andrew J.; Phillips, George N.; Thorson, Jon S.

    2013-01-01

    Sugar methyltransferases (MTs) are an important class of tailoring enzymes which catalyze the transfer of a methyl group from S-adenosyl-L-methionine to sugar-based N-, C- and O- nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using 1H-13C-gHSQC with isotopically-labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3′-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal and general acid/base-dependent O-methyltransferase and, as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering. PMID:23662776

  5. Structural and functional characterization of CalS11, a TDP-rhamnose 3'-O-methyltransferase involved in calicheamicin biosynthesis.

    PubMed

    Singh, Shanteri; Chang, Aram; Helmich, Kate E; Bingman, Craig A; Wrobel, Russell L; Beebe, Emily T; Makino, Shin-Ichi; Aceti, David J; Dyer, Kevin; Hura, Greg L; Sunkara, Manjula; Morris, Andrew J; Phillips, George N; Thorson, Jon S

    2013-07-19

    Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.

  6. Interactions among catechol-O-methyltransferase genotype, parenting, and sex predict children’s internalizing symptoms and inhibitory control: Evidence for differential susceptibility

    PubMed Central

    SULIK, MICHAEL J.; EISENBERG, NANCY; SPINRAD, TRACY L.; LEMERY-CHALFANT, KATHRYN; SWANN, GREGORY; SILVA, KASSONDRA M.; REISER, MARK; STOVER, DARYN A.; VERRELLI, BRIAN C.

    2015-01-01

    We used sex, observed parenting quality at 18 months, and three variants of the catechol-O-methyltransferase gene (Val158Met [rs4680], intron1 [rs737865], and 3′-untranslated region [rs165599]) to predict mothers’ reports of inhibitory and attentional control (assessed at 42, 54, 72, and 84 months) and internalizing symptoms (assessed at 24, 30, 42, 48, and 54 months) in a sample of 146 children (79 male). Although the pattern for all three variants was very similar, Val158Met explained more variance in both outcomes than did intron1, the 3′-untranslated region, or a haplotype that combined all three catechol-O-methyltransferase variants. In separate models, there were significant three-way interactions among each of the variants, parenting, and sex, predicting the intercepts of inhibitory control and internalizing symptoms. Results suggested that Val158Met indexes plasticity, although this effect was moderated by sex. Parenting was positively associated with inhibitory control for methionine–methionine boys and for valine–valine/valine–methionine girls, and was negatively associated with internalizing symptoms for methionine–methionine boys. Using the “regions of significance” technique, genetic differences in inhibitory control were found for children exposed to high-quality parenting, whereas genetic differences in internalizing were found for children exposed to low-quality parenting. These findings provide evidence in support of testing for differential susceptibility across multiple outcomes. PMID:25159270

  7. Catechol-O-methyltransferase polymorphisms do not play a significant role in pain perception in male Chinese Han population.

    PubMed

    Xiang, Xiaohui; Jiang, Yin; Ni, Yanjun; Fan, Min; Shen, Fang; Wang, Xuewei; Han, Jisheng; Cui, Cailian

    2012-03-01

    Polymorphisms in the human catechol-O-methyltransferase (COMT) gene have been widely studied for their role in pain and analgesia. In this study, sensitivity to potassium iontophoresis, visual analog scale measurements for fixed twofold pain threshold stimulation and pain threshold changes induced by transcutaneous electrical acupoint stimulation (TEAS) were assessed in a population of healthy Chinese males. These results were correlated with the alleles of six single nucleotide polymorphisms (SNP) or diplotypes of common haplotypes designated as low pain sensitive, average pain sensitive, and high pain sensitive in the COMT gene of these subjects. Our results reveal that the alleles of each SNP are not significantly correlated with pain perception except for the rs4633 allele in the 2 Hz TEAS session (P < 0.05). In addition, the six diplotypes of COMT haplotypes, which cover 92.5% of the Chinese population, are also not correlated with pain perception. Moreover, there were no significant differences in pain threshold changes induced by 2 and 100 Hz TEAS among the diplotypes of each SNP or the various haplotypes. These results suggest that COMT activity do not play a significant role in pain perception and TEAS-induced analgesia in the Chinese Han male population.

  8. A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine1

    PubMed Central

    Schmidlin, Laure; Poutaraud, Anne; Claudel, Patricia; Mestre, Pere; Prado, Emilce; Santos-Rosa, Maria; Wiedemann-Merdinoglu, Sabine; Karst, Francis; Merdinoglu, Didier; Hugueney, Philippe

    2008-01-01

    Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment. PMID:18799660

  9. Catechol-O-methyltransferase Val158met polymorphism interacts with early experience to predict executive functions in early childhood.

    PubMed

    Blair, Clancy; Sulik, Michael; Willoughby, Michael; Mills-Koonce, Roger; Petrill, Stephen; Bartlett, Christopher; Greenberg, Mark

    2015-11-01

    Numerous studies demonstrate that the Methionine variant of the catechol-O-methyltransferase Val158Met polymorphism, which confers less efficient catabolism of catecholamines, is associated with increased focal activation of prefrontal cortex (PFC) and higher levels of executive function abilities. By and large, however, studies of COMT Val158Met have been conducted with adult samples and do not account for the context in which development is occurring. Effects of early adversity on stress response physiology and the inverted U shape relating catecholamine levels to neural activity in PFC indicate the need to take into account early experience when considering relations between genes such as COMT and executive cognitive ability. Consistent with this neurobiology, we find in a prospective longitudinal sample of children and families (N = 1292) that COMT Val158Met interacts with early experience to predict executive function abilities in early childhood. Specifically, the Valine variant of the COMT Val158Met polymorphism, which confers more rather than less efficient catabolism of catecholamines is associated with higher executive function abilities at child ages 48 and 60 months and with faster growth of executive function for children experiencing early adversity, as indexed by cumulative risk factors in the home at child ages 7, 15, 24, and 36 months. Findings indicate the importance of the early environment for the relation between catecholamine genes and developmental outcomes and demonstrate that the genetic moderation of environmental risk is detectable in early childhood.

  10. Catechol-O-methyltransferase Val158met Polymorphism Interacts With Early Experience to Predict Executive Functions in Early Childhood

    PubMed Central

    Blair, Clancy; Sulik, Michael; Willoughby, Michael; Mills-Koonce, Roger; Petrill, Stephen; Bartlett, Christopher; Greenberg, Mark

    2017-01-01

    Numerous studies demonstrate that the Methionine variant of the catechol-O-methyltransferase Val158Met polymorphism, which confers less efficient catabolism of catecholamines, is associated with increased focal activation of prefrontal cortex (PFC) and higher levels of executive function abilities. By and large, however, studies of COMT Val158Met have been conducted with adult samples and do not account for the context in which development is occurring. Effects of early adversity on stress response physiology and the inverted U shape relating catecholamine levels to neural activity in PFC indicate the need to take into account early experience when considering relations between genes such as COMT and executive cognitive ability. Consistent with this neurobiology, we find in a prospective longitudinal sample of children and families (N=1292) that COMT Val158Met interacts with early experience to predict executive function abilities in early childhood. Specifically, the Valine variant of the COMT Val158Met polymorphism, which confers more rather than less efficient catabolism of catecholamines is associated with higher executive function abilities at child ages 48 and 60 months and with faster growth of executive function for children experiencing early adversity, as indexed by cumulative risk factors in the home at child ages 7, 15, 24, and 36 months. Findings indicate the importance of the early environment for the relation between catecholamine genes and developmental outcomes and demonstrate that the genetic moderation of environmental risk is detectable in early childhood. PMID:26251232

  11. Fewer fluctuations, higher maximum concentration and better motor response of levodopa with catechol-O-methyltransferase inhibition.

    PubMed

    Muhlack, Siegfried; Herrmann, Lennard; Salmen, Stephan; Müller, Thomas

    2014-11-01

    Catechol-O-methyltransferase inhibitor addition to levodopa/carbidopa formulations improves motor symptoms and reduces levodopa fluctuations in patients with Parkinson's disease. Objectives were to investigate the effects of entacapone and tolcapone on plasma behaviour of levodopa, its metabolite 3-O-methyldopa and on motor impairment. 22 patients orally received levodopa/carbidopa first, then levodopa/carbidopa/entacapone and finally levodopa/carbidopa plus tolcapone within a 4.5 h interval twice. Maximum concentration, time to maximum level and bioavailability of levodopa did not differ between all conditions each with 200 mg levodopa application as a whole. Catechol-O-methyltransferase inhibition caused less fluctuations and higher baseline levels of levodopa after the first intake and less 3-O-methyldopa appearance. The maximum levodopa concentrations were higher after the second levodopa intake, particularly with catechol-O-methyltransferase inhibition. The motor response to levodopa was better with catechol-O-methyltransferase inhibition than without, tolcapone was superior to entacapone. More continuous levodopa brain delivery and lower 3-O-methyldopa bioavailability caused a better motor response during catechol-O-methyltransferase inhibition.

  12. EST analysis of hop glandular trichomes identifies an O-methyltransferase that catalyzes the biosynthesis of xanthohumol.

    PubMed

    Nagel, Jana; Culley, Lana K; Lu, Yuping; Liu, Enwu; Matthews, Paul D; Stevens, Jan F; Page, Jonathan E

    2008-01-01

    The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes.

  13. EST Analysis of Hop Glandular Trichomes Identifies an O-Methyltransferase That Catalyzes the Biosynthesis of Xanthohumol[W][OA

    PubMed Central

    Nagel, Jana; Culley, Lana K.; Lu, Yuping; Liu, Enwu; Matthews, Paul D.; Stevens, Jan F.; Page, Jonathan E.

    2008-01-01

    The glandular trichomes (lupulin glands) of hop (Humulus lupulus) synthesize essential oils and terpenophenolic resins, including the bioactive prenylflavonoid xanthohumol. To dissect the biosynthetic processes occurring in lupulin glands, we sequenced 10,581 ESTs from four trichome-derived cDNA libraries. ESTs representing enzymes of terpenoid biosynthesis, including all of the steps of the methyl 4-erythritol phosphate pathway, were abundant in the EST data set, as were ESTs for the known type III polyketide synthases of bitter acid and xanthohumol biosynthesis. The xanthohumol biosynthetic pathway involves a key O-methylation step. Four S-adenosyl-l-methionine–dependent O-methyltransferases (OMTs) with similarity to known flavonoid-methylating enzymes were present in the EST data set. OMT1, which was the most highly expressed OMT based on EST abundance and RT-PCR analysis, performs the final reaction in xanthohumol biosynthesis by methylating desmethylxanthohumol to form xanthohumol. OMT2 accepted a broad range of substrates, including desmethylxanthohumol, but did not form xanthohumol. Mass spectrometry and proton nuclear magnetic resonance analysis showed it methylated xanthohumol to 4-O-methylxanthohumol, which is not known from hop. OMT3 was inactive with all substrates tested. The lupulin gland-specific EST data set expands the genomic resources for H. lupulus and provides further insight into the metabolic specialization of glandular trichomes. PMID:18223037

  14. Association between the catechol-o-methyltransferase val158met polymorphism with susceptibility and severity of carpal tunnel syndrome

    PubMed Central

    Eroğlu, P; Görükmez, O; Özemri Sağ, Ş; Yakut, T

    2015-01-01

    Abstract Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy of the upper extremity. In this study, we aimed to clarify the relationships between the catechol-O-methyltransferase (COMT) gene Val158Met (rs4680) polymorphism and development, functional and clinical status of CTS. Ninety-five women with electro diagnostically confirmed CTS and 95 healthy controls were enrolled in the study. The functional and clinical status of the patients was measured by the Turkish version of the Boston Questionnaire and intensity of pain related to the past 2 weeks was evaluated on a visual analog scale (VAS). The Val158Met polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), method. We divided patients according to the genotypes of the Val158Met polymorphism as Val/Val, Val/Met and Met/Met. There were not any significant differences in terms of Val158Met polymorphisms between patients and healthy controls (p >0.05). We also did not find any relationships between the Val158Met polymorphism and CTS (p >0.05). In conclusion, although we did not find any relationships between CTS and the Val158Met polymorphism, we could not generalize this result to the general population. Future studies are warranted to conclude precise associations. PMID:27785396

  15. Race Moderates the Association of Catechol-O-methyltransferase Genotype and Posttraumatic Stress Disorder in Preschool Children

    PubMed Central

    Humphreys, Kathryn L.; Scheeringa, Michael S.

    2014-01-01

    Abstract Objective: The present study sought to replicate previous findings of an association between the Catechol-O-methyltransferase (COMT) val158met polymorphism with posttraumatic stress disorder (PTSD) and symptomatology in a novel age group, preschool children. Methods: COMT genotype was determined in a sample of 171 3–6-year-old trauma-exposed children. PTSD was assessed with a semistructured interview. Accounting for sex, trauma type, and age, genotype was examined in relation to categorical and continuous measures of PTSD both controlling for race and within the two largest racial categories (African American [AA] and European American [EA]). Results: Race significantly moderated the association between genotype and PTSD. Specifically, the genotype associated with increased PTSD symptoms in one racial group had the opposite association in the other racial group. For AA children the met/met genotype was associated with more PTSD symptoms. However, for EA children, val allele carriers had more PTSD symptoms. Whereas every AA child with the met/met genotype met criteria for PTSD, none of the EA children with the met/met genotype did. This genetic association with COMT genotype, in both races but in opposite directions, was most associated with increased arousal symptoms. Conclusions: These findings replicate previous findings in participants of African descent, highlight the moderating effect of race on the association between COMT genotype and PTSD, and provide direct evidence that consideration of population stratification within gene-by-environment studies is valuable to prevent false negative findings. PMID:25329975

  16. Association between the catechol-o-methyltransferase val158met polymorphism with susceptibility and severity of carpal tunnel syndrome.

    PubMed

    Erkol İnal, E; Eroğlu, P; Görükmez, O; Özemri Sağ, Ş; Yakut, T

    2015-12-01

    Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy of the upper extremity. In this study, we aimed to clarify the relationships between the catechol-O-methyltransferase (COMT) gene Val158Met (rs4680) polymorphism and development, functional and clinical status of CTS. Ninety-five women with electro diagnostically confirmed CTS and 95 healthy controls were enrolled in the study. The functional and clinical status of the patients was measured by the Turkish version of the Boston Questionnaire and intensity of pain related to the past 2 weeks was evaluated on a visual analog scale (VAS). The Val158Met polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), method. We divided patients according to the genotypes of the Val158Met polymorphism as Val/Val, Val/Met and Met/Met. There were not any significant differences in terms of Val158Met polymorphisms between patients and healthy controls (p >0.05). We also did not find any relationships between the Val158Met polymorphism and CTS (p >0.05). In conclusion, although we did not find any relationships between CTS and the Val158Met polymorphism, we could not generalize this result to the general population. Future studies are warranted to conclude precise associations.

  17. Association between the Catechol-O-Methyltransferase (COMT) Val158Met Polymorphism and Manual Aiming Control in Healthy Subjects

    PubMed Central

    Lage, Guilherme M.; Miranda, Débora M.; Romano-Silva, Marco A.; Campos, Simone B.; Albuquerque, Maicon R.; Corrêa, Humberto; Malloy-Diniz, Leandro F.

    2014-01-01

    Background Prefrontal dopamine is catabolized by the catechol-O-methyltransferase (COMT) enzyme. Current evidence suggests that the val/met single nucleotide polymorphism in the COMT gene can predict the efficiency of executive cognition in humans. Individuals carrying the val allele perform more poorly because less synaptic dopamine is available. Methodology/Principal Findings We investigated the influence of the COMT polymorphism on motor performance in a task that requires different executive functions. We administered a manual aiming motor task that was performed under four different conditions of execution by 111 healthy participants. Participants were grouped according to genotype (met/met, met/val, val/val), and the motor performance among groups was compared. Overall, the results indicate that met/met carriers presented lower levels of peak velocity during the movement trajectory than the val carriers, but met/met carriers displayed higher accuracy than the val carriers. Conclusions/Significance This study found a significant association between the COMT polymorphism and manual aiming control. Few studies have investigated the genetics of motor control, and these findings indicate that individual differences in motor control require further investigation using genetic studies. PMID:24956262

  18. Functional characterization of a Mg(2+)-dependent O-methyltransferase with coumarin as preferred substrate from the liverwort Plagiochasma appendiculatum.

    PubMed

    Xu, Rui-Xue; Gao, Shuai; Zhao, Yu; Lou, Hong-Xiang; Cheng, Ai-Xia

    2016-09-01

    Coumarins (1,2-benzopyrones), which originate via the phenylpropanoid pathway, are found ubiquitously in plants and make an essential contribution to the health of the plant. Some natural coumarins have been used as human therapeutics. However, the details of their biosynthesis are still largely unknown. Scopoletin is derived from either esculetin or feruloyl CoA according to the plant species involved. Here, a gene encoding a O-methyltransferase (PaOMT2) was isolated from the liverwort species Plagiochasma appendiculatum (Aytoniaceae) through transcriptome sequencing. The purified recombinant enzyme catalyzed the methylation of esculetin, generating scopoletin and isoscopoletin. Kinetic analysis shows that the construct from the second Met in PaOMT2 had a catalytic efficiency for esculetin (Kcat/Km) of about half that of the full length PaOMT2, while the Kms of two enzymes were similar. The catalytic capacities of the studied protein suggest that two routes to scopoletin might co-exist in liverworts in that the enzyme involved in the methylation process participates in both paths, but especially the route from esculetin. The transient expression of a PaOMT2-GFP fusion in tobacco demonstrated that PaOMT2 is directed to the cytoplasm.

  19. Gender-dependent association of the functional catechol-O-methyltransferase Val158Met genotype with sensation seeking personality trait.

    PubMed

    Lang, Undine E; Bajbouj, Malek; Bajbouj, Malck; Sander, Thomas; Gallinat, Juergen

    2007-09-01

    The gene encoding cathechol-O-methyltransferase (COMT) contains a common functional missense polymorphism (Val158Met) that regulates dopamine in an allele-dependent manner. A pivotal role of dopamine neurotransmission in the prefrontal cortex has been implicated in drug-seeking behavior and related personality traits, such as sensation seeking, with some evidence for a gender-specific association. Here, we tested the hypothesis that the COMT Val158Met polymorphism modulates the personality dimension, sensation seeking, in a gender-dependent manner. Study sample included 214 male (age 38.1+/-12.6 years) and 218 female (age 36.1+/-13.6 years) healthy volunteers, who were assessed with Zuckerman's sensation-seeking scale and genotyped for the Val158Met polymorphism (dbSNP:rs4680). Univariate analysis of variance showed that the sensation seeking score was significantly affected by a COMT genotype x gender interaction (F=5.330, df=2, p=0.005). The Val158Met polymorphism was associated with the sensation seeking personality trait in women only. The highest scores in the sensation-seeking scale and in three of the four subscales were observed in female subjects with the Val/Val genotype relative to women carrying the Met allele. Our results suggest that high COMT enzyme activity associated with the Val allele predisposes to high sensation seeking scores in female subjects and add to increasing evidence for a gender specific role of COMT in normal and dysfunctional behavior.

  20. Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT)

    PubMed Central

    2015-01-01

    3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg2+. The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors. PMID:25815153

  1. Purification and Properties of an S-Adenosylmethionine: 2,4-Disubstituted Phenol O-Methyltransferase from Phanerochaete chrysosporium

    PubMed Central

    Coulter, Catherine; Kennedy, James T.; McRoberts, W. Colin; Harper, David B.

    1993-01-01

    An enzyme catalyzing the O-methylation of acetovanillone (3-methoxy-4-hydroxyacetophenone) by S-adeno-sylmethionine was isolated from Phanerochaete chrysosporium and purified 270-fold by ultrafiltration, anion-exchange chromatography, and gel filtration. The enzyme exhibited a pH optimum between 7 and 9 and was rapidly denatured at temperatures above 55°C. The Km values for acetovanillone and S-adenosylmethionine were 34 and 99 μM, respectively. S-Adenosylhomocysteine acted as a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 41 μM. The enzyme was also susceptible to inhibition by thiol reagents and low concentrations of heavy metal ions. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was monomeric and had a molecular weight of approximately 53,000. Substrate specificity studies showed that 3-methoxy- and 3,5-dimethoxy-substituted 4-hydroxy-benzaldehydes, -benzoic acids, and -acetophenones were the preferred substrates for the enzyme. The corresponding 3,4-dihydroxy compounds were methylated relatively slowly, while the 3-hydroxy-4-methoxy compounds were almost inactive as substrates. Substituents in both the 2 and 4 positions relative to the hydroxyl group appeared to be essential for significant enzyme attack of a substrate. Provided that certain steric criteria were satisfied, the nature of the substituent was not critical. Hence, xenobiotic compounds such as 2,4-dichlorophenol and 2,4-dibromophenol were methylated almost as readily as acetovanillone. However, an extended side chain in the 4 position was not compatible with activity as a substrate, and neither homovanillic, caffeic, nor ferulic acid was methylated. The substrate range of the O-methyltransferase tends to imply a role in the catabolism or detoxification of lignin degradation products such as vanillic and syringic acids. PMID:16348886

  2. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    SciTech Connect

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation.

  3. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

    PubMed

    Suriguga; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation.

  4. Floral scent production in Clarkia breweri (Onagraceae). II. Localization and developmental modulation of the enzyme S-adenosyl-L-methionine:(iso)eugenol O-methyltransferase and phenylpropanoid emission.

    PubMed Central

    Wang, J; Dudareva, N; Bhakta, S; Raguso, R A; Pichersky, E

    1997-01-01

    We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55-67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4'-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step. PMID:9159948

  5. Convergent Mechanistic Features between the Structurally Diverse N- and O-Methyltransferases: Glycine N-Methyltransferase and Catechol O-Methyltransferase.

    PubMed

    Zhang, Jianyu; Klinman, Judith P

    2016-07-27

    Although an enormous and still growing number of biologically diverse methyltransferases have been reported and identified, a comprehensive understanding of the enzymatic methyl transfer mechanism is still lacking. Glycine N-methyltransferase (GNMT), a member of the family that acts on small metabolites as the substrate, catalyzes methyl transfer from S-adenosyl-l-methionine (AdoMet) to glycine to form S-adenosyl-l-homocysteine and sarcosine. We report primary carbon ((12)C/(14)C) and secondary ((1)H3/(3)H3) kinetic isotope effects at the transferred methyl group, together with (1)H3/(3)H3 binding isotope effects for wild-type GNMT and a series of Tyr21 mutants. The data implicate a compaction effect in the methyl transfer step that is conferred by the protein structure. Furthermore, a remarkable similarity of properties is observed between GNMT and catechol O-methyltransferase, despite significant differences between these enzymes with regard to their active site structures and catalyzed reactions. We attribute these results to a catalytically relevant reduction in the methyl donor-acceptor distance that is dependent on a tyrosine side chain positioned behind the methyl-bearing sulfur of AdoMet.

  6. The relationship between childhood abuse and dissociation. Is it influenced by catechol-O-methyltransferase (COMT) activity?

    PubMed

    Savitz, Jonathan B; van der Merwe, Lize; Newman, Timothy K; Solms, Mark; Stein, Dan J; Ramesar, Rajkumar S

    2008-03-01

    Dissociation is a failure of perceptual, memorial and emotional integration that is associated with a variety of psychiatric disorders. Dissociative processes are usually attributed to the sequelae of childhood trauma although there are data to suggest that genetic influences are also important. Bipolar disorder (BD), a condition with a strong genetic basis, has also been associated with early psychological trauma. Since childhood trauma is a risk factor for both BD and dissociation, we tested for potential gene-childhood abuse interactions on dissociation in a pilot sample of BD probands and their affected and unaffected relatives (n=178). Dissociation was measured with the Dissociative Experiences Scale (DES II) and childhood maltreatment with the Childhood Trauma Questionnaire (CTQ). The BD and recurrent unipolar depression (MDE-R) groups showed higher levels of self-reported abuse and dissociation than their unaffected relatives. The low-activity Met allele of the Val66Met polymorphism of the brain-derived neurotrophic factor (BDNF) gene was associated with lower levels of self-reported dissociation. Further, the functional catechol-O-methyltransferase (COMT) Val158Met polymorphism interacted significantly with total CTQ abuse scores to impact perceived dissociation. The Val/Val genotype was associated with increasing levels of dissociation in participants exposed to higher levels of childhood trauma. The opposite was observed in people with Met/Met genotypes who displayed decreased dissociation with increasing self-reported childhood trauma. The current findings support the involvement of the COMT Val158Met polymorphism in mediating the relationship between trauma and psychopathology.

  7. Affect-modulated startle: interactive influence of catechol-O-methyltransferase Val158Met genotype and childhood trauma.

    PubMed

    Klauke, Benedikt; Winter, Bernward; Gajewska, Agnes; Zwanzger, Peter; Reif, Andreas; Herrmann, Martin J; Dlugos, Andrea; Warrings, Bodo; Jacob, Christian; Mühlberger, Andreas; Arolt, Volker; Pauli, Paul; Deckert, Jürgen; Domschke, Katharina

    2012-01-01

    The etiology of emotion-related disorders such as anxiety or affective disorders is considered to be complex with an interaction of biological and environmental factors. Particular evidence has accumulated for alterations in the dopaminergic and noradrenergic system--partly conferred by catechol-O-methyltransferase (COMT) gene variation--for the adenosinergic system as well as for early life trauma to constitute risk factors for those conditions. Applying a multi-level approach, in a sample of 95 healthy adults, we investigated effects of the functional COMT Val158Met polymorphism, caffeine as an adenosine A2A receptor antagonist (300 mg in a placebo-controlled intervention design) and childhood maltreatment (CTQ) as well as their interaction on the affect-modulated startle response as a neurobiologically founded defensive reflex potentially related to fear- and distress-related disorders. COMT val/val genotype significantly increased startle magnitude in response to unpleasant stimuli, while met/met homozygotes showed a blunted startle response to aversive pictures. Furthermore, significant gene-environment interaction of COMT Val158Met genotype with CTQ was discerned with more maltreatment being associated with higher startle potentiation in val/val subjects but not in met carriers. No main effect of or interaction effects with caffeine were observed. Results indicate a main as well as a GxE effect of the COMT Val158Met variant and childhood maltreatment on the affect-modulated startle reflex, supporting a complex pathogenetic model of the affect-modulated startle reflex as a basic neurobiological defensive reflex potentially related to anxiety and affective disorders.

  8. The effects of catechol O-methyltransferase genotype on brain activation elicited by affective stimuli and cognitive tasks.

    PubMed

    Heinz, Andreas; Smolka, Michael N

    2006-01-01

    Catechol-O-methyltransferase (COMT) degrades the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. A functional polymorphism in the COMT gene (val158 met) accounts for a four-fold variation in enzyme activity. The low activity met158 allele has been associated with improved working memory, executive functioning, and attentional control, but also with a higher risk of anxiety-related behaviors. In spite of the strong effect of the COMT genotype on enzyme activity, its effects on behavior are moderate, accounting for only 4% of variance in task performance. Studies of individuals with intermediate phenotypes during activities such as task-dependent brain activation, may more sensitively detect gene effects on the brain. A series of studies using functional magnetic resonance imaging (fMRI) assessed the effects of the COMT val158 met genotype on central processing during working memory, attentional control, and emotional tasks. fMRI revealed a more focused response in the prefrontal cortex (PFC) of met158 allele carriers during a working memory task. A comparable effect during the performance of an attentional control task in the cingulate cortex was also observed. These data indicate that met158 allele load is associated with improved processing efficiency in the PFC and cingulate, which might be due to lower prefrontal dopamine (DA) metabolism, higher DA concentrations, and an increased neuronal signal-to-noise ratio during information processing. During performance of an emotional task, reactivity to unpleasant visual stimuli was positively correlated with the number of met158 alleles in the amygdala, as well as in other limbic and paralimbic nodes. This increased limbic reactivity to unpleasant stimuli might be the underlying cause of the lower emotional resilience against negative mood states observed in individuals with a higher met158 allele load. Thus the met158 allele seems to be beneficial during the performance of working memory and

  9. An assay for human erythrocyte catechol-O-methyltransferase activity using a catechol estrogen as the substrate.

    PubMed

    Bates, G W; Edman, C D; Porter, J C; Johnston, J M; MacDonald, P C

    1979-05-16

    A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and non-radiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy[3H]estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 microliter of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37 degrees C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 microliter of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 x 10(-7) M and 6.7 x 10(-9) mol . ml-1 erythrocytes . h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 +/- 0.17 (mean +/- S.E.) nmol 2-methoxyestrone . ml-1 erythrocytes . h-1.

  10. Changes In Cell Wall Polymers And Degradability In Maize Mutants Lacking 3'- And 5'-O-Methyltransferases Involved In Lignin Biosynthesis.

    PubMed

    Fornalé, Silvia; Rencoret, Jorge; García-Calvo, Laura; Encina, Antonio; Rigau, Joan; Gutiérrez, Ana; Del Río, José Carlos; Caparros-Ruiz, David

    2016-12-23

    Caffeoyl Coenzyme A 3-O-Methyltransferase (CCoAOMT) and Caffeic acid-O-Methyltransferase (COMT) are key enzymes in the biosynthesis of coniferyl and sinapyl alcohols, the precursors of guaiacyl (G) and syringyl (S) lignin subunits. The function of these enzymes was characterised in single and double mutant maize plants. In this work, we determined that the comt (brown-midrib 3) mutant plants display a reduction of the flavonolignin unit derived from tricin (a dimethylated flavone), demonstrating that COMT is a key enzyme involved in the synthesis of this compound. In contrast, the ccoaomt1 mutants displays wild type amount of tricin, suggesting that CCoAOMT1 is not essential for the synthesis of this compound. Based on our data, we suggest that CCoAOMT1 is involved in lignin biosynthesis at least in midribs. The phenotype of ccoaomt1 mutant plants displays no alterations, and their lignin content and composition remain unchanged. On the other hand, the ccoaomt1 comt mutant displays phenotypic and lignin alterations similar to the ones already described for the comt mutant. Although stems from the three mutants display a similar increase of hemicelluloses, the effect on cell wall degradability varies, being the cell walls of the ccoaomt1 the most degradable. This suggests that the positive effect of lignin reduction on cell wall degradability of comt and ccoaomt1 comt mutants is counteracted by changes occurring in lignin composition, such as the decreased S/G ratio. In addition, the role of the flavonolignin unit derived from tricin in cell wall degradability is also discussed.

  11. The Catechol-O-Methyltransferase Val158Met Polymorphism Contributes to the Risk of Breast Cancer in the Chinese Population: An Updated Meta-Analysis

    PubMed Central

    Wan, Guo-Xing; Cao, Yu-Wen; Li, Wen-Qin; Li, Yu-Cong; Li, Feng

    2014-01-01

    Purpose Catechol-O-methyltransferase (COMT) enzyme plays a central role in estrogen-induced carcinogenesis. Emerging evidence from association studies has revealed that the functional Val158Met polymorphism (rs4680 G>A) of the Catechol-O-methyltransferase gene (COMT) has been implicated in susceptibility to breast cancer in the Chinese population, while results of individual published studies remain inconclusive and inconsistent. To assess this association in the Chinese population, a meta-analysis was performed. Methods Eligible studies were searched on MEDLINE, Embase, Cochrane Library, China National Knowledge Infrastructure, and the Chinese Biomedicine Database. Odds ratios (ORs) with their corresponding 95% confidence intervals (CIs) were pooled to assess the association between COMT polymorphisms and the risk of breast cancer using RevMan 5.2 and Stata 12.0 software. Results The meta-analysis included 14 eligible studies, with a total of 4,626 breast cancer cases and 5,637 controls. Overall, the COMT Val158Met polymorphism (rs4680 G>A) was significantly associated with an increased risk of breast cancer in several genetic models (A/A vs. G/G: OR, 1.59, 95% CI, 1.12-2.27; A/A vs. G/A+G/G: OR, 1.62, 95% CI, 1.14-2.29; A vs. G: OR, 1.15, 95% CI, 1.00-1.32), and a subgroup analysis according to menopausal status showed that this association was especially evident among premenopausal Chinese women (A/A vs. G/G: OR, 1.87, 95% CI, 0.99-3.54; A/A vs. G/A+G/G: OR, 1.94, 95% CI, 1.03-3.63). Conclusion The results of this meta-analysis indicated that COMT Val158Met variants contribute to breast cancer susceptibility in the Chinese population, particularly among premenopausal women. PMID:25013436

  12. Structure-based drug design of catechol-O-methyltransferase inhibitors for CNS disorders

    PubMed Central

    Ma, Zhiguo; Liu, Hongming; Wu, Baojian

    2014-01-01

    Catechol-O-methyltransferase (COMT) is of great importance in pharmacology because it catalyzes the metabolism (methylation) of endogenous and xenobiotic catechols. Moreover, inhibition of COMT is the drug target in the management of central nervous system (CNS) disorders such as Parkinson's disease due to its role in regulation of the dopamine level in the brain. The X-ray crystal structures for COMT have been available since 1994. The active sites for cofactor and substrate/inhibitor binding are well resolved to an atomic level, providing valuable insights into the catalytic mechanisms as well as the role of magnesium ions in catalysis. Determination of how the substrates/inhibitors bind to the protein leads to a structure-based approach that has resulted in potent and selective inhibitors. This review focuses on the design of two types of inhibitors (nitrocatechol-type and bisubstrate inhibitors) for COMT using the protein structures. PMID:23713800

  13. Methylation of sulfhydryl groups: a new function for a family of small molecule plant O-methyltransferases

    PubMed Central

    Coiner, Heather; Schröder, Gudrun; Wehinger, Elke; Liu, Chang-Jun; Noel, Joseph P.; Schwab, Wilfried; Schröder, Joachim

    2010-01-01

    Summary In plants, type I and II S-adenosyl-L-methionine-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology-based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56–58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 μM) and furfuryl thiol (Km 60 μM) were the best substrates (6–7-fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5-fold lower than with thiols. The enzyme was named C. roseus S-methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 so far is the only recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S-methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups. PMID:16623883

  14. Catechol-O-methyltransferase val158met genotype determines effect of reboxetine on emotional memory in healthy male volunteers

    PubMed Central

    Gibbs, Ayana A.; Bautista, Carla E.; Mowlem, Florence D.; Naudts, Kris H.; Duka, Dora T.

    2014-01-01

    Background Catechol-O-methyltransferase (COMT) metabolizes catecholamines in the prefrontal cortex (PFC). A common polymorphism in the COMT gene (COMT val158met) has pleiotropic effects on cognitive and emotional processing. The met allele has been associated with enhanced cognitive processing but impaired emotional processing relative to the val allele. Methods We genotyped healthy, white men in relation to the COMT val158met polymorphism. They were given a single 4 mg dose of the selective noradrenaline reuptake inhibitor (NRI) reboxetine or placebo in a randomized, double-blind between-subjects model and then completed an emotional memory task 2 hours later. Results We included 75 men in the study; 41 received reboxetine and 34 received placebo. In the placebo group, met/met carriers did not demonstrate the usual memory advantage for emotional stimuli that was observed in val carriers. Reboxetine restored this emotional enhancement of memory in met/met carriers, but had no significant effect in val carriers. Limitations We studied only men, thus limiting the generalizability of our findings. We also relied on self-reported responses to screening questions to establish healthy volunteer status, and in spite of the double-blind design, participants were significantly better than chance at identifying their intervention allocation. Conclusion Emotional memory is impaired in healthy met homozygotes and selectively improved in this group by reboxetine. This has potential translational implications for the use of reboxetine, which is currently licensed as an antidepressant in several countries, and edivoxetine, a new selective NRI currently in development. PMID:24467942

  15. Gonadectomy and hormone replacement exert region- and enzyme isoform-specific effects on monoamine oxidase and catechol-O-methyltransferase activity in prefrontal cortex and neostriatum of adult male rats.

    PubMed

    Meyers, B; D'Agostino, A; Walker, J; Kritzer, M F

    2010-02-03

    Sex differences and gonadal hormone influences are well known for diverse aspects of forebrain amine and indolamine neurotransmitter systems, the cognitive and affective functions they govern and their malfunction in mental illness. This study explored whether hormone regulation/dysregulation of these systems could be related to gonadal steroid effects on catechol-O-methyltransferase and monoamine oxidase which are principal enzymatic controllers of forebrain dopamine, serotonin and norepinephrine levels. Driven by male over female differences in cortical enzyme activities, by male-specific associations between monoamine oxidase and catechol-O-methyltransferase gene polymorphisms and cognitive and dysfunction in disease and by male-specific consequences of gene knockouts in mice, the question of hormone sensitivity was addressed here using a male rat model where prefrontal dopamine levels and related behaviors are also known to be affected. Specifically, quantitative O-methylation and oxidative deamination assays were used to compare the activities of catechol-O-methyltransferase's soluble and membrane-bound isoforms and of monoamine oxidase's A and B isoforms in the pregenual medial prefrontal cortex and dorsal striatum of male rats that were sham operated, gonadectomized or gonadectomized and supplemented with testosterone propionate or with estradiol for 28 days. These studies revealed significant effects of hormone replacement but not gonadectomy on the soluble but not the membrane-bound isorfom of catechol-O-methyltransferase in both striatum and cortex. A significant, cortex-specific testosterone-but not estradiol-attenuated effect (increase) of gonadectomy on monoamine oxidase's A but not B isoform was also observed. Although none of these actions suggest potential roles in the regulation/dysregulation of prefrontal dopamine, the suppressive effects of testosterone on cortical monoamine oxidase-A that were observed could have bearing on the increased

  16. Design, synthesis and in vitro/in vivo evaluation of orally bioavailable prodrugs of a catechol-O-methyltransferase inhibitor.

    PubMed

    Rautio, Jarkko; Leppänen, Jukka; Lehtonen, Marko; Laine, Krista; Koskinen, Mikko; Pystynen, Jarmo; Savolainen, Jouko; Sairanen, Mikko

    2010-04-15

    Compound 1 is an investigational, nanomolar inhibitor of catechol-O-methyltransferase (COMT) that suffers from poor oral bioavailability, most probably due to its low lipophilicity throughout most of the gastrointestinal tract and, to a lesser extent, its rapid systemic clearance. Several lipophilic esters were designed as prodrugs and synthesized in an attempt to optimize presystemic drug absorption. A modest twofold increase in 6-h exposure of 1 was observed with two prodrugs, compared to that of 1, after oral treatment in rats.

  17. Catechol-O-methyltransferase val(158)met Polymorphism Interacts with Sex to Affect Face Recognition Ability.

    PubMed

    Lamb, Yvette N; McKay, Nicole S; Singh, Shrimal S; Waldie, Karen E; Kirk, Ian J

    2016-01-01

    The catechol-O-methyltransferase (COMT) val158met polymorphism affects the breakdown of synaptic dopamine. Consequently, this polymorphism has been associated with a variety of neurophysiological and behavioral outcomes. Some of the effects have been found to be sex-specific and it appears estrogen may act to down-regulate the activity of the COMT enzyme. The dopaminergic system has been implicated in face recognition, a form of cognition for which a female advantage has typically been reported. This study aimed to investigate potential joint effects of sex and COMT genotype on face recognition. A sample of 142 university students was genotyped and assessed using the Faces I subtest of the Wechsler Memory Scale - Third Edition (WMS-III). A significant two-way interaction between sex and COMT genotype on face recognition performance was found. Of the male participants, COMT val homozygotes and heterozygotes had significantly lower scores than met homozygotes. Scores did not differ between genotypes for female participants. While male val homozygotes had significantly lower scores than female val homozygotes, no sex differences were observed in the heterozygotes and met homozygotes. This study contributes to the accumulating literature documenting sex-specific effects of the COMT polymorphism by demonstrating a COMT-sex interaction for face recognition, and is consistent with a role for dopamine in face recognition.

  18. Catechol-O-methyltransferase, a new target for pancreatic cancer therapy

    PubMed Central

    Wu, Wenming; Wu, Qiao; Hong, Xiafei; Zhou, Li; Zhang, Jie; You, Lei; Wang, Wenze; Wu, Huanwen; Dai, Hongmei; Zhao, Yupei

    2015-01-01

    Catechol-O-methyltransferase (COMT) is an important molecule in different types of cancers. Its biological effect and therapeutic significance, however, rarely been investigated fully in pancreatic cancer. Immunohistologically, high COMT expression was significantly correlated with the longer overall survival of patients (P < 0.05), indicating its protective nature. The effects of COMT on cell growth, apoptosis, and invasion were evaluated using overexpression and silencing methods. In detail, we carried out experiments using one stably transduced and two transiently transfected pancreatic cancer cell lines in vitro, and one stably transduced cell line in vivo mice xenograft models. In vitro experiments showed that COMT inhibited cell proliferation, enhanced gemcitabine-induced apoptosis, and inhibited cell invasion in stably transduced and transiently transfected cell lines by regulating the PI3K/Akt pathway, p53, and E-cadherin. The COMT overexpressed and silenced cell lines showed significantly inhibited and enhanced growth capacities in in vivo xenograft models, respectively. In conclusion, COMT suppressed pancreatic cancer and its high expression predicted longer survival time. The interaction of COMT with the PI3K/Akt pathway makes it a potential target for therapy. PMID:25711924

  19. Catechol-O-Methyltransferase val158met Polymorphism Predicts Placebo Effect in Irritable Bowel Syndrome

    PubMed Central

    Hall, Kathryn T.; Lembo, Anthony J.; Kirsch, Irving; Ziogas, Dimitrios C.; Douaiher, Jeffrey; Jensen, Karin B.; Conboy, Lisa A.; Kelley, John M.; Kokkotou, Efi; Kaptchuk, Ted J.

    2012-01-01

    Identifying patients who are potential placebo responders has major implications for clinical practice and trial design. Catechol-O-methyltransferase (COMT), an important enzyme in dopamine catabolism plays a key role in processes associated with the placebo effect such as reward, pain, memory and learning. We hypothesized that the COMT functional val158met polymorphism, was a predictor of placebo effects and tested our hypothesis in a subset of 104 patients from a previously reported randomized controlled trial in irritable bowel syndrome (IBS). The three treatment arms from this study were: no-treatment (“waitlist”), placebo treatment alone (“limited”) and, placebo treatment “augmented” with a supportive patient-health care provider interaction. The primary outcome measure was change from baseline in IBS-Symptom Severity Scale (IBS-SSS) after three weeks of treatment. In a regression model, the number of methionine alleles in COMT val158met was linearly related to placebo response as measured by changes in IBS-SSS (p = .035). The strongest placebo response occurred in met/met homozygotes treated in the augmented placebo arm. A smaller met/met associated effect was observed with limited placebo treatment and there was no effect in the waitlist control. These data support our hypothesis that the COMT val158met polymorphism is a potential biomarker of placebo response. PMID:23110189

  20. Performance of hydrophobic interaction ligands for human membrane-bound catechol-O-methyltransferase purification.

    PubMed

    Santos, Fátima Milhano; Pedro, Augusto Quaresma; Soares, Rui Filipe; Martins, Rita; Bonifácio, Maria João; Queiroz, João António; Passarinha, Luís António

    2013-06-01

    Despite of membrane catechol-O-methyltransferase (MBCOMT, EC 2.1.1.6) physiological importance on catecholamines' O-methylation, no studies allowed their total isolation. Therefore, for the first time, we compare the performance of three hydrophobic adsorbents (butyl-, epoxy-, and octyl-Sepharose) in purification of recombinant human COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates to develop a sustainable chromatographic process. Hydrophobic matrices were evaluated in terms of selectivity and hMBCOMT's binding and elution conditions. Results show that hMBCOMT's adsorption was promoted on octyl and butyl at ≤375 mM NaH2 PO4, while on epoxy higher concentrations (>850 mM) were required. Additionally, hMBCOMT's elution was promoted on epoxy, butyl, and octyl using respectively 0.1-0.5, 0.25-1, and 1% of Triton X-100. On butyl media, a stepwise strategy using 375 and 0 mM NaH2PO4, followed by three elution steps at 0.25, 0.7 and 1% Triton X-100, allowed selective hMBCOMT isolation. In conclusion, significant amounts of MBCOMT were purified with high selectivity on a single chromatography procedure, despite its elution occurs on multiple peaks. Although successful applications of hydrophobic interaction chromatography in purification of membrane proteins are uncommon, we proved that traditional hydrophobic matrices can open a promising unexplored field to fulfill specific requirements for kinetic and pharmacological trials.

  1. Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

    SciTech Connect

    Mastrangelo, Eloise; Bollati, Michela; Milani, Mario; Lamballeire, Xavier de; Brisbare, Nadege; Dalle, Karen; Lantez, Violaine; Egloff, Marie-Pierre; Coutard, Bruno; Canard, Bruno; Gould, Ernest; Forrester, Naomi; Bolognesi, Martino

    2006-08-01

    Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed, purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.

  2. Catechol-O-methyltransferase val158met polymorphism predicts placebo effect in irritable bowel syndrome.

    PubMed

    Hall, Kathryn T; Lembo, Anthony J; Kirsch, Irving; Ziogas, Dimitrios C; Douaiher, Jeffrey; Jensen, Karin B; Conboy, Lisa A; Kelley, John M; Kokkotou, Efi; Kaptchuk, Ted J

    2012-01-01

    Identifying patients who are potential placebo responders has major implications for clinical practice and trial design. Catechol-O-methyltransferase (COMT), an important enzyme in dopamine catabolism plays a key role in processes associated with the placebo effect such as reward, pain, memory and learning. We hypothesized that the COMT functional val158met polymorphism, was a predictor of placebo effects and tested our hypothesis in a subset of 104 patients from a previously reported randomized controlled trial in irritable bowel syndrome (IBS). The three treatment arms from this study were: no-treatment ("waitlist"), placebo treatment alone ("limited") and, placebo treatment "augmented" with a supportive patient-health care provider interaction. The primary outcome measure was change from baseline in IBS-Symptom Severity Scale (IBS-SSS) after three weeks of treatment. In a regression model, the number of methionine alleles in COMT val158met was linearly related to placebo response as measured by changes in IBS-SSS (p = .035). The strongest placebo response occurred in met/met homozygotes treated in the augmented placebo arm. A smaller met/met associated effect was observed with limited placebo treatment and there was no effect in the waitlist control. These data support our hypothesis that the COMT val158met polymorphism is a potential biomarker of placebo response.

  3. Molecular Cloning and Characterization of O-Methyltransferase from Mango Fruit (Mangifera indica cv. Alphonso).

    PubMed

    Chidley, Hemangi G; Oak, Pranjali S; Deshpande, Ashish B; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2016-05-01

    Flavour of ripe Alphonso mango is invariably dominated by the de novo appearance of lactones and furanones during ripening. Of these, furanones comprising furaneol (4-hydroxy-2,5-dimethyl-3(2H)-furanone) and mesifuran (2,5-dimethyl-4-methoxy-3(2H)-furanone) are of particular importance due to their sweet, fruity caramel-like flavour characters and low odour detection thresholds. We isolated a 1056 bp complete open reading frame of a cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase from Alphonso mango. The recombinantly expressed enzyme, MiOMTS showed substrate specificity towards furaneol and protocatechuic aldehyde synthesizing mesifuran and vanillin, respectively, in an in vitro assay reaction. A semi-quantitative PCR analysis showed fruit-specific expression of MiOMTS transcripts. Quantitative real-time PCR displayed ripening-related expression pattern of MiOMTS in both pulp and skin of Alphonso mango. Also, early and significantly enhanced accumulation of its transcripts was detected in pulp and skin of ethylene-treated fruits. Ripening-related and fruit-specific expression profile of MiOMTS and substrate specificity towards furaneol is a suggestive of its involvement in the synthesis of mesifuran in Alphonso mango. Moreover, a significant trigger in the expression of MiOMTS transcripts in ethylene-treated fruits point towards the transcriptional regulation of mesifuran biosynthesis by ethylene.

  4. Characterization of Non-Nitrocatechol Pan and Isoform Specific Catechol-O-methyltransferase Inhibitors and Substrates

    PubMed Central

    2011-01-01

    Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity. PMID:22860182

  5. Catechol-O-methyltransferase in complex with substituted 3'-deoxyribose bisubstrate inhibitors.

    PubMed

    Ellermann, Manuel; Lerner, Christian; Burgy, Guillaume; Ehler, Andreas; Bissantz, Caterina; Jakob-Roetne, Roland; Paulini, Ralph; Allemann, Oliver; Tissot, Heloïse; Grünstein, Dan; Stihle, Martine; Diederich, Francois; Rudolph, Markus G

    2012-03-01

    The biological activity of catechol neurotransmitters such as dopamine in the synapse is modulated by transporters and enzymes. Catechol-O-methyltransferase (COMT; EC 2.1.1.6) inactivates neurotransmitters by catalyzing the transfer of a methyl group from S-adenosylmethionine to catechols in the presence of Mg²⁺. This pathway also inactivates L-DOPA, the standard therapeutic for Parkinson's disease. Depletion of catechol neurotransmitters in the prefrontal cortex has been linked to schizophrenia. The inhibition of COMT therefore promises improvements in the treatment of these diseases. The concept of bisubstrate inhibitors for COMT has been described previously. Here, ribose-modified bisubstrate inhibitors were studied. Three high-resolution crystal structures of COMT in complex with novel ribose-modified bisubstrate inhibitors confirmed the predicted binding mode but displayed subtle alterations at the ribose-binding site. The high affinity of the inhibitors can be convincingly rationalized from the structures, which document the possibility of removing and/or replacing the ribose 3'-hydroxyl group and provide a framework for further inhibitor design.

  6. Catechol-O-methyltransferase val158met Polymorphism Interacts with Sex to Affect Face Recognition Ability

    PubMed Central

    Lamb, Yvette N.; McKay, Nicole S.; Singh, Shrimal S.; Waldie, Karen E.; Kirk, Ian J.

    2016-01-01

    The catechol-O-methyltransferase (COMT) val158met polymorphism affects the breakdown of synaptic dopamine. Consequently, this polymorphism has been associated with a variety of neurophysiological and behavioral outcomes. Some of the effects have been found to be sex-specific and it appears estrogen may act to down-regulate the activity of the COMT enzyme. The dopaminergic system has been implicated in face recognition, a form of cognition for which a female advantage has typically been reported. This study aimed to investigate potential joint effects of sex and COMT genotype on face recognition. A sample of 142 university students was genotyped and assessed using the Faces I subtest of the Wechsler Memory Scale – Third Edition (WMS-III). A significant two-way interaction between sex and COMT genotype on face recognition performance was found. Of the male participants, COMT val homozygotes and heterozygotes had significantly lower scores than met homozygotes. Scores did not differ between genotypes for female participants. While male val homozygotes had significantly lower scores than female val homozygotes, no sex differences were observed in the heterozygotes and met homozygotes. This study contributes to the accumulating literature documenting sex-specific effects of the COMT polymorphism by demonstrating a COMT-sex interaction for face recognition, and is consistent with a role for dopamine in face recognition. PMID:27445927

  7. No association between catechol-o-methyltransferase Val108/158Met polymorphism and schizophrenia or its clinical symptomatology in a Mexican population.

    PubMed

    Tovilla-Zárate, Carlos; Medellín, Beatriz Camarena; Fresán, Ana; López-Narváez, Lilia; Castro, Thelma Beatriz Gonzalez; Juárez Rojop, Isela; Ramírez-Bello, Julián; Genis, Alma; Nicolini, Humberto

    2013-02-01

    The gene coding for catecol-o-methyltransferase (COMT), participant in the metabolism of catecholamines, has long been implicated as a candidate gene for schizophrenia. We determined the relation of the COMT Val108/158Met polymorphism with schizophrenia or its symptomatology (negative, disorganized and psychotic dimension). We conducted a case-control study comprising 186 patients with schizophrenia and 247 controls. The diagnosis of schizophrenia was established using the DSM-IV criteria for this illness. The clinical symptomatology was assessed through the Scale for the Assessment of Negative Symptoms and the Scale for the Assessment of Positive Symptoms. No significant differences were found in the distribution of alleles (χ2 = 0.01, df = 1, p = 0.90) or genotypes (χ2 = 1.66, df = 2, p = 0.43) between schizophrenic patients and the control group. Multivariate analysis showed that the COMT Val108/158Met polymorphism has no influence in the clinical symptomatology of schizophrenia. Our results showed no association between COMT Val108/158Met and schizophrenia or evidence for an association between COMT and the clinical symptomatology of this illness. This suggests that the COMT gene may not contribute to the risk for schizophrenia among the Mexican population.

  8. Genetic Polymorphism of 1019C/G (rs6295) Promoter of Serotonin 1A Receptor and Catechol-O-Methyltransferase in Panic Disorder

    PubMed Central

    Ishiguro, Shin; Aoki, Akiko; Ueda, Mikito; Hayashi, Yuki; Akiyama, Kazufumi; Kato, Kazuko; Shimoda, Kazutaka

    2017-01-01

    Objective Family and twin studies have suggested genetic liability for panic disorder (PD) and therefore we sought to determine the role of noradrenergic and serotonergic candidate genes for susceptibility for PD in a Japanese population. Methods In this age- and gender-matched case-control study involving 119 PD patients and 119 healthy controls, we examined the genotype distributions and allele frequencies of the serotonin transporter gene linked polymorphic region (5-HTTLPR), −1019C/G (rs6295) promoter polymorphism of the serotonin receptor 1A (5-HT1A), and catechol-O-methyltransferase (COMT) gene polymorphism (rs4680) and their association with PD. Results No significant differences were evident in the allele frequencies or genotype distributions of the COMT (rs4680), 5-HTTLPR polymorphisms or the −1019C/G (rs6295) promoter polymorphism of 5-HT1A between PD patients and controls. Although there were no significant associations of these polymorphisms with in subgroups of PD patients differentiated by gender or in subgroup comorbid with agoraphobia (AP), significant difference was observed in genotype distributions of the −1019C/G (rs6295) promoter polymorphism of 5-HT1A between PD patients without AP and controls (p=0.047). Conclusion In this association study, the 1019C/G (rs6295) promoter polymorphism of the 5-HT1A receptor G/G genotype was associated with PD without AP in a Japanese population. PMID:28096880

  9. Isolation and Characterization of O-methyltransferases Involved in the Biosynthesis of Glaucine in Glaucium flavum1

    PubMed Central

    Chang, Limei; Hagel, Jillian M.; Facchini, Peter J.

    2015-01-01

    Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4′-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C6′ coupling and four O-methyl groups at C6, C7, C3′, and C4′ as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3′ and 4′ hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4′OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4′-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3′-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3′- and 7′-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O

  10. Conscientiousness is modified by genetic variation in catechol-O-methyltransferase to reduce symptom complaints in IBS patients

    PubMed Central

    Hall, Kathryn T; Tolkin, Benjamin R; Chinn, Garrett M; Kirsch, Irving; Kelley, John M; Lembo, Anthony J; Kaptchuk, Ted J; Kokkotou, Efi; Davis, Roger B; Conboy, Lisa A

    2015-01-01

    Background Attention to and perception of physical sensations and somatic states can significantly influence reporting of complaints and symptoms in the context of clinical care and randomized trials. Although anxiety and high neuroticism are known to increase the frequency and severity of complaints, it is not known if other personality dimensions or genes associated with cognitive function or sympathetic tone can influence complaints. Genetic variation in catechol-O-methyltransferase (COMT) is associated with anxiety, personality, pain, and response to placebo treatment. We hypothesized that the association of complaint reporting with personality might be modified by variation in the COMT val158met genotype. Methods We administered a standard 25-item complaint survey weekly over 3-weeks to a convenience sample of 187 irritable bowel syndrome patients enrolled in a placebo intervention trial and conducted a repeated measures analysis. Results We found that complaint severity rating, our primary outcome, was negatively associated with the personality measures of conscientiousness (β = −0.31 SE 0.11, P = 0.003) and agreeableness (β = −0.38 SE 0.12, P = 0.002) and was positively associated with neuroticism (β = 0.24 SE 0.09, P = 0.005) and anxiety (β = 0.48 SE 0.09, P < 0.0001). We also found a significant interaction effect of COMT met alleles (β = −32.5 SE 14.1, P = 0.021). in patients genotyped for COMT val158met (N  = 87) specifically COMT × conscientiousness (β = 0.73 SE 0.26, P = 0.0042) and COMT × anxiety (β = −0.42 SE 0.16, P = 0.0078) interaction effects. Conclusion These findings potentially broaden our understanding of the factors underlying clinical complaints to include the personality dimension of conscientiousness and its modification by COMT. PMID:25722948

  11. Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

    SciTech Connect

    Cooke, H.; Guenther, E; Luo, Y; Shen, B; Bruner, S

    2009-01-01

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

  12. Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

    PubMed

    Cooke, Heather A; Guenther, Elizabeth L; Luo, Yinggang; Shen, Ben; Bruner, Steven D

    2009-10-13

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

  13. CbCTB2, an O-methyltransferase is essential for biosynthesis of the phytotoxin cercosporin and infection of sugar beet by Cercospora beticola

    PubMed Central

    2013-01-01

    Background Cercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beets (Beta vulgaris) worldwide. Cercosporin, a light-inducible toxin, is essential for necrosis of the leaf tissue and development of the typical leaf spots on sugar beet leaves. Results In this study we show that the O-methyltransferase gene CTB2 is essential for cercosporin production and pathogenicity in two C. beticola isolates. We established a transformation system for C. beticola protoplasts, disrupted CTB2, and transformed the Δctb2 strains as well as a wild type strain with the DsRed reporter gene. The Δctb2 strains had lost their pigmentation and toxin measurements demonstrated that the Δctb2 strains were defective in cercosporin production. Infection of sugar beets with the wild type and Δctb2 DsRed strains showed that the deletion strain was severely impaired in plant infection. Histological analysis revealed that the CTB2-deficient isolate cannot enter the leaf tissue through stomata like the wild type. Conclusions Taken together, these observations indicate that cercosporin has a dual function in sugar beet infection: in addition to the well-known role in tissue necrosis, the toxin is required for the early phase of sugar beet infection. PMID:23517289

  14. Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase.

    PubMed

    Patra, Niladri; Ioannidis, Efthymios I; Kulik, Heather J

    2016-01-01

    Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21-22 kcal/mol, in good agreement with experiment (18-19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are

  15. Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase

    PubMed Central

    Patra, Niladri; Ioannidis, Efthymios I.

    2016-01-01

    Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21–22 kcal/mol, in good agreement with experiment (18–19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are

  16. Catechol-O-methyltransferase genotype predicts pain severity in hospitalized burn patients

    PubMed Central

    Orrey, Danielle C.; Bortsov, Andrey V.; Hoskins, Janelle M.; Shupp, Jeffrey W.; Jones, Samuel W.; Cicuto, Bryan J.; Hwang, James; Jordan, Marion H.; Holmes, James H.; Haith, Linwood R.; Roane, Brandon M.; Diatchenko, Luda; Cairns, Bruce A.; McLean, Samuel A.

    2011-01-01

    Background Increasing evidence suggests that stress system activation after burn injury may contribute to burn-related pain. If this is the case, then genetic variations influencing the function of important stress system components, such as the enzyme catechol-O-methyltransferase (COMT), may predict pain severity after thermal burn injury. Methods We evaluated the association between COMT genotype and pain intensity in 57 individuals hospitalized after thermal burn injury. Consenting participants at four burn centers were genotyped and completed daily 0-10 numeric rating scale pain assessments on two consecutive days including evaluation of waking, least, and worst pain. The association between COMT genotype and individual pain outcomes was calculated using a linear mixed model adjusting for sociodemographic and burn injury characteristics. Results Overall pain (combination of least, worst, and waking pain scores) was significantly higher in patients with a COMT pain vulnerable genotype (6.3 (.4) vs. 5.4 (.4), p=.037). Individuals with a COMT pain vulnerable genotype also had significantly higher “least pain” scores (3.8 (.5) vs. 2.6 (.4), p=.017) and significantly higher pain on awakening (6.8 (.5) vs. 5.3 (.4), p=.004). Differences in worst pain according to genotype group were not significant. COMT pain vulnerable genotype was a stronger predictor of overall pain severity than burn size, burn depth, or time from admission to pain interview assessment. Conclusions These findings suggest that genetic factors influencing stress system function may have an important influence on pain severity after burn injury. Further studies of genetic predictors of pain after burn injury are needed. PMID:22210062

  17. Genetic Polymorphisms of Catechol-O-Methyltransferase Modify the Neurobehavioral Effects of Mercury in Children

    PubMed Central

    Woods, James S.; Heyer, Nicholas J.; Russo, Joan E.; Martin, Michael D.; Pillai, Pradeep B.; Bammler, Theodor K.; Farin, Federico M.

    2014-01-01

    Mercury (Hg) is neurotoxic and children may be particularly susceptible to this effect. A current major challenge is identification of children who may be uniquely susceptible to Hg toxicity because of genetic disposition. This study examined the hypothesis that genetic variants of catechol-O-methyltransferase (COMT) that are reported to alter neurobehavioral functions that are also affected by Hg in adults might modify the adverse neurobehavioral effects of Hg exposure in children. Five hundred and seven children, 8–12 yr of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of Hg from dental amalgam tooth fillings. Subjects were evaluated at baseline and at seven subsequent annual intervals for neurobehavioral performance and urinary Hg levels. Following the clinical trial, genotyping assays were performed for single-nucleotide polymorphisms (SNPs) of COMT rs4680, rs4633, rs4818, and rs6269 on biological samples provided by 330 of the trial participants. Regression-modeling strategies were employed to evaluate associations between allelic status, Hg exposure, and neurobehavioral test outcomes. Similar analysis was performed using haplotypes of COMT SNPs. Among girls, few interactions for Hg exposure and COMT variants were found. In contrast, among boys, numerous gene–Hg interactions were observed between individual COMT SNPs, as well as with a common COMT haplotype affecting multiple domains of neurobehavioral function. These findings suggest increased susceptibility to the adverse neurobehavioral effects of Hg among children with common genetic variants of COMT, and may have important implications for strategies aimed at protecting children from the potential health risks associated with Hg exposure. PMID:24593143

  18. Purification and properties of a new S-adenosyl-L-methionine:flavonoid 4'-O-methyltransferase from carnation (Dianthus caryophyllus L.).

    PubMed

    Curir, Paolo; Lanzotti, Virginia; Dolci, Marcello; Dolci, Paola; Pasini, Carlo; Tollin, Gordon

    2003-08-01

    A new enzyme, S-adenosyl-l-methionine:flavonoid 4'-O-methyltransferase (EC 2.1.1.-) (F 4'-OMT), has been purified 1 399-fold from the tissues of carnation (Dianthus caryophyllus L). The enzyme, with a molecular mass of 43-45 kDa and a pI of 4.15, specifically methylates the hydroxy substituent in 4'-position of the flavones, flavanones and isoflavones in the presence of S-adenosyl-l-methionine. A high affinity for the flavone kaempferol was observed (Km = 1.7 micro m; Vmax = 95.2 micro mol.min-1.mg-1), while other 4'-hydroxylated flavonoids proved likewise to be suitable substrates. Enzyme activity had no apparent Mg++ requirement but was inhibited by SH-group reagents. The optimum pH value for F 4'-OMT activity was found to be around neutrality. Kinetic analysis of the enzyme bi-substrate reaction indicates a Ping-Pong mechanism and excludes the formation of a ternary complex. The F 4'-OMT activity was increased, in both in vitro and in vivo carnation tissues, by the inoculation with Fusarium oxysporum f. sp. dianthi. The enzyme did not display activity towards hydroxycinnamic acid derivatives, some of which are involved, as methylated monolignols, in lignin biosynthesis; the role of this enzyme could be therefore mainly defensive, rather than structural, although its precise function still needs to be ascertained.

  19. Allelic and genotype frequencies of catechol-O-methyltransferase (Val158Met) and CYP2D6*10 (Pro34Ser) single nucleotide polymorphisms in the Philippines

    PubMed Central

    Baclig, Michael O; Predicala, Rey Z; Mapua, Cynthia A; Lozano-Kühne, Jingky P; Daroy, Maria Luisa G; Natividad, Filipinas F; Javier, Francis O

    2012-01-01

    A hospital-based cross-sectional study was conducted to determine the allelic and genotype frequencies in the genes encoding for catechol-O-methyltransferase and CYP2D6*10 among healthy volunteers and patients clinically diagnosed with cancer pain. PCR-RFLP was used to identify COMT and CYP2D6*10 genotypes. Allelic frequencies among healthy volunteer Filipinos were 0.83 and 0.17 for the COMT Val and COMT Met alleles, respectively. Calculated frequencies in Hardy-Weinberg equilibrium (HWE) were 73% for COMT Val/Val, 26% for COMT Val/Met, and 1% for COMT Met/Met genotype. For CYP2D6*10, allelic frequencies in HWE among volunteers were 0.46 for the C allele and 0.54 for the T allele. Twenty percent were identified as homozygous for the wild-type C/C genotype, 56% were identified as heterozygous for the C/T genotype, and 24% were identified as homozygous for the T/T variant genotype. No significant differences in COMT and CYP2D6*10 allele frequencies between cancer patients and healthy volunteers were noted. Our data demonstrated that the allele frequencies of COMT and CYP2D6*10 in the Filipino healthy volunteers were similar with other Asians but markedly different from Caucasian populations. PMID:22724048

  20. Allelic and genotype frequencies of catechol-O-methyltransferase (Val158Met) and CYP2D6*10 (Pro34Ser) single nucleotide polymorphisms in the Philippines.

    PubMed

    Baclig, Michael O; Predicala, Rey Z; Mapua, Cynthia A; Lozano-Kühne, Jingky P; Daroy, Maria Luisa G; Natividad, Filipinas F; Javier, Francis O

    2012-01-01

    A hospital-based cross-sectional study was conducted to determine the allelic and genotype frequencies in the genes encoding for catechol-O-methyltransferase and CYP2D6*10 among healthy volunteers and patients clinically diagnosed with cancer pain. PCR-RFLP was used to identify COMT and CYP2D6*10 genotypes. Allelic frequencies among healthy volunteer Filipinos were 0.83 and 0.17 for the COMT Val and COMT Met alleles, respectively. Calculated frequencies in Hardy-Weinberg equilibrium (HWE) were 73% for COMT Val/Val, 26% for COMT Val/Met, and 1% for COMT Met/Met genotype. For CYP2D6*10, allelic frequencies in HWE among volunteers were 0.46 for the C allele and 0.54 for the T allele. Twenty percent were identified as homozygous for the wild-type C/C genotype, 56% were identified as heterozygous for the C/T genotype, and 24% were identified as homozygous for the T/T variant genotype. No significant differences in COMT and CYP2D6*10 allele frequencies between cancer patients and healthy volunteers were noted. Our data demonstrated that the allele frequencies of COMT and CYP2D6*10 in the Filipino healthy volunteers were similar with other Asians but markedly different from Caucasian populations.

  1. Differential effect of catechol-O-methyltransferase Val158Met genotype on emotional recognition abilities in healthy men and women.

    PubMed

    Weiss, Elisabeth M; Stadelmann, Edith; Kohler, Christian G; Brensinger, Colleen M; Nolan, Karen A; Oberacher, Herbert; Parson, Walther; Pitterl, Florian; Niederstätter, Harald; Kemmler, Georg; Hinterhuber, Hartmann; Marksteiner, Josef

    2007-09-01

    The catechol-O-methyltransferase (COMT) Val158Met polymorphism modulates executive functions and working memory and recent neuroimaging studies implicate an association with emotional processing. We examined the relationship between the COMT Val158Met polymorphism and facial emotion recognition and differentiation in 100 healthy individuals. Compared to Met homozygosity, Val homozygosity was associated with better and faster recognition of negative facial expressions such as anger and sad. Our study provides evidence for a possible influence of the COMT polymorphism on emotion recognition abilities in healthy subjects. Additional research is needed to further define the neurocognitive phenotypes associated with COMT polymorphisms.

  2. Current understanding of the interplay between catechol-O-methyltransferase genetic variants, sleep, brain development and cognitive performance in schizophrenia.

    PubMed

    Tucci, Valter; Lassi, Glenda; Kas, Martien J

    2012-05-01

    Abnormal sleep is an endophenotype of schizophrenia. Here we provide an overview of the genetic mechanisms that link specific sleep physiological processes to schizophrenia-related cognitive defects. In particular, we will review the possible relationships between catechol-O-methyltransferase (COMT), sleep regulation and schizophrenia development. Recent studies validate the hypothesis that COMT mutations may trigger disturbances during adolescence that affect sleep and cortical development. Anomalies in cortical development during this critical developmental phase may increase the susceptibility for schizophrenia. In conclusion, in view of therapeutic efficacy, we can envisage indications for future investigations into the role of COMT for sleep regulation, cognitive performance and sleep-related cognitive deficits.

  3. The blood-brain barrier-permeable catechol-O-methyltransferase inhibitor dinitrocatechol suppresses experimental autoimmune encephalomyelitis.

    PubMed

    Polak, Paul E; Lin, Shao Xia; Pelligrino, Dale; Feinstein, Douglas L

    2014-11-15

    Reduced levels of noradrenaline (NA) in CNS of multiple sclerosis patients could be due to metabolism by catechol-O-methyltransferase (COMT). In mice immunized with myelin oligodendrocyte glycoprotein peptide, the BBB-permeable COMT inhibitor dinitrocatechol (DNC) reduced clinical signs, while entacapone, a non-BBB-permeable inhibitor, had no effect. Spinal cord NA levels were slightly increased by DNC, and there was an inverse correlation between NA levels and average clinical signs. Spinal cord COMT mRNA levels were not increased during EAE, but were found increased in the frontal cortex of MS patients. These results suggest that COMT inhibitors could provide benefit to MS patients.

  4. Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17β on mRNA expression.

    PubMed

    Chaube, R; Rawat, A; Inbaraj, R M; Bobe, J; Guiguen, Y; Fostier, A; Joy, K P

    2016-12-08

    Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the

  5. Polymorphisms in Catechol-O-methyltransferase Modify Treatment Effects of Aspirin on Risk of Cardiovascular Disease

    PubMed Central

    Hall, Kathryn T.; Nelson, Christopher P.; Davis, Roger B.; Buring, Julie E.; Kirsch, Irving; Mittleman, Murray A.; Loscalzo, Joseph; Samani, Nilesh J.; Ridker, Paul M; Kaptchuk, Ted J.; Chasman, Daniel I.

    2014-01-01

    Objective Catechol-O-methyltransferase (COMT), a key enzyme in catecholamine metabolism, is implicated in cardiovascular, sympathetic, and endocrine pathways. This study aimed to confirm preliminary association of COMT genetic variation with incident cardiovascular disease (CVD). It further aimed to evaluate whether aspirin, a commonly used CVD prevention agent, modified the potential association of COMT with incident CVD. Approach and Results We examined COMT polymorphism rs4680 (MAF=0.47), encoding a non-synonymous methionine (met)-to-valine (val) substitution, in the Women's Genome Health Study (WGHS), a large population-based cohort of women with randomized allocation to aspirin or vitamin E compared with placebo and 10 years follow-up. Rs4680 effects were confirmed with COMT polymorphism rs4818 and also examined in CARDIoGRAM/C4D, consortia for genome-wide association studies of coronary artery disease. Among WGHS women allocated to placebo (135 events/N=5811), the rs4680 val allele was protective against incident CVD relative to the met, (HR[95%CI]=0.66[0.51-0.84], p=0.0007); an association also observed in CARDIoGRAM and C4D (combined p=2.4×10-5). In the WGHS, the rs4680 association was abolished by randomized allocation to aspirin, such that val/val women experienced higher CVD rates with aspirin allocation compared to placebo (HR[95%CI]=1.85[1.05-3.25], p=0.033) while met/met women experienced lower rates (HR[95%CI]=0.60[0.39-0.93], p=0.023). Allocation to vitamin E also conferred higher but non-significant CVD rates on val/val (HR[95%CI]=1.50 [0.83-2.70], p=0.180) compared with significantly lower rates on met/met (HR[95%CI]=0.53[0.34-0.84], p=0.006) women. Rs4818 results were similar. Conclusions Common COMT polymorphisms were associated with incident CVD, and this association was modified by randomized allocation to aspirin or vitamin E. Replication of these findings is required. PMID:25035343

  6. Crystal structures of human 108V and 108M catechol O-methyltransferase

    SciTech Connect

    Rutherford, K.; Le Trong, I.; Stenkamp, R.E.; Parson, W.W.

    2008-08-01

    Catechol O-methyltransferase (COMT) plays important roles in the metabolism of catecholamine neurotransmitters and catechol estrogens. The development of COMT inhibitors for use in the treatment of Parkinson's disease has been aided by crystallographic structures of the rat enzyme. However, the human and rat proteins have significantly different substrate specificities. Additionally, human COMT contains a common valine-methionine polymorphism at position 108. The methionine protein is less stable than the valine polymorph, resulting in decreased enzyme activity and protein levels in vivo. Here we describe the crystal structures of the 108V and 108M variants of the soluble form of human COMT bound with S-adenosylmethionine (SAM) and a substrate analog, 3,5-dinitrocatechol. The polymorphic residue 108 is located in the {alpha}5-{beta}3 loop, buried in a hydrophobic pocket {approx}16 {angstrom} from the SAM-binding site. The 108V and 108M structures are very similar overall [RMSD of C{sup {alpha}} atoms between two structures (C{sup {alpha}} RMSD) = 0.2 {angstrom}], and the active-site residues are superposable, in accord with the observation that SAM stabilizes 108M COMT. However, the methionine side chain is packed more tightly within the polymorphic site and, consequently, interacts more closely with residues A22 ({alpha}2) and R78 ({alpha}4) than does valine. These interactions of the larger methionine result in a 0.7-{angstrom} displacement in the backbone structure near residue 108, which propagates along {alpha}1 and {alpha}5 toward the SAM-binding site. Although the overall secondary structures of the human and rat proteins are very similar (C{sup {alpha}} RMSD = 0.4 {angstrom}), several nonconserved residues are present in the SAM-(I89M, I91M, C95Y) and catechol- (C173V, R201M, E202K) binding sites. The human protein also contains three additional solvent-exposed cysteine residues (C95, C173, C188) that may contribute to intermolecular disulfide bond

  7. Meta-analysis reveals a lack of association between a common catechol-O-methyltransferase (COMT) polymorphism val158met and fibromyalgia

    PubMed Central

    Zhang, Lei; Zhu, Junwei; Chen, Yong; Zhao, Jianning

    2014-01-01

    This study is to evaluate the association between the catechol-O-methyltransferase (COMT) gene val158met polymorphism and FM risk. We performed a meta-analysis of 8 case-control studies that included 589 FM cases and 527 case-free controls. We assessed the strength of the association, using odds ratios (ORs) with 95% confidence intervals (CIs). Overall, this meta-analysis showed that the COMT gene val158met polymorphism was not associated with FM risk in all genetic models, i.e., allele (met vs. val: OR=1.46, 95% CI=0.80-2.66, P heterpgeneity<0.001), homozygous (met/met vs. val/val: OR=1.72, 95% CI=0.61-4.87, P heterpgeneity<0.001), heterozygous (val/met vs. val/val: OR=1.25, 95% CI=0.82-1.92, P heterpgeneity=0.050), recessive (met/met vs. val/val+val/met: OR=1.52, 95% CI=0.60-3.86, P heterpgeneity<0.001) and dominant model (met/met+val/met vs. val/val: OR=1.52, 95% CI=0.80-2.90, P heterpgeneity<0.001). Similarly, there were no significant associations in the subgroup analyses by ethnicity and HWE. No publication bias was found in the present study. This meta-analysis suggests that the COMT gene val158met polymorphism is not associated with FM risk. Further large and well-designed studies are needed to confirm this association. PMID:25674213

  8. Interactions between genetic polymorphism of cytochrome P450-1B1, sulfotransferase 1A1, catechol-o-methyltransferase and tobacco exposure in breast cancer risk.

    PubMed

    Saintot, Monique; Malaveille, Christian; Hautefeuille, Agnès; Gerber, Mariette

    2003-11-20

    Genetic polymorphisms of enzymes involved in the metabolism of xenobiotics and estrogens might play a role in breast carcinogenesis related to environmental exposures. In a case-only study on 282 women with breast cancer, we studied the interaction effects (ORi) between smoking habits and the gene polymorphisms of Cytochrome P450 1B1 (Val432Leu CYP1B1), Phenol-sulfotransferase 1A1 (Arg213His SULT1A1) and Catechol-O-methyltransferase (Val158Met COMT). The smokers carrying the Val CYP1B1 allele associated with a high hydroxylation activity had a higher risk of breast cancer than never smokers with the Leu/Leu genotype (ORi=2.32, 95%CI: 1.00-5.38). Also, the smokers carrying the His SULT1A1 allele associated with a low sulfation activity had a 2-fold excess risk compared to never smokers carrying Arg/Arg SULT1A1 common genotype (ORi= 2.55, 95%CI: 1.21-5.36). The His SULT1A1 allele increased the risk only in premenopausal patients. The Met COMT allele with a lower methylation activity than Val COMT did not modify the risk among smokers. The excess risk due to joint effect could result from a higher exposure to activated tobacco-compounds for women homo/heterozygous for the Val CYP1B1 allele. Also, a lower sulfation of the tobacco carcinogens among women with His SULT1A1 could increase exposure to genotoxic compounds. Alternatively, the Val CYP1B1 or His SULT1A1 allele with modified ability to metabolize estrogens could increase the level of genotoxic catechol estrogen (i.e., 4-hydroxy-estradiol) among smokers. Our study showed that gene polymorphisms of CYP1B1 and SULT1A1 induce an individual susceptibility to breast cancer among current smokers.

  9. Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

    PubMed

    Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

    2012-08-01

    Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV.

  10. Amino acid regulation of gene expression.

    PubMed Central

    Fafournoux, P; Bruhat, A; Jousse, C

    2000-01-01

    The impact of nutrients on gene expression in mammals has become an important area of research. Nevertheless, the current understanding of the amino acid-dependent control of gene expression is limited. Because amino acids have multiple and important functions, their homoeostasis has to be finely maintained. However, amino-acidaemia can be affected by certain nutritional conditions or various forms of stress. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes. The aim of the present review is to examine the role of amino acids in regulating mammalian gene expression and protein turnover. It has been reported that some genes involved in the control of growth or amino acid metabolism are regulated by amino acid availability. For instance, limitation of several amino acids greatly increases the expression of the genes encoding insulin-like growth factor binding protein-1, CHOP (C/EBP homologous protein, where C/EBP is CCAAT/enhancer binding protein) and asparagine synthetase. Elevated mRNA levels result from both an increase in the rate of transcription and an increase in mRNA stability. Several observations suggest that the amino acid regulation of gene expression observed in mammalian cells and the general control process described in yeast share common features. Moreover, amino acid response elements have been characterized in the promoters of the CHOP and asparagine synthetase genes. Taken together, the results discussed in the present review demonstrate that amino acids, by themselves, can, in concert with hormones, play an important role in the control of gene expression. PMID:10998343

  11. Biosynthesis of t-Anethole in Anise: Characterization of t-Anol/Isoeugenol Synthase and an O-Methyltransferase Specific for a C7-C8 Propenyl Side Chain1[W][OA

    PubMed Central

    Koeduka, Takao; Baiga, Thomas J.; Noel, Joseph P.; Pichersky, Eran

    2009-01-01

    The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-l-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 μm for coumaryl acetate and 230 μm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 μm for isoeugenol and 54.5 μm for S-[methyl-14C]adenosyl-l-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol. PMID:18987218

  12. Biosynthesis of t-anethole in anise: characterization of t-anol/isoeugenol synthase and an O-methyltransferase specific for a C7-C8 propenyl side chain.

    PubMed

    Koeduka, Takao; Baiga, Thomas J; Noel, Joseph P; Pichersky, Eran

    2009-01-01

    The phenylpropene t-anethole imparts the characteristic sweet aroma of anise (Pimpinella anisum, family Apiaceae) seeds and leaves. Here we report that the aerial parts of the anise plant accumulate t-anethole as the plant matures, with the highest levels of t-anethole found in fruits. Although the anise plant is covered with trichomes, t-anethole accumulates inside the leaves and not in the trichomes or the epidermal cell layer. We have obtained anise cDNA encoding t-anol/isoeugenol synthase 1 (AIS1), an NADPH-dependent enzyme that can biosynthesize t-anol and isoeugenol (the latter not found in anise) from coumaryl acetate and coniferyl acetate, respectively. In addition, we have obtained a cDNA encoding S-[methyl-14C]adenosyl-l-methionine:t-anol/isoeugenol O-methyltransferase 1 (AIMT1), an enzyme that can convert t-anol or isoeugenol to t-anethole or methylisoeugenol, respectively, via methylation of the para-OH group. The genes encoding AIS1 and AIMT1 were expressed throughout the plant and their transcript levels were highest in developing fruits. The AIS1 protein is 59% identical to petunia (Petunia hybrida) isoeugenol synthase 1 and displays apparent Km values of 145 microm for coumaryl acetate and 230 microm for coniferyl acetate. AIMT1 prefers isoeugenol to t-anol by a factor of 2, with Km values of 19.3 microm for isoeugenol and 54.5 microm for S-[methyl-14C]adenosyl-l-methionine. The AIMT1 protein sequence is approximately 40% identical to basil (Ocimum basilicum) and Clarkia breweri phenylpropene O-methyltransferases, but unlike these enzymes, which do not show large discrimination between substrates with isomeric propenyl side chains, AIMT1 shows a 10-fold preference for t-anol over chavicol and for isoeugenol over eugenol.

  13. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

    PubMed Central

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang-Jun

    2016-01-01

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications. PMID:27349324

  14. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

    DOE PAGES

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; ...

    2016-06-28

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens’ lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in themore » yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Furthermore, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.« less

  15. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase.

    PubMed

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang-Jun

    2016-06-28

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.

  16. The BioC O-Methyltransferase Catalyzes Methyl Esterification of Malonyl-Acyl Carrier Protein, an Essential Step in Biotin Synthesis*

    PubMed Central

    Lin, Steven; Cronan, John E.

    2012-01-01

    Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682–688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-l-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-l-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis. PMID:22965231

  17. Catechol-O-methyltransferase inhibition protects against 3,4-dihydroxyphenylalanine (DOPA) toxicity in primary mesencephalic cultures: new insights into levodopa toxicity.

    PubMed

    Blessing, Heike; Bareiss, Markus; Zettlmeisl, Heinz; Schwarz, Johannes; Storch, Alexander

    2003-01-01

    Inhibition of catechol-O-methyltransferase (COMT) has protective effects on levodopa (L-DOPA), but not D-DOPA toxicity towards dopamine (DA) neurons in rat primary mesencephalic cultures [Mol. Pharmacol. 57 (2000) 589]. Here, we extend our recent studies to elucidate the mechanisms of these protective effects. Thus, we investigated the effects of all main L-DOPA/DA metabolites on survival of tyrosine hydroxylase immunoreactive (THir) neurons in primary rat mesencephalic cultures. 3-O-Methyldopa, homovanillic acid, dihydroxyphenyl acetate and 3-methoxytyramine had no effects at concentrations up to 300 micro M after 24h, whereas DA was more toxic than L-DOPA with toxicity at concentrations of >or=1 micro M. The coenzyme of COMT, S-adenosyl-L-methionine (SAM), and its demethylated product S-adenosylhomocystein caused no relevant alteration of THir neuron survival or L-DOPA toxicity. In contrast, inhibition of SAM synthesis by selenomethionine showed time- and dose-dependent increase of THir neuron survival, but did not affect L-DOPA toxicity. L-DOPA-induced lipid peroxidation in mesencephalic cultures was not modified by the COMT inhibitor Ro 41-0960 (1 micro M). Increased contamination of the cultures with glial cells attenuated L- and D-DOPA toxicity, but caused significant enhancement of protection by COMT inhibitors against L-DOPA toxicity only. Investigations of L-DOPA uptake in rat striatal cultures using HPLC revealed a significant reduction of extracellular L-DOPA concentrations by Ro 41-0960. Our data confirm that L-DOPA toxicity towards DA neurons is mediated by an autooxidative process, which is attenuated by glial cells. In addition, we demonstrate a second mechanism of L-DOPA toxicity in vitro mediated by a COMT- and glia-dependent pathway, which is blocked by COMT inhibitors, most likely due to enhanced glial uptake of L-DOPA.

  18. Integrated proteomic analysis of major isoaspartyl-containing proteins in the urine of wild type and protein L-isoaspartate O-methyltransferase-deficient mice.

    PubMed

    Dai, Shujia; Ni, Wenqin; Patananan, Alexander N; Clarke, Steven G; Karger, Barry L; Zhou, Zhaohui Sunny

    2013-02-19

    The formation of isoaspartyl residues (isoAsp or isoD) via either aspartyl isomerization or asparaginyl deamidation alters protein structure and potentially biological function. This is a spontaneous and nonenzymatic process, ubiquitous both in vivo and in nonbiological systems, such as in protein pharmaceuticals. In almost all organisms, protein L-isoaspartate O-methyltransferase (PIMT, EC2.1.1.77) recognizes and initiates the conversion of isoAsp back to aspartic acid. Additionally, alternative proteolytic and excretion pathways to metabolize isoaspartyl-containing proteins have been proposed but not fully explored, largely due to the analytical challenges for detecting isoAsp. We report here the relative quantitation and site profiling of isoAsp in urinary proteins from wild type and PIMT-deficient mice, representing products from excretion pathways. First, using a biochemical approach, we found that the total isoaspartyl level of proteins in urine of PIMT-deficient male mice was elevated. Subsequently, the major isoaspartyl protein species in urine from these mice were identified as major urinary proteins (MUPs) by shotgun proteomics. To enhance the sensitivity of isoAsp detection, a targeted proteomic approach using electron transfer dissociation-selected reaction monitoring (ETD-SRM) was developed to investigate isoAsp sites in MUPs. A total of 38 putative isoAsp modification sites in MUPs were investigated, with five derived from the deamidation of asparagine that were confirmed to contribute to the elevated isoAsp levels. Our findings lend experimental evidence for the hypothesized excretion pathway for isoAsp proteins. Additionally, the developed method opens up the possibility to explore processing mechanisms of isoaspartyl proteins at the molecular level, such as the fate of protein pharmaceuticals in circulation.

  19. Genetic variation in the beta2-adrenergic receptor but not catecholamine-O-methyltransferase predisposes to chronic pain: results from the 1958 British Birth Cohort Study.

    PubMed

    Hocking, Lynne J; Smith, Blair H; Jones, Gareth T; Reid, David M; Strachan, David P; Macfarlane, Gary J

    2010-04-01

    More than 1 in 10 adults in the general population experience chronic widespread body pain (CWP), which lies at one end of a continuous spectrum of pain ranging in both severity and duration. Neuroendocrine factors can modify the effect of known psychological and psychosocial risk factors for progression along the spectrum of pain and development of CWP, and genetic variants that affect neuroendocrine and neural processing potentially affect susceptibility to chronic pain development. We have examined variants across genes encoding the beta2-adrenergic receptor (ADRB2) and catecholamine-O-methyltransferase (COMT) - key neuroendocrine signalling factors - in a large population-based sample to determine whether these may be involved in pain progression and CWP development. A nested association study was conducted using individuals from the 1958 British Birth Cohort Study who had been assessed for pain status. Genotypes were available for nine single nucleotide polymorphisms (SNPs) across ADRB2 and 11 SNPs across COMT. ADRB2 SNPs rs12654778 and rs1042713 were associated either with CWP alone (p=0.02 for both) or with position along pain spectrum (pain status; p=0.04). Common functional ADRB2 haplotype combinations were also associated with pain status (p(model)=0.002) and, further, with both extent and duration of pain (p(model)=0.003 and p(model)=0.002, respectively). There were no associations of either CWP or pain status with COMT genotypes or haplotypes. These results are the first to suggest that functional ADRB2 variants are involved in regulating pain status at a population level. A role for COMT in chronic pain development was not identified, though could not be excluded.

  20. Effect of catechol-O-methyltransferase polymorphism on response to propranolol therapy in chronic musculoskeletal pain: A randomized, double–blind, placebo-controlled, crossover pilot study

    PubMed Central

    Tchivileva, Inna E.; Lim, Pei Feng; Smith, Shad B.; Slade, Gary D.; Diatchenko, Luda; McLean, Samuel A.; Maixner, William

    2010-01-01

    Introduction Three common haplotypes in the gene encoding catechol-O-methyltransferase (COMT) have been associated with pain modulation and the risk of developing chronic musculoskeletal pain, namely temporomandibular disorder (TMD). Haplotypes coding for higher enzymatic activity were correlated with lower pain perception. Rodent studies showed that COMT inhibition increases pain sensitivity via β2/3-adrenergic receptors. We hypothesized that the non-selective β-adrenergic antagonist propranolol will reduce clinical and experimental pain in TMD patients in a manner dependent on the subjects’ COMT diplotype. Methods 40 female Caucasian participants meeting the Research Diagnostic Criteria for TMD were genotyped for COMT polymorphisms and completed a randomized, double–blind, placebo-controlled, two-period crossover pilot study. Each period consisted of a baseline assessment week followed by an intervention week (propranolol or placebo). Changes in clinical pain ratings, psychological status, and responses to heat and pressure stimuli between baseline and intervention weeks were compared across periods. Results The number of patients reporting a reduction in pain intensity rating was greater during propranolol treatment (p=0.014) compared with placebo. Propranolol significantly reduced a composite pain index (p=0.02) but did not decrease other clinical and experimental pain ratings. When stratified by the COMT high activity haplotype, a beneficial effect of propranolol on pain perception was noted in subjects not carrying this haplotype, a diminished benefit was observed in the heterozygotes, and no benefit was noted in the homozygotes. Conclusion COMT haplotypes may serve as genetic predictors of propranolol treatment outcome, identifying a subgroup of TMD patients who will benefit from propranolol therapy. PMID:20216107

  1. Immediate reward bias in humans: fronto-parietal networks and a role for the catechol-O-methyltransferase 158(Val/Val) genotype.

    PubMed

    Boettiger, Charlotte A; Mitchell, Jennifer M; Tavares, Venessa C; Robertson, Margaret; Joslyn, Geoff; D'Esposito, Mark; Fields, Howard L

    2007-12-26

    The tendency to choose lesser immediate benefits over greater long-term benefits characterizes alcoholism and other addictive disorders. However, despite its medical and socioeconomic importance, little is known about its neurobiological mechanisms. Brain regions that are activated when deciding between immediate or delayed rewards have been identified (McClure et al., 2004, 2007), as have areas in which responses to reward stimuli predict a paper-and-pencil measure of temporal discounting (Hariri et al., 2006). These studies assume "hot" and "cool" response selection systems, with the hot system proposed to generate impulsive choices in the presence of a proximate reward. However, to date, brain regions in which the magnitude of activity during decision making reliably predicts intertemporal choice behavior have not been identified. Here we address this question in sober alcoholics and non-substance-abusing control subjects and show that immediate reward bias directly scales with the magnitude of functional magnetic resonance imaging bold oxygen level-dependent (BOLD) signal during decision making at sites within the posterior parietal cortex (PPC), dorsal prefrontal cortex (dPFC), and rostral parahippocampal gyrus regions. Conversely, the tendency of an individual to wait for a larger, delayed reward correlates directly with BOLD signal in the lateral orbitofrontal cortex. In addition, genotype at the Val158Met polymorphism of the catechol-O-methyltransferase gene predicts both impulsive choice behavior and activity levels in the dPFC and PPC during decision making. These genotype effects remained significant after controlling for alcohol abuse history. These results shed new light on the neurobiological underpinnings of temporal discounting behavior and identify novel behavioral and neural consequences of genetic variation in dopamine metabolism.

  2. Protective Role of Maternal P.VAL158MET Catechol-O-Methyltransferase Polymorphism against Early-Onset Preeclampsia and its Complications

    PubMed Central

    Mirković, Ljiljana; Ignjatović, Svetlana; Tomašević, Dragana; Lukić, Jelena; Topalov, Drina; Soldatović, Ivan; Majkić-Singh, Nada

    2016-01-01

    Summary Background Up until now there have been contradictory data about the association between p.Val158Met catechol-O-methyltransferase (COMT) polymorphism and risk of preeclampsia (PE). The goal of this study was to assess the potential correlation between p.Val158Met COMT polymorphism and risk of early-onset PE, risk of a severe form of early-onset PE, as well as risk of small-for-gestational-age (SGA) complicating PE. Methods The study included 47 early-onset PE patients and 47 control cases. Forty-seven early-onset PE patients were grouped by disease severity (33 patients with a severe form and 14 patients without severe features) and secondly by size for gestational age (12 patients with appropriate-for-gestational-age (AGA) and 35 patients with SGA size). p.Val158Met polymorphism was genotyped by PCR-RFLP analysis. Results Allele analysis showed significant difference in COMT allele distribution between early-onset PE and control group as well as early-onset PE SGA and controls (p=0.04057 and p=0.0411 respectively). A statistically significant distribution difference between the severe form and form without severe features of early-onset PE patients was not observed (p>0.05). The highest difference observed was in the allele recessive model where COMT MetMet genotype was associated with decreased risk of early-onset PE (OR=0.281; 95%CI = 0.092–0.7836) and PE complications including severe early-onset PE (OR= 0.304; 95%CI=0.086–0.944) and SGA early-onset PE (OR=0.284; 95%CI=0.081–0.874). Conclusions COMT may be used as a candidate gene for early-onset PE and its severe form and SGA complications. PMID:28356882

  3. Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O-methyltransferase activity in poplar monolignol biosynthesis

    PubMed Central

    Wang, Jack P.; Chuang, Ling; Loziuk, Philip L.; Chen, Hao; Lin, Ying-Chung; Shi, Rui; Qu, Guan-Zheng; Muddiman, David C.; Sederoff, Ronald R.; Chiang, Vincent L.

    2015-01-01

    Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn2+-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser123 or Ser125 in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser123 or Ser125 and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser123 phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser123 or Ser125. PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa. PMID:26109572

  4. Significant association of catechol-O-methyltransferase (COMT) haplotypes with nicotine dependence in male and female smokers of two ethnic populations.

    PubMed

    Beuten, Joke; Payne, Thomas J; Ma, Jennie Z; Li, Ming D

    2006-03-01

    The catechol-O-methyltransferase (COMT) gene plays a prominent role in dopaminergic circuits central to drug reward. Allelic variants within the COMT gene are therefore potential candidates for examining interindividual differences in vulnerability to nicotine dependence (ND). We analyzed five single nucleotide polymorphisms (SNPs), including the Val/Met variant (rs4680), which results in a three- to four-fold difference in enzyme activity within COMT, for association with the three ND measures, SQ, HSI, and FTND, in 602 nuclear families of African-American (AA) or European-American (EA) origin. The Val/Met variant showed a significant association with the three ND measures in the pooled and EA samples and with FTND in the AA sample. Haplotype analysis revealed a major protective A-G-T haplotype (frequency 23.6%) for rs740603-rs4680-rs174699 in the AA sample (minimum Z=-3.35; P=0.0005 for FTND), a major protective T-G-T haplotype (frequency 15.2%; minimum Z=-2.92; P=0.003 for FTND) in the EA sample, and a high-risk C-A-T haplotype (frequency 16.9%; minimum Z=3.16; P=0.002 for SQ) in the AA sample for rs933271-rs4680-rs174699. Furthermore, we found that the significant haplotypes within COMT were gender-specific and the significantly associated A-G-T is protective in AA females only, whereas T-G-T is protective in EA males only. Moreover, we found a major high-risk T-A-T haplotype (frequency 56.7%) that showed significant association with the three ND measures in EA males. Further examination of two protective haplotypes, A-G-T in AAs and T-G-T in EAs, indicated that the low COMT enzyme activity Met allele is protective to become nicotine dependent. In summary, our results provide evidence for a role of COMT in the susceptibility to ND and further confirm that its effect is ethnic and gender specific.

  5. Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase.

    PubMed

    Kimos, Martha; Burton, Maggi; Urbain, David; Caudron, Didier; Martini, Murielle; Famelart, Michel; Gillard, Michel; Barrow, James; Wood, Martyn

    2016-06-01

    Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson's disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)-based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.

  6. Catechol-O-methyltransferase (COMT) Genotype Affects Age-Related Changes in Plasticity in Working Memory: A Pilot Study

    PubMed Central

    Riemer, Thomas G.; Schulte, Stefanie; Onken, Johanna; Heinz, Andreas; Rapp, Michael A.

    2014-01-01

    Objectives. Recent work suggests that a genetic variation associated with increased dopamine metabolism in the prefrontal cortex (catechol-O-methyltransferase Val158Met; COMT) amplifies age-related changes in working memory performance. Research on younger adults indicates that the influence of dopamine-related genetic polymorphisms on working memory performance increases when testing the cognitive limits through training. To date, this has not been studied in older adults. Method. Here we investigate the effect of COMT genotype on plasticity in working memory in a sample of 14 younger (aged 24–30 years) and 25 older (aged 60–75 years) healthy adults. Participants underwent adaptive training in the n-back working memory task over 12 sessions under increasing difficulty conditions. Results. Both younger and older adults exhibited sizeable behavioral plasticity through training (P < .001), which was larger in younger as compared to older adults (P < .001). Age-related differences were qualified by an interaction with COMT genotype (P < .001), and this interaction was due to decreased behavioral plasticity in older adults carrying the Val/Val genotype, while there was no effect of genotype in younger adults. Discussion. Our findings indicate that age-related changes in plasticity in working memory are critically affected by genetic variation in prefrontal dopamine metabolism. PMID:24772423

  7. No association between germline variation in catechol-O-methyltransferase and colorectal cancer survival in postmenopausal women

    PubMed Central

    Passarelli, Michael N.; Newcomb, Polly A.; Makar, Karen W.; Burnett-Hartman, Andrea N.; Phipps, Amanda I.; David, Sean P.; Hsu, Li; Harrison, Tabitha A.; Hutter, Carolyn M.; Duggan, David J.; White, Emily; Chan, Andrew T.; Peters, Ulrike

    2013-01-01

    Objective Sex-steroid hormones play a role in colorectal cancer (CRC) development, but little is known about their influence on tumor progression and metastasis. Because catechol-O-methyltransferase activity (COMT; 22q11.21) is an important component of estrogen-mediated carcinogenesis, we hypothesized that germline variation in COMT may be associated with CRC survival. Methods We identified 10 single-nucleotide polymorphisms (SNPs) that tagged variation across both isoforms of COMT in 2,458 women with CRC from the Nurses’ Health Study (NHS), Postmenopausal Hormones Supplementary Study to the Colon Cancer Family Registry (PMH-CCFR), VITamins And Lifestyle (VITAL) Study, and Women’s Health Initiative (WHI). All four studies participate in the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). Results Over a median follow-up of 7 years across all studies, there were 799 deaths, including 566 from CRC. Accounting for multiple comparisons, no associations between the SNPs and CRC-specific or overall survival reached statistical significance, including the well-characterized Val108/158Met polymorphism (rs4680; hazard ratio per minor allele [HR], 1.04; 95% confidence interval [CI], 0.92–1.17 for CRC-specific survival and 1.01; 0.90–1.14 for overall survival). Conclusions In this large study of women with CRC, we found no evidence that common inherited variation in COMT is associated with survival-time after diagnosis. PMID:23880798

  8. How Large Should the QM Region Be in QM/MM Calculations? The Case of Catechol O-Methyltransferase.

    PubMed

    Kulik, Heather J; Zhang, Jianyu; Klinman, Judith P; Martínez, Todd J

    2016-11-10

    Hybrid quantum mechanical-molecular mechanical (QM/MM) simulations are widely used in studies of enzymatic catalysis. Until recently, it has been cost prohibitive to determine the asymptotic limit of key energetic and structural properties with respect to increasingly large QM regions. Leveraging recent advances in electronic structure efficiency and accuracy, we investigate catalytic properties in catechol O-methyltransferase, a prototypical methyltransferase critical to human health. Using QM regions ranging in size from reactants-only (64 atoms) to nearly one-third of the entire protein (940 atoms), we show that properties such as the activation energy approach within chemical accuracy of the large-QM asymptotic limits rather slowly, requiring approximately 500-600 atoms if the QM residues are chosen simply by distance from the substrate. This slow approach to asymptotic limit is due to charge transfer from protein residues to the reacting substrates. Our large QM/MM calculations enable identification of charge separation for fragments in the transition state as a key component of enzymatic methyl transfer rate enhancement. We introduce charge shift analysis that reveals the minimum number of protein residues (approximately 11-16 residues or 200-300 atoms for COMT) needed for quantitative agreement with large-QM simulations. The identified residues are not those that would be typically selected using criteria such as chemical intuition or proximity. These results provide a recipe for a more careful determination of QM region sizes in future QM/MM studies of enzymes.

  9. Metabolic Disposition of Luteolin Is Mediated by the Interplay of UDP-Glucuronosyltransferases and Catechol-O-Methyltransferases in Rats.

    PubMed

    Wang, Liping; Chen, Qingwei; Zhu, Lijun; Li, Qiang; Zeng, Xuejun; Lu, Linlin; Hu, Ming; Wang, Xinchun; Liu, Zhongqiu

    2017-03-01

    Luteolin partially exerts its biologic effects via its metabolites catalyzed by UDP-glucuronosyltransferases (UGTs) and catechol-O-methyltransferases (COMTs). However, the interplay of UGTs and COMTs in mediating luteolin disposition has not been well clarified. In this study, we investigated the glucuronidation and methylation pathways of luteolin mediated by the interplay of UGTs and COMTs in vivo and in vitro. A total of nine luteolin metabolites was detected in rat plasma and bile by liquid chromatography-tandem mass spectrometry, namely, three glucuronides, two methylated metabolites, and four methylated glucuronides. Luteolin-3'-glucuronide (Lut-3'-G) exhibited the highest systemic exposure among these metabolites. Kinetics studies in rat liver S9 fractions suggested two pathways, as follows: 1) Luteolin was glucuronidated to luteolin-7-glucuronide, luteolin-4'-glucuronide, and Lut-3'-G by UGTs, and then Lut-7-G was methylated to chrysoeriol-7-glucuronide and diosmetin-7-glucuronide by COMTs. 2) Alternatively, luteolin was methylated to chrysoeriol and diosmetin by COMTs, and then chrysoeriol and diosmetin were glucuronidated by UGTs to their respective glucuronides. The methylation rate of luteolin was significantly increased by the absence of glucuronidation, whereas the glucuronidation rate was increased by the absence of methylation, but to a lesser extent. In conclusion, two pathways mediated by the interplay of UGTs and COMTs are probably involved in the metabolic disposition of luteolin. The glucuronidation and methylation of luteolin compensate for each other, although glucuronidation is the predominant pathway.

  10. Catechol-O-methyltransferase is dispensable for vascular protection by estradiol in mouse models of atherosclerosis and neointima formation.

    PubMed

    Wilhelmson, Anna S; Bourghardt-Fagman, Johan; Gogos, Joseph A; Fogelstrand, Per; Tivesten, Asa

    2011-12-01

    Estradiol is converted to the biologically active metabolite 2-methoxyestradiol via the activity of the enzyme catechol-O-methyltransferase (COMT). Exogenous administration of both estradiol and 2-methoxyestradiol reduces experimental atherosclerosis and neointima formation, and COMT-dependent formation of 2-methoxyestradiol likely mediates the antimitogenic effect of estradiol on smooth muscle cells in vitro. This study evaluated whether 2-methoxyestradiol mediates the vasculoprotective actions of estradiol in vivo. Wild-type (WT) and COMT knockout (COMTKO) mice on an apolipoprotein E-deficient background were gonadectomized and treated with estradiol or placebo. Exogenous estradiol reduced atherosclerotic lesion formation in both females (WT, -78%; COMTKO, -82%) and males (WT, -48%; COMTKO, -53%) and was equally effective in both genotypes. We further evaluated how exogenous estradiol affected neointima formation after ligation of the carotid artery in ovariectomized female mice; estradiol reduced intimal hyperplasia to a similar extent in both WT (-80%) and COMTKO (-77%) mice. In ovarian-intact female COMTKO mice, atherosclerosis was decreased (-25%) compared with WT controls. In conclusion, the COMT enzyme is dispensable for vascular protection by exogenous estradiol in experimental atherosclerosis and neointima formation in vivo. Instead, COMT deficiency in virgin female mice with intact endogenous production of estradiol results in relative protection against atherosclerosis.

  11. A novel prokaryotic expression system for biosynthesis of recombinant human membrane-bound catechol-O-methyltransferase.

    PubMed

    Pedro, A Q; Bonifácio, M J; Queiroz, J A; Maia, C J; Passarinha, L A

    2011-11-10

    Membrane proteins constitute 20-30% of all proteins encoded by the genome of various organisms. While large amounts of purified proteins are required for pharmaceutical and crystallization attempts, there is an unmet need for the development of novel heterologous membrane protein overexpression systems. Specifically, we tested the application of Brevibacillus choshinensis cells for the biosynthesis of human membrane bound catechol-O-methyltransferase (hMBCOMT). In terms of the upstream stage moderate to high expression was obtained for complex media formulation with a value near 45 nmol/h/mg for hMBCOMT specific activity achieved at 20 h culture with 37°C and 250 rpm. Subsequently, the efficiency for reconstitution of hMBCOMT is markedly null in the presence of ionic detergents, such as sodium dodecyl sulphate (SDS). In general, for non-ionic and zwiterionic detergents, until a detergent critic micellar concentration (CMC) of 1.0 mM, hMBCOMT shows more biological activity at lower detergent concentrations while for detergent CMC higher than 1 mM, higher detergent concentrations seem to be ideal for hMBCOMT solubilization. Indeed, from the detergents tested, the non-ionic digitonin at 0.5% (w/v) appears to be the most suitable for hMBCOMT solubilization.

  12. Methylation of catechins and procyanidins by rat and human catechol-O-methyltransferase: metabolite profiling and molecular modeling studies.

    PubMed

    Weinert, Christoph H; Wiese, Stefanie; Rawel, Harshadrai M; Esatbeyoglu, Tuba; Winterhalter, Peter; Homann, Thomas; Kulling, Sabine E

    2012-02-01

    Catechins and procyanidins are major polyphenols in plant-derived foods. Despite intensive studies in recent years, neither their biochemical nor their toxicological properties have been clarified sufficiently. This study aimed to compare the methylation of catechins and procyanidins by the enzyme catechol-O-methyltransferase (COMT) in vitro. We conducted incubations with rat liver cytosol and human placental cytosol including S-adenosyl-l-methionine. The set of substrates comprised the catechins (-)-epicatechin (EC) and (+)-catechin (CAT), the procyanidin dimers B1, B2, B3, B4, B5, and B7 as well as procyanidin trimer C1. After extraction, metabolites were analyzed by means of liquid chromatography-electrospray ionization-mass spectrometry and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. EC and CAT were converted to two monomethylated metabolites each by human and rat COMT, with the 3'-O-methyl derivatives being consistently the main metabolites. Furthermore, the flavanyl units of procyanidins were methylated consecutively, leading to monomethylated and dimethylated dimeric metabolites as well as monomethylated, dimethylated, and trimethylated C1 metabolites. The methylation status of each flavanyl unit was determined by means of mass spectrometric quinone-methide fragmentation patterns. In addition, molecular modeling studies were performed with the aim to predict the preferred site of methylation and to verify the experimental data. In conclusion, our results indicate that the degree and position of methylation depend clearly on the three-dimensional structure of the entire substrate molecule.

  13. Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: interaction with cofactors and effect of osmolytes on unfolding.

    PubMed

    Chatterjee, Tanaya; Pal, Aritrika; Chakravarty, Devlina; Dey, Sucharita; Saha, Rudra P; Chakrabarti, Pinak

    2013-04-01

    Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.

  14. A structure-activity relationship study of catechol- O-methyltransferase inhibitors combining molecular docking and 3D QSAR methods

    NASA Astrophysics Data System (ADS)

    Tervo, Anu J.; Nyrönen, Tommi H.; Rönkkö, Toni; Poso, Antti

    2003-12-01

    A panel of 92 catechol- O-methyltransferase (COMT) inhibitors was used to examine the molecular interactions affecting their biological activity. COMT inhibitors are used as therapeutic agents in the treatment of Parkinson's disease, but there are limitations in the currently marketed compounds due to adverse side effects. This study combined molecular docking methods with three-dimensional structure-activity relationships (3D QSAR) to analyse possible interactions between COMT and its inhibitors, and to incite the design of new inhibitors. Comparative molecular field analysis (CoMFA) and GRID/GOLPE models were made by using bioactive conformations from docking experiments, which yielded q2 values of 0.594 and 0.636, respectively. The docking results, the COMT X-ray structure, and the 3D QSAR models are in agreement with each other. The models suggest that an interaction between the inhibitor's catechol oxygens and the Mg2+ ion in the COMT active site is important. Both hydrogen bonding with Lys144, Asn170 and Glu199, and hydrophobic contacts with Trp38, Pro174 and Leu198 influence inhibitor binding. Docking suggests that a large R1 substituent of the catechol ring can form hydrophobic contacts with side chains of Val173, Leu198, Met201 and Val203 on the COMT surface. Our models propose that increasing steric volume of e.g. the diethylamine tail of entacapone is favourable for COMT inhibitory activity.

  15. Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol*

    PubMed Central

    Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

    2015-01-01

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta. PMID:26378240

  16. In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression

    SciTech Connect

    Rimando A. M.; Liu C.; Pan, Z.; Polashock, J. J.; Dayan, F. E., Mizuno, C. S.; Snook, M. E.; Baerson, S. R.

    2012-04-01

    Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

  17. Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

    SciTech Connect

    Liu, C.; Deavours, B; Richard, S; Ferrer, J; Blount, J; Huhman, D; Dixon, R; Noel, J

    2006-01-01

    In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.

  18. Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

    SciTech Connect

    Liu, C.-J.; Deavours, B.E.; Richard, S.B.; Ferrer, J.-L.; Blount, J.W.; Huhman, D.; Dixon, R.A.; Noel, J.

    2007-07-10

    In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for the 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.

  19. Genetic contribution of catechol-O-methyltransferase polymorphism (Val158Met) in children with chronic tension-type headache.

    PubMed

    Fernández-de-las-Peñas, César; Ambite-Quesada, Silvia; Rivas-Martínez, Inés; Ortega-Santiago, Ricardo; de-la-Llave-Rincón, Ana Isabel; Fernández-Mayoralas, Daniel M; Pareja, Juan A

    2011-10-01

    Our aim was to investigate the relationship between Val158Met polymorphisms, headache, and pressure hypersensitivity in children with chronic tension-type headache (CTTH). A case-control study with blinded assessor was conducted. Seventy children with CTTH associated with pericranial tenderness and 70 healthy children participated. After amplifying Val158Met polymorphism by polymerase chain reactions, we assessed genotype frequencies and allele distributions. We classified children according to their Val158Met polymorphism: Val/Val, Val/Met, Met/Met. Pressure pain thresholds (PPT) were bilaterally assessed over the temporalis, upper trapezius, second metacarpal, and tibialis anterior muscles. The distribution of Val158Met genotypes was not significantly different (p = 0.335), between children with CTTH and healthy children, and between boys and girls (p = 0.872). Children with CTTH with the Met/Met genotype showed a longer headache history compared with those with Met/Val (p = 0.001) or Val/Val (p = 0.002) genotype. Children with CTTH with Met/Met genotype showed lower PPT over upper trapezius and temporalis muscles than children with CTTH with Met/Val or Val/Val genotype (p < 0.01). The Val158Met catechol-O-methyltransferase (COMT) polymorphism does not appear to be involved in predisposition to suffer from CTTH in children; nevertheless, this genetic factor may be involved in the phenotypic expression, as pressure hypersensitivity was greater in those CTTH children with the Met/Met genotype.

  20. Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase.

    PubMed

    Pfeiffer, Erika; Wefers, Daniel; Hildebrand, Andreas A; Fleck, Stefanie C; Metzler, Manfred

    2013-08-01

    The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.

  1. Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alchohol

    DOE PAGES

    Cai, Yuanheng; Shanklin, John; Mohammad -Wadud Bhuiya; ...

    2015-09-16

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create anmore » enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. Lastly, the resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.« less

  2. How Large Should the QM Region Be in QM/MM Calculations? The Case of Catechol O-Methyltransferase

    PubMed Central

    2016-01-01

    Hybrid quantum mechanical–molecular mechanical (QM/MM) simulations are widely used in studies of enzymatic catalysis. Until recently, it has been cost prohibitive to determine the asymptotic limit of key energetic and structural properties with respect to increasingly large QM regions. Leveraging recent advances in electronic structure efficiency and accuracy, we investigate catalytic properties in catechol O-methyltransferase, a prototypical methyltransferase critical to human health. Using QM regions ranging in size from reactants-only (64 atoms) to nearly one-third of the entire protein (940 atoms), we show that properties such as the activation energy approach within chemical accuracy of the large-QM asymptotic limits rather slowly, requiring approximately 500–600 atoms if the QM residues are chosen simply by distance from the substrate. This slow approach to asymptotic limit is due to charge transfer from protein residues to the reacting substrates. Our large QM/MM calculations enable identification of charge separation for fragments in the transition state as a key component of enzymatic methyl transfer rate enhancement. We introduce charge shift analysis that reveals the minimum number of protein residues (approximately 11–16 residues or 200–300 atoms for COMT) needed for quantitative agreement with large-QM simulations. The identified residues are not those that would be typically selected using criteria such as chemical intuition or proximity. These results provide a recipe for a more careful determination of QM region sizes in future QM/MM studies of enzymes. PMID:27704827

  3. Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.

    PubMed

    Cai, Yuanheng; Bhuiya, Mohammad-Wadud; Shanklin, John; Liu, Chang-Jun

    2015-10-30

    Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.

  4. Catechol metabolites of zeranol and 17β-estradiol: a comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase.

    PubMed

    Fleck, Stefanie C; Hildebrand, Andreas A; Pfeiffer, Erika; Metzler, Manfred

    2012-04-05

    α-Zearalanol (α-ZAL, zeranol) is a highly estrogenic macrocyclic β-resorcylic acid lactone, which is used as a growth promotor for cattle in various countries. We have recently reported that α-ZAL and its major metabolite zearalanone (ZAN) are hydroxylated at the aromatic ring by microsomes from human liver in vitro, thereby forming two catechol metabolites each. Thus, the oxidative metabolism of α-ZAL and ZAN resembles that of the endogenous steroidal estrogens 17β-estradiol (E2) and estrone (E1), which also give rise to two catechols each. As these catechol metabolites are believed to mediate the carcinogenicity of E2 and E1 by causing oxidative DNA damage and DNA adducts, their methylation by catechol-O-methyltransferase (COMT) is an important inactivation pathway. Here we report that hepatic microsomes from five species generate catechol metabolites of α-ZAL and ZAN, the highest amounts being formed by human liver microsomes, followed by rat, mouse, steer and swine. The microsomal extracts and the individual catechols of α-ZAL, ZAN, E2 and E1 were found to induce oxidative DNA damage, as measured by the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in a cell-free system. The ranking of pro-oxidant activity was 15-HO-ZAN>15-HO-α-ZAL≈4-HO-E2/E1≈2-HO-E2/E1>13-HO-ZAN>13-HO-α-ZAL. With respect to the rate of methylation by human hepatic COMT, the ranking was 2-HO-E2/E1>4-HO-E2/E1>15-HO-α-ZAL/ZAN>13-HO-α-ZAL/ZAN. Thus, some catechol metabolites of α-ZAL and ZAN are better pro-oxidants and poorer substrates of COMT than the catechols of E2 and E1. These findings warrant further investigations into the genotoxic potential of α-ZAL, which may constitute another biological activity in addition to its well-known estrogenicity.

  5. Differential Effects of the Catechol-O-Methyltransferase Val158Met Genotype on the Cognitive Function of Schizophrenia Patients and Healthy Japanese Individuals

    PubMed Central

    Tsuchimine, Shoko; Yasui-Furukori, Norio; Kaneda, Ayako; Kaneko, Sunao

    2013-01-01

    Background The functional polymorphism Val158Met in the catechol-O-methyltransferase (COMT) gene has been associated with differences in prefrontal cognitive functions in patients with schizophrenia and healthy individuals. Several studies have indicated that the Met allele is associated with better performance on measures of cognitive function. We investigated whether the COMT Val158Met genotype was associated with cognitive function in 149 healthy controls and 118 patients with schizophrenia. Methods Cognitive function, including verbal memory, working memory, motor speed, attention, executive function and verbal fluency, was assessed by the Brief Assessment of Cognition in Schizophrenia (BACS-J). We employed a one-way analysis of variance (ANOVA) and a multiple regression analysis to determine the associations between the COMT Val158Met genotype and the BACS-J measurements. Results The one-way ANOVA revealed a significant difference in the scores on the Tower of London, a measure of executive function, between the different Val158Met genotypes in the healthy controls (p = 0.023), and a post-hoc analysis showed significant differences between the scores on the Tower of London in the val/val genotype group (18.6 ± 2.4) compared to the other two groups (17.6 ± 2.7 for val/met and 17.1 ± 3.2 for met/met; p = 0.027 and p = 0.024, respectively). Multiple regression analyses revealed that executive function was significantly correlated with the Val158Met genotype (p = 0.003). However, no evidence was found for an effect of the COMT on any cognitive domains of the BACS-J in the patients with schizophrenia. Conclusion These data support the hypothesis that the COMT Val158Met genotype maintains an optimal level of dopamine activity. Further studies should be performed that include a larger sample size and include patients on and off medication, as these patients would help to confirm our findings. PMID:24282499

  6. Dopamine D3 Receptor Ser9Gly and Catechol-O-methyltransferase Val158Met Polymorphisms and Acute Pain in Sickle Cell Disease

    PubMed Central

    Jhun, Ellie; He, Ying; Yao, Yingwei; Molokie, Robert E.; Wilkie, Diana J.; Wang, Zaijie Jim

    2014-01-01

    Background Pain in sickle cell disease (SCD) is characterized by episodes of acute pain, primarily responsible for acute health care utilization, and persistent chronic pain. Pain severity and frequency vary significantly among SCD patients. In this study, we investigated the possible contribution of monoamine gene polymorphisms to pain variation. Methods Adult subjects with SCD completed PAINReportIt®, a computerized McGill Pain Questionnaire, from which we calculated the Composite Pain Index. Utilization data were obtained from the medical record and biweekly telephone calls for 12 months. Utilization is defined as admissions to the emergency department and/or the acute care center resulting from a sickle cell pain crisis. We performed genotyping for catechol-O-methyltransferase (COMT) Val158Met (rs4680) and dopamine D3 receptor(DRD3) Ser9Gly (rs6280) polymorphisms, which were analyzed for associations with pain phenotypes. Results Binary logistic models revealed that DRD3 Ser9Gly heterozygote patients were more likely not to have an acute pain crisis (odds ratio [OR] [95% confidence interval (CI)], 4.37 [1.39, 22.89]; p=0.020), which remained so when demographic variables were considered (OR [95% CI], 4.53 [1.41, 28.58]; p=0.016). COMT Val158Met Met allele showed lower probability for zero utilization (OR [95% CI], 0.32 [0.12, 0.83]; p=0.020) than the Val allele. In the negative binomial regression analysis, subjects with COMT Met/Met genotype had utilization incident rate ratio [95% CI] of 2.20 [1.21, 3.99] over those with Val/Val (p=0.010). Conclusions These exploratory findings suggest that DRD3 Ser9Gly and COMT Val158Met may contribute to pain heterogeneity in SCD, as suggested by the different rates of acute pain crisis. Specifically, SCD patients with the DRD3 homozygote genotypes, COMT 158 Met allele or Met/Met genotype are more likely to have acute care utilization, an indicator of acute pain. These results, however, will need to be further examined in

  7. Functional Analysis of Genetic Variation in Catechol-O-Methyltransferase (COMT): Effects on mRNA, Protein, and Enzyme Activity in Postmortem Human Brain

    PubMed Central

    Chen, Jingshan; Lipska, Barbara K.; Halim, Nader; Ma, Quang D.; Matsumoto, Mitsuyuki; Melhem, Samer; Kolachana, Bhaskar S.; Hyde, Thomas M.; Herman, Mary M.; Apud, Jose; Egan, Michael F.; Kleinman, Joel E.; Weinberger, Daniel R.

    2004-01-01

    Catechol-O-methyltransferase (COMT) is a key enzyme in the elimination of dopamine in the prefrontal cortex of the human brain. Genetic variation in the COMT gene (MIM 116790) has been associated with altered prefrontal cortex function and higher risk for schizophrenia, but the specific alleles and their functional implications have been controversial. We analyzed the effects of several single-nucleotide polymorphisms (SNPs) within COMT on mRNA expression levels (using reverse-transcriptase polymerase chain reaction analysis), protein levels (using Western blot analysis), and enzyme activity (using catechol methylation) in a large sample (n = 108) of postmortem human prefrontal cortex tissue, which predominantly expresses the -membrane-bound isoform. A common coding SNP, Val158Met (rs4680), significantly affected protein abundance and enzyme activity but not mRNA expression levels, suggesting that differences in protein integrity account for the difference in enzyme activity between alleles. A SNP in intron 1 (rs737865) and a SNP in the 3′ flanking region (rs165599)—both of which have been reported to contribute to allelic expression differences and to be associated with schizophrenia as part of a haplotype with Val—had no effect on mRNA expression levels, protein immunoreactivity, or enzyme activity. In lymphocytes from 47 subjects, we confirmed a similar effect on enzyme activity in samples with the Val/Met genotype but no effect in samples with the intron 1 or 3′ SNPs. Separate analyses revealed that the subject's sex, as well as the presence of a SNP in the P2 promoter region (rs2097603), had small effects on COMT enzyme activity. Using site-directed mutagenesis of mouse COMT cDNA, followed by in vitro translation, we found that the conversion of Leu at the homologous position into Met or Val progressively and significantly diminished enzyme activity. Thus, although we cannot exclude a more complex genetic basis for functional effects of COMT, Val is a

  8. Polymethylated Myricetin in Trichomes of the Wild Tomato Species Solanum habrochaites and Characterization of Trichome-Specific 3′/5′- and 7/4′-Myricetin O-Methyltransferases1[C][W][OA

    PubMed Central

    Schmidt, Adam; Li, Chao; Shi, Feng; Jones, A. Daniel; Pichersky, Eran

    2011-01-01

    Flavonoids are a class of metabolites found in many plant species. They have been reported to serve several physiological roles, such as in defense against herbivores and pathogens and in protection against harmful ultraviolet radiation. They also serve as precursors of pigment compounds found in flowers, leaves, and seeds. Highly methylated, nonglycosylated derivatives of the flavonoid myricetin flavonoid, have been previously reported from a variety of plants, but O-methyltransferases responsible for their synthesis have not yet been identified. Here, we show that secreting glandular trichomes (designated types 1 and 4) and storage glandular trichomes (type 6) on the leaf surface of wild tomato (Solanum habrochaites accession LA1777) plants contain 3,7,3′-trimethyl myricetin, 3,7,3′,5′-tetramethyl myricetin, and 3,7,3′,4′,5′-pentamethyl myricetin, with gland types 1 and 4 containing severalfold more of these compounds than type 6 glands and with the tetramethylated compound predominating in all three gland types. We have also identified transcripts of two genes expressed in the glandular trichomes and showed that they encode enzymes capable of methylating myricetin at the 3′ and 5′ and the 7 and 4′ positions, respectively. Both genes are preferentially expressed in secreting glandular trichome types 1 and 4 and to a lesser degree in storage trichome type 6, and the levels of the proteins they encode are correspondingly higher in types 1 and 4 glands compared with type 6 glands. PMID:21343428

  9. Inhibition of human catechol-O-methyltransferase-mediated dopamine O-methylation by daphnetin and its Phase II metabolites.

    PubMed

    Liang, Si-Cheng; Ge, Guang-Bo; Xia, Yang-Liu; Pei-Pei, Dong; Ping, Wang; Qi, Xiao-Yi; Cai-Xia, Tu; Ling, Yang

    2016-07-20

    1. Finding and developing inhibitors of catechol-O-methyltransferase (COMT) from natural products is highly recommended. Daphnetin, a naturally occurring catechol from the family thymelaeaceae, has a chemical structure similar to several potent COMT inhibitors reported previously. Here the potential of daphnetin and its Phase II metabolites as inhibitors of COMT was investigated with human liver cytosol (HLC). 2. Daphnetin and its methylated metabolite (8-O-methyldaphnetin) were found to inhibit COMT-mediated dopamine O-methylation in a dose-dependent manner. The IC50 values for daphnetin (0.51∼0.53 μM) and 8-O-methyldaphnetin (22.5∼24.3 μM) were little affected by changes in HLC concentrations. Further kinetic analysis showed the differences in inhibition type and parameters (Ki) between daphnetin (competitive, 0.37 μM) and 8-O-methyldaphnetin (noncompetitive, 25.7 μM). Other metabolites, including glucuronidated and sulfated species, showed negligible inhibition against COMT. By using in vitro-in vivo extrapolation (IV-IVE), a 24.3-fold increase in the exposure of the COMT substrates was predicted when they are co-administrated with daphnetin. 3. With high COMT-inhibiting activity, daphnetin could serve as a lead compound for the design and development of new COMT inhibitors. Also, much attention should be paid to the clinical impact of combination of daphnetin and herbal preparations containing daphnetin with the drugs primarily cleared by COMT.

  10. Catechol-O-Methyltransferase Val158Met Polymorphism on the Relationship between White Matter Hyperintensity and Cognition in Healthy People

    PubMed Central

    Liu, Mu-En; Huang, Chu-Chung; Yang, Albert C.; Tu, Pei-Chi; Yeh, Heng-Liang; Hong, Chen-Jee; Liou, Ying-Jay; Chen, Jin-Fan; Chou, Kun-Hsien; Lin, Ching-Po; Tsai, Shih-Jen

    2014-01-01

    Background White matter lesions can be easily observed on T2-weighted MR images, and are termed white matter hyperintensities (WMH). Their presence may be correlated with cognitive impairment; however, the relationship between regional WMH volume and catechol-O-methyltransferase (COMT) Val158Met polymorphism in healthy populations remains unclear. Methods We recruited 315 ethnic Chinese adults with a mean age of 54.9±21.8 years (range: 21–89 y) to examine the genetic effect of COMT on regional WMH and the manner in which they interact to affect cognitive function in a healthy adult population. Cognitive tests, structural MRI scans, and genotyping of COMT were conducted for each participant. Results Negative correlations between the Digit Span Forward (DSF) score and frontal WMH volumes (r = −.123, P = .032, uncorrected) were noted. For the genetic effect of COMT, no significant difference in cognitive performance was observed among 3 genotypic groups. However, differences in WMH volumes over the subcortical region (P = .016, uncorrected), whole brain (P = .047, uncorrected), and a trend over the frontal region (P = .050, uncorrected) were observed among 3 COMT genotypic groups. Met homozygotes and Met/Val heterozygotes exhibited larger WMH volumes in these brain regions than the Val homozygotes. Furthermore, a correlation between the DSF and regional WMH volume was observed only in Met homozygotes. The effect size (cohen’s f) revealed a small effect. Conclusions The results indicate that COMT might modulate WMH volumes and the effects of WMH on cognition. PMID:24551149

  11. Kinetic and inhibition studies on catechol-O-methyltransferase affinity labelling by N-(3,4-dihydroxyphenyl)maleimide.

    PubMed Central

    Piedrafita, F J; Fernandez-Alvarez, E; Nieto, O; Tipton, K F

    1992-01-01

    Initial velocity and product inhibition studies have been performed on soluble catechol-O-methyltransferase which has been partially purified from pig liver. The results are consistent with an ordered reaction mechanism, in which S-adenosyl-L-methionine (AdoMet) is the leading substrate. The enzyme is irreversibly inhibited by maleimide derivatives in a biphasic manner, which suggests a differential reaction with two thiol groups. N-(3,4-Dihydroxyphenyl)maleimide, which has a reactive moiety (maleimide ring) and an affinity moiety (catechol ring), acts as an affinity labelling compound on the more reactive SH group; AdoMet and Mg2+ protect against this modification. Total protection of this SH group results in a pseudo-first-order inhibition of the enzyme, with the apparent rate constant being proportional to the inhibitor concentration. All the other maleimide derivatives studied inhibited the enzyme by reacting with one of the two SH groups in a non-specific manner. The reaction of the other, more reactive, SH group was either specific (active-site-directed) or non-specific, depending on the substituent present in the affinity moiety and also on the length of an intermediate chain of methylene groups present between this moiety and the reactive maleimide ring. In the presence of both AdoMet and Mg2+, 3,5-dinitrocatechol, a reversible inhibitor of the enzyme which is competitive with respect to the catechol substrate, protects the enzyme from inactivation by any of the maleimide derivatives. The adducts of these maleimide derivatives formed with dithiothreitol inhibit the enzyme reversibly, showing inhibition patterns that are consistent with the mechanism deduced from the initial velocity and product inhibition studies. PMID:1417755

  12. Association study of a functional catechol-o-methyltransferase polymorphism and cognitive function in patients with dementia.

    PubMed

    Nedić, Gordana; Borovecki, Fran; Klepac, Natasa; Mubrin, Zdenko; Hajnsek, Sanja; Nikolac, Matea; Muck-Seler, Dorotea; Pivac, Nela

    2011-01-01

    A functional catechol-o-methyltransferase (COMT Val158/108Met) polymorphism, a valine (Val) to methionine (Met) substitution, has been associated with cognitive processing in the normal brain, older age, mild cognitive impairment and in various dementias. COMT is involved in the breakdown of dopamine and other catecholamines, especially in the frontal cortex; hence the carriers of Met allele, with the lower enzymatic activity, are expected to perform better on particular neuro-cognitive tests. The study included 46 patients with dementia and 65 healthy older subjects. The neurological status was assessed, using the Mini Mental Status Examination (MMSE), and the batery of different neurological tests. In DNA samples COMT polymorphism was genotyped. Patients with dementia exhibited significant genotype-induced differences in scores for MMSE, Visual Association Test (VAT) duration of numbers test, VAT time of response to numbers test, VAT average response to numbers test and WPLCR/PPLR unanswered. Carriers of Met/Met genotype had significantly lower scores of MMSE, significantly longer time to respond to VAT duration of numbers test, VAT time of response to numbers test and VAT average response to numbers test, and significantly greater number of unanswered questions to WPLCR/PPLR when compared to Met/Val or Val/Val genotypes. Our preliminary data showed significantly impaired performance in several neuro-cognitive tests in carriers of Met/Met genotype in patients with dementia compared to either Met/Val or Val/Val genotype carriers. Although Met/Met genotype with more dopamine available in the frontal cortex should be associated with better neuro-cognitive test results than Met/Val or Val/Val genotype, our data on patients with dementia did not confirm this hypothesis. Further study on larger sample of patients is needed to clarify the role of COMT polymorphism in cognitive functions.

  13. Voxelwise eigenvector centrality mapping of the human functional connectome reveals an influence of the catechol-O-methyltransferase val158met polymorphism on the default mode and somatomotor network.

    PubMed

    Markett, Sebastian; Montag, Christian; Heeren, Behrend; Saryiska, Rayna; Lachmann, Bernd; Weber, Bernd; Reuter, Martin

    2016-06-01

    Functional connections between brain regions constitute the substrate of the human functional connectome, whose topography has been discussed as an endophenotype for psychiatric disorders. Genetic influences on the entire connectome, however, have been rarely investigated so far. We tested for connectome-wide influences of the val158met (rs4860) polymorphism on the catechol-O-methyltransferase (COMT) gene by applying formal network analysis and eigenvector centrality mapping on the voxel level to resting-state functional magnetic imaging data. This approach finds brain regions that are central in the network by aggregating local and global connectivity patterns, most importantly without the requirement to select regions or networks of interest. The COMT variant linked to high enzyme activity increased network centrality in distributed brain areas that are known to constitute the brain's default mode network. Further results also indicated a COMT influence on areas implicated in the somatomotor network. These findings are in line with the polymorphism's alleged role in cognitive processing and its role in psychotic disorders. The study is the first to demonstrate the influence of a functional and behaviorally relevant genetic variant on connectome-wide functional connectivity and is an important step toward establishing the functional connectome as an endophenotype for psychiatric and behavioral phenotypes.

  14. Genetic moderation of the effects of cannabis: catechol-O-methyltransferase (COMT) affects the impact of Δ9-tetrahydrocannabinol (THC) on working memory performance but not on the occurrence of psychotic experiences.

    PubMed

    Tunbridge, Elizabeth M; Dunn, Graham; Murray, Robin M; Evans, Nicole; Lister, Rachel; Stumpenhorst, Katharina; Harrison, Paul J; Morrison, Paul D; Freeman, Daniel

    2015-11-01

    Cannabis use can induce cognitive impairments and psychotic experiences. A functional polymorphism in the catechol-O-methyltransferase (COMT) gene (Val(158)Met) appears to influence the immediate cognitive and psychotic effects of cannabis, or ∆(9)-tetrahydrocannabinol (THC), its primary psychoactive ingredient. This study investigated the moderation of the impact of experimentally administered THC by COMT. Cognitive performance and psychotic experiences were studied in participants without a psychiatric diagnosis, using a between-subjects design (THC vs. placebo). The effect of COMT Val(158)Met genotype on the cognitive and psychotic effects of THC, administered intravenously in a double-blind, placebo-controlled manner to 78 participants who were vulnerable to paranoia, was examined. The results showed interactive effects of genotype and drug group (THC or placebo) on working memory, assayed using the Digit Span Backwards task. Specifically, THC impaired performance in COMT Val/Val, but not Met, carriers. In contrast, the effect of THC on psychotic experiences, measured using the Community Assessment of Psychic Experiences (CAPE) positive dimension, was unaffected by COMT genotype. This study is the largest to date examining the impact of COMT genotype on response to experimentally administered THC, and the first using a purely non-clinical cohort. The data suggest that COMT genotype moderates the cognitive, but not the psychotic, effects of acutely administered THC.

  15. Mapping of glutamic acid decarboxylase (GAD) genes

    SciTech Connect

    Edelhoff, S.; Adler, D.A.; Disteche, C.M.; Grubin, C.E.; Karlsen, A.E.; Lernmark, A.; Foster, D. )

    1993-07-01

    Glutamic acid decarboxylase (GAD) catalyzes the synthesis of [gamma]-aminobutyric acid (GABA), which is known as a major inhibitory neurotransmitter in the central nervous system (CNS), but is also present outside the CNS. Recent studies showed that GAD is the major target of autoantibodies associated with the development of insulin-dependent diabetes mellitus and of the rare stiff man syndrome. Studies of GAD expression have demonstrated multiple transcripts, suggesting several isoforms of GAD. In this study, three different genes were mapped by in situ hybridization to both human and mouse chromosomes. The GAD1 gene was mapped to human chromosome 2q31 and to mouse chromosome 2D in a known region of conservation between human and mouse. GAD2, previously mapped to human chromosome 10p11.2-p12, was mapped to mouse chromosome 2A2-B, which identifies a new region of conservation between human and mouse chromosomes. A potential GAD3 transcript was mapped to human chromosome 22q13 and to mouse chromosome 15E in a known region of conservation between human and mouse. It is concluded that the GAD genes may form a family with as many as three related members. 30 refs., 5 figs.

  16. Catechol-O-methyltransferase inhibition with tolcapone reduces the "wearing off" phenomenon and levodopa requirements in fluctuating parkinsonian patients

    PubMed Central

    Baas, H; Beiske, A; Ghika, J; Jackson, M; Oertel, W; Poewe, W; Ransmayr, G

    1997-01-01

    BACKGROUND—More than 50% of patients with Parkinson's disease develop motor response fluctuations (the "wearing off" phenomenon) after more than five years of levodopa therapy. Inhibition of catechol-O-methyltransferase by tolcapone has been shown to increase levodopa bioavailability and plasma elimination half life, thereby prolonging the efficacy of levodopa.
OBJECTIVES—The primary objective was to evaluate the efficacy of tolcapone in reducing "wearing off" in levodopa treated, fluctuating parkinsonian patients. Secondary objectives included assessment of reduction in levodopa requirements, improvement in patients' clinical status, duration of improvements, and tolerability of tolcapone.
METHODS—In this multicentre, randomised, double blind, placebo controlled trial, 58 patients received placebo, 60 received 100 mg tolcapone three times daily (tid), and 59 received 200 mg tolcapone tid, in addition to levodopa/benserazide.
RESULTS—After three months with 200 mg tolcapone tid, "off" time decreased by 26.2% of the baseline value, "on" time increased by 20.6% (P<0.01 v placebo), and the mean total daily levodopa dose decreased by 122 mg from the baseline dose of 676 mg (P<0.01). These responses were maintained up to nine months. With 100 mg tolcapone tid, "off" time decreased by 31.5% (P<0.05), "on" time increased by 21.3% (P<0.01), and the mean total daily levodopa dose decreased by 109 mg from the baseline dose of 668 mg (P<0.05). With 200 mg tolcapone tid, unified Parkinson's disease rating scale motor and total scores were significantly reduced, and quality of life (sickness impact profile) scores were significantly improved. Both dosages were well tolerated. Dyskinesia was the most often reported levodopa induced adverse event. Diarrhoea was the most often reported non-dopaminergic adverse event and the most frequent reason for withdrawal from the study: four patients in the 100mg tolcapone tid group and six in the 200 mg tid group withdrew

  17. Genetic Analysis of Strawberry Fruit Aroma and Identification of O-Methyltransferase FaOMT as the Locus Controlling Natural Variation in Mesifurane Content1[C][W][OA

    PubMed Central

    Zorrilla-Fontanesi, Yasmín; Rambla, José-Luis; Cabeza, Amalia; Medina, Juan J.; Sánchez-Sevilla, José F.; Valpuesta, Victoriano; Botella, Miguel A.; Granell, Antonio; Amaya, Iraida

    2012-01-01

    Improvement of strawberry (Fragaria × ananassa) fruit flavor is an important goal in breeding programs. To investigate genetic factors controlling this complex trait, a strawberry mapping population derived from genotype ‘1392’, selected for its superior flavor, and ‘232’ was profiled for volatile compounds over 4 years by headspace solid phase microextraction coupled to gas chromatography and mass spectrometry. More than 300 volatile compounds were detected, of which 87 were identified by comparison of mass spectrum and retention time to those of pure standards. Parental line ‘1392’ displayed higher volatile levels than ‘232’, and these and many other compounds with similar levels in both parents segregated in the progeny. Cluster analysis grouped the volatiles into distinct chemically related families and revealed a complex metabolic network underlying volatile production in strawberry fruit. Quantitative trait loci (QTL) detection was carried out over 3 years based on a double pseudo-testcross strategy. Seventy QTLs covering 48 different volatiles were detected, with several of them being stable over time and mapped as major QTLs. Loci controlling γ-decalactone and mesifurane content were mapped as qualitative traits. Using a candidate gene approach we have assigned genes that are likely responsible for several of the QTLs. As a proof of concept we show that one homoeolog of the O-methyltransferase gene (FaOMT) is the locus responsible for the natural variation of mesifurane content. Sequence analysis identified 30 bp in the promoter of this FaOMT homoeolog containing putative binding sites for basic/helix-loop-helix, MYB, and BZIP transcription factors. This polymorphism fully cosegregates with both the presence of mesifurane and the high expression of FaOMT during ripening. PMID:22474217

  18. Mouse Vk gene classification by nucleic acid sequence similarity.

    PubMed

    Strohal, R; Helmberg, A; Kroemer, G; Kofler, R

    1989-01-01

    Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates of V gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i.e., sequence similarity) and their organization in gene families. While mouse Igh heavy chain V region (VH) gene families are relatively well-established, a corresponding systematic classification of Igk light chain V region (Vk) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of the Vk germline gene repertoire and Vk gene usage in a variety of responses to foreign and self antigens, provides a classification of mouse Vk genes in gene families composed of members with greater than 80% overall nucleic acid sequence similarity. This classification differed in several aspects from that of VH genes: only some Vk gene families were as clearly separated (by greater than 25% sequence dissimilarity) as typical VH gene families; most Vk gene families were closely related and, in several instances, members from different families were very similar (greater than 80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications for Vk gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization between VH and Vk genes.

  19. Association of the Catechol O-Methyltransferase Val158-Met Polymorphism and Reduced Interference Control in Korean Children with Attention-Deficit Hyperactivity Disorder

    PubMed Central

    Park, Subin; Park, Jong-Eun; Yoo, Hee Jeong; Kim, Jae-Won; Cheong, Jae Hoon; Han, Doug Hyun; Kim, Yeni

    2015-01-01

    Objective We tested for association of the catechol-O-methyltransferase (COMT) Val158-Met (rs4680) polymorphism with attention-deficit hyperactivity disorder (ADHD) using family-based test in Korean trios. Methods A total of 181 subjects with ADHD along with both of their biological parents were recruited from University Hospitals in Korea. We performed a transmission disequilibrium test (TDT) on 181 trios. Results In the TDT, we found the over-transmission of the Val allele in children with ADHD (χ2=4.21, p=0.040). Conclusion These results suggest that the COMT Val158-Met polymorphism is associated with ADHD among the Korean population. However, this study must be replicated in larger populations. PMID:26508970

  20. Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

    SciTech Connect

    Culman, J.; Torda, T.; Weise, V.K.

    1987-08-01

    A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.

  1. Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

    SciTech Connect

    Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang -Jun

    2016-06-28

    Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens’ lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Furthermore, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.

  2. Structural characterization of CalO1: a putative orsellinic acid methyltransferase in the calicheamicin-biosynthetic pathway

    SciTech Connect

    Chang, Aram; Singh, Shanteri; Bingman, Craig A.; Thorson, Jon S.; Phillips, Jr, George N.

    2011-11-07

    The X-ray structure determination at 2.4 {angstrom} resolution of the putative orsellinic acid C3 O-methyltransferase (CalO1) involved in calicheamicin biosynthesis is reported. Comparison of CalO1 with a homology model of the functionally related calicheamicin orsellinic acid C2 O-methyltransferase (CalO6) implicates several residues that are likely to contribute to the regiospecificity of alkylation. Consistent with the proposed requirement of an acyl-carrier-protein-bound substrate, this structural study also reveals structural determinants within CalO1 that are anticipated to accommodate an association with an acyl carrier protein.

  3. Gene Expressions for Signal Transduction under Acidic Conditions

    PubMed Central

    Fukamachi, Toshihiko; Ikeda, Syunsuke; Wang, Xin; Saito, Hiromi; Tagawa, Masatoshi; Kobayashi, Hiroshi

    2013-01-01

    Although it is now well known that some diseased areas, such as cancer nests, inflammation loci, and infarction areas, are acidified, little is known about cellular signal transduction, gene expression, and cellular functions under acidic conditions. Our group showed that different signal proteins were activated under acidic conditions compared with those observed in a typical medium of around pH 7.4 that has been used until now. Investigations of gene expression under acidic conditions may be crucial to our understanding of signal transduction in acidic diseased areas. In this study, we investigated gene expression in mesothelioma cells cultured at an acidic pH using a DNA microarray technique. After 24 h culture at pH 6.7, expressions of 379 genes were increased more than twofold compared with those in cells cultured at pH 7.5. Genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors numbered 35, 32, and 17 among the 379 genes, respectively. Since the functions of 78 genes are unknown, it can be argued that cells may have other genes for signaling under acidic conditions. The expressions of 37 of the 379 genes were observed to increase after as little as 2 h. After 24 h culture at pH 6.7, expressions of 412 genes were repressed more than twofold compared with those in cells cultured at pH 7.5, and the 412 genes contained 35, 76, and 7 genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors, respectively. These results suggest that the signal pathways in acidic diseased areas are different, at least in part, from those examined with cells cultured at a pH of around 7.4. PMID:24705103

  4. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy.

    PubMed

    Desgagné-Penix, Isabel; Facchini, Peter J

    2012-10-01

    Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)-norcoclaurine to papaverine involves 3'-hydroxylation, four O-methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)-reticuline, and the enzymes involved. We used virus-induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N-methyltransferase dramatically increased papaverine levels at the expense of N-methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N-desmethylated compounds rather than through (S)-reticuline. Suppression of genes encoding (S)-3'-hydroxy-N-methylcoclaurine 4-O-methyltransferase and norreticuline 7-O-methyltransferase, which accept certain N-desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N-methylcoclaurine 3'-hydroxylase or reticuline 7-O-methyltransferase, which are specific for N-methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)-coclaurine as a key branch-point intermediate. The differential detection of N-desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine.

  5. Determination of the structure and catalytic mechanism of sorghum bicolor caffeoyl-CoA O-methyltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although cold acclimation is a key process in plants from temperate climates, the mechanisms sensing low temperature remain obscure. Here, we show that the accumulation of the organic acid fumaric acid, mediated by the cytosolic fumarase FUM2, is essential for cold acclimation of metabolism in the c...

  6. Gene quantification by the NanoGene assay is resistant to inhibition by humic acids.

    PubMed

    Kim, Gha-Young; Wang, Xiaofang; Ahn, Hosang; Son, Ahjeong

    2011-10-15

    NanoGene assay is a magnetic bead and quantum dot nanoparticles based gene quantification assay. It relies on a set of probe and signaling probe DNAs to capture the target DNA via hybridization. We have demonstrated the inhibition resistance of the NanoGene assay using humic acids laden genomic DNA (gDNA). At 1 μg of humic acid per mL, quantitiative PCR (qPCR) was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 60% of its quantification capability. To further increase the inhibition resistance of NanoGene assay at high concentration of humic acids, we have identified the specific mechanisms that are responsible for the inhibition. We examined five potential mechanisms with which the humic acids can partially inhibit our NanoGene assay. The mechanisms examined were (1) adsorption of humic acids on the particle surface; (2) particle aggregation induced by humic acids; (3) fluorescence quenching of quantum dots by humic acids during hybridization; (4) humic acids mimicking of target DNA; and (5) nonspecific binding between humic acids and target gDNA. The investigation showed that no adsorption of humic acids onto the particles' surface was observed for the humic acids' concentration. Particle aggregation and fluorescence quenching were also negligible. Humic acids also did not mimic the target gDNA except 1000 μg of humic acids per mL and hence should not contribute to the partial inhibition. Four of the above mechanisms were not related to the inhibition effect of humic acids particularly at the environmentally relevant concentrations (<100 μg/mL). However, a substantial amount of nonspecific binding was observed between the humic acids and target gDNA. This possibly results in lesser amount of target gDNA being captured by the probe and signaling DNA.

  7. Polymorphisms in Dopamine System Genes Are Associated with Individual Differences in Attention in Infancy

    ERIC Educational Resources Information Center

    Holmboe, Karla; Nemoda, Zsofia; Fearon, R. M. Pasco; Csibra, Gergely; Sasvari-Szekely, Maria; Johnson, Mark H.

    2010-01-01

    Knowledge about the functional status of the frontal cortex in infancy is limited. This study investigated the effects of polymorphisms in four dopamine system genes on performance in a task developed to assess such functioning, the Freeze-Frame task, at 9 months of age. Polymorphisms in the catechol-O-methyltransferase ("COMT") and the…

  8. Novel acid resistance genes from the metagenome of the Tinto River, an extremely acidic environment.

    PubMed

    Guazzaroni, María-Eugenia; Morgante, Verónica; Mirete, Salvador; González-Pastor, José E

    2013-04-01

    Microorganisms that thrive in acidic environments are endowed with specialized molecular mechanisms to survive under this extremely harsh condition. In this work, we performed functional screening of six metagenomic libraries from planktonic and rhizosphere microbial communities of the Tinto River, an extremely acidic environment, to identify genes involved in acid resistance. This approach has revealed 15 different genes conferring acid resistance to Escherichia coli, most of which encoding putative proteins of unknown function or previously described proteins not known to be related to acid resistance. Moreover, we were able to assign function to one unknown and three hypothetical proteins. Among the recovered genes were the ClpXP protease, the transcriptional repressor LexA and nucleic acid-binding proteins such as an RNA-binding protein, HU and Dps. Furthermore, nine of the retrieved genes were cloned and expressed in Pseudomonas putida and Bacillus subtilis and, remarkably, most of them were able to expand the capability of these bacteria to survive under severe acid stress. From this set of genes, four presented a broad-host range as they enhance the acid resistance of the three different organisms tested. These results expand our knowledge about the different strategies used by microorganisms to survive under extremely acid conditions.

  9. Association of catechol-O-methyltransferase Val(108/158) Met genetic polymorphism with schizophrenia, P50 sensory gating, and negative symptoms in a Chinese population.

    PubMed

    Mao, Qiao; Tan, Yun-Long; Luo, Xing-Guang; Tian, Li; Wang, Zhi-Ren; Tan, Shu-Ping; Chen, Song; Yang, Gui-Gang; An, Hui-Mei; Yang, Fu-De; Zhang, Xiang-Yang

    2016-08-30

    Catechol-O-methyltransferase (COMT), an enzyme involved in the degradation and inactivation of the neurotransmitter dopamine, is associated with the sensory gating phenomenon, protecting the cerebral cortex from information overload. The COMT Val(108/158)Met polymorphism is essential for prefrontal cortex processing capacity and efficiency. The current study was designed to investigate the role of COMT Val(108/158)Met polymorphism in development, sensory gating deficit, and symptoms of schizophrenia in Han Chinese population. P50 gating was determined in 139 schizophrenic patients and 165 healthy controls. Positive and Negative Syndrome Scale (PANSS) was used to assess the clinical symptomatology in 370 schizophrenic subjects. COMT Val(108/158)Met polymorphism was genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP). No significant differences in COMT allele and genotype distributions were observed between schizophrenic patients and control groups. Although P50 deficits were present in patients, there was no evidence for an association between COMT Val(108/158)Met polymorphism and the P50 biomarker. Moreover, PANSS negative subscore was significantly higher in Val allele carriers than in Met/Met individuals. The present findings suggest that COMT Val(108/158)Met polymorphism may not contribute to the risk of schizophrenia and to the P50 deficits, but may contribute to the negative symptoms of schizophrenia among Han Chinese.

  10. Association between the Catechol-O-Methyltransferase (COMT) Val158Met Polymorphism and Alexithymia in Patients with Obsessive-Compulsive Disorder

    PubMed Central

    Koh, Min Jung; Kang, Jee In; Namkoong, Kee; Lee, Su Young

    2016-01-01

    Purpose Alexithymia, defined as a deficit in the ability to recognize and describe one's own feelings, may be related to the development and maintenance of obsessive-compulsive symptoms. The aim of this study was to evaluate the association between the catechol-O-methyltransferase (COMT) Val158Met polymorphism and alexithymia in patients with obsessive-compulsive disorder (OCD). Materials and Methods We recruited 244 patients with OCD (169 males, 75 females). Alexithymia was assessed using the 20-item Toronto Alexithymia Scale (TAS-20), and genotyping of the COMT Val158Met polymorphism was evaluated. Results Patients with the COMT Val/Val genotype had significantly higher total and "difficulty identifying feelings" (DIF) subdimension scores than those with the Val/Met or Met/Met genotypes. Patients with the COMT Val/Val genotype had significantly higher "difficulty describing feelings" (DDF) subdimension scores than those with the COMT Val/Met genotype. However, there were no differences in the scores for the "externally oriented thinking" (EOT) subdimension among the three genotypes. Conclusion These results indicate that the high-activity Val allele of the COMT Val158Met polymorphism is associated with increased alexithymic traits in patients with OCD. The present finding suggests that alexithymia is an endophenotype of OCD that is mediated by the COMT Val158Met polymorphism. PMID:26996573

  11. Catechol-O-methyltransferase Val(108/158)Met polymorphism affects fronto-limbic connectivity during emotional processing in bipolar disorder.

    PubMed

    Vai, B; Riberto, M; Poletti, S; Bollettini, I; Lorenzi, C; Colombo, C; Benedetti, F

    2016-12-30

    Catechol-O-methyltransferase (COMT) inactivates catecholamines, Val/Val genotype was associated to an increased amygdala (Amy) response to negative stimuli and can influence the symptoms severity and the outcome of bipolar disorder, probably mediated by the COMT polymorphism (rs4680) interaction between cortical and subcortical dopaminergic neurotransmission. The aim of this study is to explore how rs4680 and implicit emotional processing of negative emotional stimuli could interact in affecting the Amy connectivity in bipolar depression. Forty-five BD patients (34 Met carriers vs. 11 Val/Val) underwent fMRI scanning during implicit processing of fearful and angry faces. We explore the effect of rs4680 on the strength of functional connectivity from the amygdalae to whole brain. Val/Val and Met carriers significantly differed for the connectivity between Amy and dorsolateral prefrontal cortex (DLPFC) and supramarginal gyrus. Val/Val patients showed a significant positive connectivity for all of these areas, where Met carriers presented a significant negative one for the connection between DLPFC and Amy. Our findings reveal a COMT genotype-dependent difference in corticolimbic connectivity during affective regulation, possibly identifying a neurobiological underpinning of clinical and prognostic outcome of BD. Specifically, a worse antidepressant recovery and clinical outcome previously detected in Val/Val patients could be associated to a specific increased sensitivity to negative emotional stimuli.

  12. Effect of 3 Single-Dose Regimens of Opicapone on Levodopa Pharmacokinetics, Catechol-O-Methyltransferase Activity and Motor Response in Patients With Parkinson Disease.

    PubMed

    Rocha, José-Francisco; Ferreira, Joaquim J; Falcão, Amílcar; Santos, Ana; Pinto, Roberto; Nunes, Teresa; Almeida, Luis; Soares-da-Silva, Patrício

    2016-05-01

    This study determined the effects of single doses of opicapone (OPC), a novel third-generation catechol-O-methyltransferase (COMT) inhibitor, on levodopa and 3-O-methyl-levodopa (3-OMD) pharmacokinetics (PK), COMT activity and motor fluctuations in patients with Parkinson disease (PD). Subjects received, in a double-blind manner, 25, 50, and 100 mg OPC or placebo (PLC) in 4 separate treatment periods. The washout period between doses was at least 10 days. During each period, the OPC/PLC capsules were to be coadministered with the morning dose of 100/25 mg levodopa/carbidopa (LC) or levodopa/benserazide (LB) on day 3. In relation to PLC, levodopa exposure increased 3.7%, 16.4%, and 34.8% following 25, 50, or 100 mg OPC, respectively. Maximum S-COMT inhibition (Emax ) ranged from 67.8% (25 mg OPC) to 100% (100 mg OPC). Peak and extent of S-COMT inhibition were dose-dependent. Maximum decrease in the plasma 3-OMD was observed following administration of 100 mg OPC. Opicapone administered concomitantly with standard-release 100/25 mg LC or LB improved motor performance. Treatments were generally well tolerated and safe. It was concluded that OPC is a new COMT inhibitor that significantly decreased COMT activity and increased systemic exposure to levodopa in PD patients with motor fluctuations.

  13. The catechol-o-methyltransferase Val158Met polymorphism modulates the intrinsic functional network centrality of the parahippocampal cortex in healthy subjects

    PubMed Central

    Zhang, Xiaolong; Li, Jin; Qin, Wen; Yu, Chunshui; Liu, Bing; Jiang, Tianzi

    2015-01-01

    The influence of catechol-o-methyltransferase (COMT) Val158Met on brain activation and functional connectivity has been widely reported. However, voxel-wise effects of this genotype on resting-state brain networks remain unclear. Here, we used resting-state fMRI and eigenvector centrality to examine the effects of COMT Val158Met genotypes on the connection patterns of the brain network and working memory (WM) in healthy, young Val/Val and Met carrier subjects. There were significant differences in the performance level on the 2-back WM task between the different COMT genotypes: Val/Val individuals exhibited a higher correct rate compared to the Met carriers. A two-sample t test was used to examine the differences in the eigenvector centrality maps, using age and gender as covariates of no interest, between the Val/Val and Met carriers. We found that the Val/Val individuals exhibited significantly higher eigenvector centrality compared to the Met carriers in the left parahippocampal cortex. Furthermore, a significantly positive correlation between the mean eigenvector centrality of the significant cluster and the correct rate of the 2-back WM task was observed. By using a voxel-wise data-driven method, our findings may provide plausible implications regarding individual differences in the genetic contribution of COMT Val158Met to the brain network and cognition. PMID:26054510

  14. Melatonin Synthesis: Acetylserotonin O-Methyltransferase (ASMT) Is Strongly Expressed in a Subpopulation of Pinealocytes in the Male Rat Pineal Gland.

    PubMed

    Rath, Martin F; Coon, Steven L; Amaral, Fernanda G; Weller, Joan L; Møller, Morten; Klein, David C

    2016-05-01

    The rat pineal gland has been extensively used in studies of melatonin synthesis. However, the cellular localization of melatonin synthesis in this species has not been investigated. Here we focus on the localization of melatonin synthesis using immunohistochemical methods to detect the last enzyme in melatonin synthesis, acetylserotonin O-methyltransferase (ASMT), and in situ hybridization techniques to study transcripts encoding ASMT and two other enzymes in melatonin synthesis, tryptophan hydroxylase (TPH)-1 and aralkylamine N-acetyltransferase. In sections of the rat pineal gland, marked cell-to-cell differences were found in ASMT immunostaining intensity and in the abundance of Tph1, Aanat, and Asmt transcripts. ASMT immunoreactivity was localized to the cytoplasm in pinealocytes in the parenchyma of the superficial pineal gland, and immunopositive pinealocytes were also detected in the pineal stalk and in the deep pineal gland. ASMT was found to inconsistently colocalize with S-antigen, a widely used pinealocyte marker; this colocalization was seen in cells throughout the pineal complex and also in displaced pinealocyte-like cells of the medial habenular nucleus. Inconsistent colocalization between ASMT and TPH protein was also detected in the pineal gland. ASMT protein was not detected in extraepithalamic parts of the central nervous system or in peripheral tissues. The findings in this report are of special interest because they provide reason to suspect that melatonin synthesis varies significantly among individual pinealocytes.

  15. Levodopa-related cysteinyl-glycine and cysteine reduction with and without catechol-O-methyltransferase inhibition in Parkinson's disease patients.

    PubMed

    Müller, Thomas; Muhlack, Siegfried

    2014-06-01

    Oxidative stress is influenced by the thiol homeostasis, which regulates the redox milieu via glutathione. Components of glutathione metabolism are cysteine and cysteinyl-glycine. Both substrates decay following levodopa application or dopamine-related oxidative stress. Objective was to investigate the impact of an acute levodopa application with and without catechol-O-methyltransferase inhibitor on cysteine- and cysteinyl-glycine plasma levels. On two investigation days, 13 patients with Parkinson's disease took one retarded release 200-mg levodopa/50 mg carbidopa-containing tablet or one 150-mg levodopa/50-mg carbidopa/200-mg entacapone formulation under standardized conditions. Levodopa, 3-O-methyldopa, cysteine and cysteinyl-glycine were measured at baseline, 80 and 140 min following levodopa administration. Cysteine and cysteinyl-glycine similarly decreased, levodopa was nearly equal during both conditions. Entacapone lowered 3-O-methyldopa. Cysteine decay may be due to an elevated glutathione generation, which consumes cysteine. Cysteinyl-glycine decrease results from the alternative glutathione transformation to its oxidized form glutathione dissulfide after free radical scavenging.

  16. Catechol-O-methyltransferase inhibition alters pain and anxiety-related volitional behaviors through activation of β-adrenergic receptors in the rat.

    PubMed

    Kline, R H; Exposto, F G; O'Buckley, S C; Westlund, K N; Nackley, A G

    2015-04-02

    Reduced catechol-O-methyltransferase (COMT) activity resulting from genetic variation or pharmacological depletion results in enhanced pain perception in humans and nociceptive behaviors in animals. Using phasic mechanical and thermal reflex tests (e.g. von Frey, Hargreaves), recent studies show that acute COMT-dependent pain in rats is mediated by β-adrenergic receptors (βARs). In order to more closely mimic the characteristics of human chronic pain conditions associated with prolonged reductions in COMT, the present study sought to determine volitional pain-related and anxiety-like behavioral responses following sustained as well as acute COMT inhibition using an operant 10-45°C thermal place preference task and a light/dark preference test. In addition, we sought to evaluate the effects of sustained COMT inhibition on generalized body pain by measuring tactile sensory thresholds of the abdominal region. Results demonstrated that acute and sustained administration of the COMT inhibitor OR486 increased pain behavior in response to thermal heat. Further, sustained administration of OR486 increased anxiety behavior in response to bright light, as well as abdominal mechanosensation. Finally, all pain-related behaviors were blocked by the non-selective βAR antagonist propranolol. Collectively, these findings provide the first evidence that stimulation of βARs following acute or chronic COMT inhibition drives cognitive-affective behaviors associated with heightened pain that affects multiple body sites.

  17. Structure related effects of flavonoid aglycones on cell cycle progression of HepG2 cells: Metabolic activation of fisetin and quercetin by catechol-O-methyltransferase (COMT).

    PubMed

    Poór, Miklós; Zrínyi, Zita; Kőszegi, Tamás

    2016-10-01

    Dietary flavonoids are abundant in the Plant Kingdom and they are extensively studied because of their manifold pharmacological activities. Recent studies highlighted that cell cycle arrest plays a key role in their antiproliferative effect in different tumor cells. However, structure-activity relationship of flavonoids is poorly characterized. In our study the influence of 18 flavonoid aglycones (as well as two metabolites) on cell cycle distribution was investigated. Since flavonoids are extensively metabolized by liver cells, HepG2 tumor cell line was applied, considering the potential metabolic activation/inactivation of flavonoids. Our major observations are the followings: (1) Among the tested compounds diosmetin, fisetin, apigenin, lutelin, and quercetin provoked spectacular extent of G2/M phase cell cycle arrest. (2) Inhibition of catechol-O-methyltransferase enzyme by entacapone decreased the antiproliferative effects of fisetin and quercetin. (3) Geraldol and isorhamnetin (3'-O-methylated metabolites of fisetin and quercetin, respectively) demonstrated significantly higher antiproliferative effect on HepG2 cells compared to the parent compounds. Based on these results, O-methylated flavonoid metabolites or their chemically modified derivatives may be suitable candidates of tumor therapy in the future.

  18. An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis[C][W][OA

    PubMed Central

    Zhang, Kewei; Bhuiya, Mohammad-Wadud; Pazo, Jorge Rencoret; Miao, Yuchen; Kim, Hoon; Ralph, John; Liu, Chang-Jun

    2012-01-01

    Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds. PMID:22851762

  19. Development of Blood-Brain Barrier Permeable Nitrocatechol-Based Catechol O-Methyltransferase Inhibitors with Reduced Potential for Hepatotoxicity.

    PubMed

    Silva, Tiago; Mohamed, Tarek; Shakeri, Arash; Rao, Praveen P N; Martínez-González, Loreto; Pérez, Daniel I; Martínez, Ana; Valente, Maria João; Garrido, Jorge; Uriarte, Eugenio; Serrão, Paula; Soares-da-Silva, Patrício; Remião, Fernando; Borges, Fernanda

    2016-08-25

    Recent efforts have been focused on the development of centrally active COMT inhibitors, which can be valuable assets for neurological disorders such as Parkinson's disease, due to the severe hepatotoxicity risk associated with tolcapone. New nitrocatechol COMT inhibitors based on naturally occurring caffeic acid and caffeic acid phenethyl ester were developed. All nitrocatechol derivatives displayed potent inhibition of peripheral and cerebral COMT within the nanomolar range. Druglike derivatives 13, 15, and 16 were predicted to cross the blood-brain barrier in vitro and were significantly less toxic than tolcapone and entacapone when incubated at 50 μM with rat primary hepatocytes. Moreover, their unique acidity and electrochemical properties decreased the chances of formation of reactive quinone-imines and, as such, the potential for hepatotoxicity. The binding mode of 16 confirmed that the major interactions with COMT were established via the nitrocatechol ring, allowing derivatization of the side chain for future lead optimization efforts.

  20. Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

    NASA Astrophysics Data System (ADS)

    Dong, Xuewei; He, Qingfang; Peng, Zhenying; Yu, Jinhui; Bian, Fei; Li, Youzhi; Bi, Yuping

    2016-07-01

    Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

  1. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

  2. Genomic organization of the human lysosomal acid lipase gene (LIPA)

    SciTech Connect

    Aslandis, C.; Klima, H.; Lackner, K.J.; Schmitz, G. )

    1994-03-15

    Defects in the human lysosomal acid lipase gene are responsible for cholesteryl ester storage disease (CESD) and Wolman disease. Exon skipping as the cause for CESD has been demonstrated. The authors present here a summary of the exon structure of the entire human lysosomal acid lipase gene consisting of 10 exons, together with the sizes of genomic EcoRI and SacI fragments hybridizing to each exon. In addition, the DNA sequence of the putative promoter region is presented. The EMBL accession numbers for adjacent intron sequences are given. 7 refs., 2 figs., 1 tab.

  3. A new approach on the purification of recombinant human soluble catechol-O-methyltransferase from an Escherichia coli extract using hydrophobic interaction chromatography.

    PubMed

    Passarinha, L A; Bonifácio, M J; Soares-da-Silva, P; Queiroz, J A

    2008-01-11

    Catechol-O-methyltransferase (COMT) is a significant target in protein engineering due to its role not only in normal brain function but also to its possible involvement in some human disorders. In this work, a new approach was employed for the purification of recombinant human soluble COMT (hSCOMT) using hydrophobic interaction chromatography, as the main isolation method, from an Escherichia coli culture broth. A simplified overall process flow is proposed. Indeed, with an optimized heterologous expression system for recombinant hSCOMT production, such as E. coli, it was possible to produce and recover the active monomeric enzyme directly from the cell crude culture broth either by a freeze/thaw or ultrasonication lysis step. The recombinant enzyme present in the bacterial soluble fraction, exhibited similar affinity for epinephrine (K(m) 276 [215; 337] microM) and the methyl donor (S-adenosyl-L-methionine, SAMe) (K(m) 36 [30; 41]microM) as human SCOMT. After the precipitation step by 55% of ammonium sulphate, a HIC step on the butyl-sepharose resin was found to be highly effective in selectively eluting a range of contaminating key proteins present in the concentrate soluble extract. Consequently, the partially purified eluate from HIC could then be loaded and polished by gel filtration in order to increase the process efficiency. The final product appeared as a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The procedure resulted in a global 10.9-fold purification with a specific activity of 5500 nmol/h/mg of protein. The widespread applicability of the process, here described, to different COMT sources could make this protocol highly useful for all studies requiring purified and active COMT proteins.

  4. Active site mapping and substrate specificity of bacterial Hen1, a manganese-dependent 3' terminal RNA ribose 2'O-methyltransferase.

    PubMed

    Jain, Ruchi; Shuman, Stewart

    2011-03-01

    The RNA methyltransferase Hen1 and the RNA end-healing/sealing enzyme Pnkp comprise an RNA repair system encoded by an operon-like cassette present in bacteria from eight different phyla. Clostridium thermocellum Hen1 (CthHen1) is a manganese-dependent RNA ribose 2'O-methyltransferase that marks the 3' terminal nucleoside of broken RNAs and protects repair junctions from iterative damage by transesterifying endonucleases. Here we used the crystal structure of the homologous plant Hen1 to guide a mutational analysis of CthHen1, the results of which provide new insights to RNA end recognition and catalysis. We illuminated structure-activity relations at eight essential constituents of the active site implicated in binding the 3' dinucleotide of the RNA methyl acceptor (Arg273, Arg414), the manganese cofactor (Glu366, Glu369, His370, His418), and the AdoMet methyl donor (Asp291, Asp316). We investigated the effects of varying the terminal nucleobase, RNA size, RNA content, and RNA secondary structure on methyl acceptor activity. Key findings are as follows. CthHen1 displayed a fourfold preference for guanosine as the terminal nucleoside. RNA size had little impact in the range of 12-24 nucleotides, but activity declined sharply with a 9-mer. CthHen1 was adept at methylating a polynucleotide composed of 23 deoxyribonucleotides and one 3' terminal ribonucleotide, signifying that it has no strict RNA specificity beyond the 3' nucleoside. CthHen1 methylated RNA ends in the context of duplex secondary structures. These properties distinguish bacterial Hen1 from plant and metazoan homologs.

  5. Crystal structure and activity of protein L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of AdoHcy binding.

    PubMed

    Chatterjee, Tanaya; Mukherjee, Debadrita; Banerjee, Mousumi; Chatterjee, Barun K; Chakrabarti, Pinak

    2015-10-01

    The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39-58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 10(6) M(-1), quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.

  6. Biopsychosocial Influence on Exercise-induced Delayed Onset Muscle Soreness at the Shoulder: Pain Catastrophizing and Catechol-O-Methyltransferase (COMT) Diplotype Predict Pain Ratings

    PubMed Central

    George, Steven Z.; Dover, Geoffrey C.; Wallace, Margaret R.; Sack, Brandon K.; Herbstman, Deborah M.; Aydog, Ece; Fillingim, Roger B.

    2009-01-01

    Objective The experience of pain is believed to be influenced by psychologic and genetic factors. A previous study suggested pain catastrophizing and catechol-O-methyltransferase (COMT) genotype influenced clinical pain ratings for patients seeking operative treatment of shoulder pain. This study investigated whether these same psychologic and genetic factors predicted responses to induced shoulder pain. Methods Participants (n=63) completed self-report questionnaires and had COMT genotype determined before performing a standardized fatigue protocol to induce delayed onset muscle soreness. Then, shoulder pain ratings, self-report of upper-extremity disability ratings, and muscle torque production were reassessed 24, 48, and 72 hours later. Results This cohort consisted of 35 women and 28 men, with a mean age of 20.9 years (SD=1.7). The frequency of COMT diplotypes was 42 with “high COMT enzyme activity” (low pain sensitivity group) and 21 with “low COMT enzyme activity” (average pain sensitivity/high pain sensitivity group). A hierarchical regression model indicated that an interaction between pain catastrophizing and COMT diplotype was the strongest unique predictor of 72-hour pain ratings. The same interaction was not predictive of self-report of disability or muscle torque production at 72 hours. The pain catastrophizing × COMT diplotype interaction indicated that participants with high pain catastrophizing and low COMT enzyme activity (average pain sensitivity/high pain sensitivity group) were more likely (relative risk=3.5, P=0.025) to have elevated pain intensity ratings (40/100 or higher). Discussion These findings from an experimental model converge with those from a surgical cohort and provide additional evidence that the presence of elevated pain catastrophizing and COMT diplotype indicative of low COMT enzyme activity have the potential to increase the risk of developing chronic pain syndromes. PMID:18936597

  7. Conjugation of catechols by recombinant human sulfotransferases, UDP-glucuronosyltransferases, and soluble catechol O-methyltransferase: structure-conjugation relationships and predictive models.

    PubMed

    Taskinen, Jyrki; Ethell, Brian T; Pihlavisto, Pia; Hood, Alan M; Burchell, Brian; Coughtrie, Michael W H

    2003-09-01

    Conjugation of a structurally diverse set of 53 catechol compounds was studied in vitro using six recombinant human sulfotransferases (SULTs), five UDP-glucuronosyltransferases (UGT) and the soluble form of catechol O-methyltransferase (S-COMT) as catalyst. The catechol set comprised endogenous compounds, such as catecholamines and catecholestrogens, drugs, natural plant constituents, and other catechols with diverse substituent properties and substitution patterns. Most of the catechols studied were substrates of S-COMT and four SULT isoforms (1A1, 1A2, 1A3, and 1B1), but the rates of conjugation varied considerably, depending on the substrate structure and the enzyme form. SULT1E1 sulfated fewer catechols. Only low activities were observed for SULT1C2. UGT1A9 glucuronidated catechols representing various structural classes, and almost half of the studied compounds were glucuronidated at a high rate. The other UGT enzymes (1A1, 1A6, 2B7, and 2B15) showed narrower substrate specificity for catechols, but each glucuronidated some catechols at a high rate. Dependence of specificity and rate of conjugation on the molecular structure of the substrate was characterized by structure-activity relationship analysis and quantitative structure-activity relationship modeling. Twelve structural descriptors were used to characterize lipophilicity/polar interaction properties, steric properties, and electronic effects of the substituents modifying the catechol structure. PLS models explaining more than 80% and predicting more than 70% of the variance in conjugation activity were derived for the representative enzyme forms SULT1A3, UGT1A9, and S-COMT. Several structural factors governing the conjugation of catechol hormones, metabolites, and drugs were identified. The results have significant implications for predicting the metabolic fate of catechols.

  8. The impact of the catechol-O-methyltransferase genotype on the acute responsiveness of vascular reactivity to a green tea extract.

    PubMed

    Miller, Rosalind J; Jackson, Kim G; Dadd, Tony; Mayes, Andrew E; Brown, A Louise; Minihane, Anne M

    2011-04-01

    The beneficial effects of green tea catechins, such as the proposed improvement in endothelial function, may be influenced by phase II metabolism during and after absorption. The methylation enzyme, catechol-O-methyltransferase (COMT), has a missense mutation rs4680 (G to A), proposed to result in a 40 % reduction in enzyme activity. In the present pilot study, twenty subjects (ten of each homozygous COMT genotype) were recruited. Green tea extract capsules (836 mg green tea catechins) were given in a fasted state, and a high-carbohydrate breakfast was given after 60 min. Blood samples and vascular function measurements were taken at regular intervals. The change in digital volume pulse stiffness index (SI) from baseline was shown to be different between genotype groups at 120 and 240 min, with a lower SI in the GG individuals (P ≤ 0·044). The change in blood pressure from baseline also differed between genotype groups, with a greater increase in systolic (P = 0·023) and diastolic (P = 0·034) blood pressure at 120 min in the GG group. The GG [corrected] group was shown to have a greater increase in insulin concentrations at 120 min (P = 0·019) and 180 min (P = 0·008) compared with baseline, despite similar glucose profiles. No genotypic differences were found in vascular reactivity measured using laser Doppler iontophoresis, total nitrite, lipids, plasma total antioxidant capacity or inflammatory markers after ingestion of the green tea extract. In conclusion, SI and insulin response to the glucose load differed between the COMT genotype groups, and this may be suggestive of a green tea extract and genotype interaction.

  9. Enantioselectivity in the methylation of the catecholic phase I metabolites of methylenedioxy designer drugs and their capability to inhibit catechol-O-methyltransferase-catalyzed dopamine 3-methylation.

    PubMed

    Meyer, Markus R; Maurer, Hans H

    2009-06-01

    The designer drugs R,S-3,4-methylenedioxy-methamphetamine (MDMA, Ecstasy), R,S-3,4-methylenedioxy-ethylamphetamine (MDEA, Eve), and R,S-N-methyl-benzodioxolyl-butanamine (MBDB, Eden) are chiral compounds, and their in vitro and in vivo metabolism is enantioselective with a preference for the S-enantiomer caused in part by P450-mediated demethylenation. As the elimination of the catecholamine metabolites could also be enantioselective, the aim of the present study was to investigate the O-methylation to the corresponding methoxy derivatives catalyzed by the soluble or membrane-bound form of the catechol-O-methyltransferase (COMT). As all three compounds showed substrate inhibition effects during the incubation, their inhibition potential was quantified using the methylation of dopamine as a marker reaction. For investigation of the catechol-O-methylation catalyzed by the soluble form of the COMT (sCOMT), incubations with human liver cytosol (HLC) were performed. Human liver microsomes (HLM) were used for investigation of the membrane-bound form. For inhibition studies, 3-hydroxytyramine (dopamine) was incubated in HLC. The respective catechols were added at various concentrations to check whether they influence the methylation of 3-hydroxytyramine. Our data showed that the S-enantiomers of all studied catecholamines were preferably O-methylated by both types of COMT. Comparing the resulting kinetics of the HLC and HLM assays, the affinity for all substrates was 10-fold higher for the membrane-bound COMT, whereas the turnover rate was 10-fold higher for the soluble COMT. Uncompetitive inhibition of dopamine methylation could be observed for all tested catechols. In conclusion, elimination of the catecholamine metabolites of MDMA, MDEA, and MBDB was shown to be enantioselective and might therefore contribute to the different pharmacokinetic properties observed for both enantiomers. Furthermore, the catecholic metabolites were identified to be uncompetitive inhibitors

  10. Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis.

    PubMed

    Tokuoka, Masafumi; Kikuchi, Tomoki; Shinohara, Yasutomo; Koyama, Akifumi; Iio, Shin-Ichiro; Kubota, Takaaki; Kobayashi, Jun'ichi; Koyama, Yasuji; Totsuka, Akira; Shindo, Hitoshi; Sato, Kazuo

    2015-01-01

    Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.

  11. Control of mammalian gene expression by amino acids, especially glutamine.

    PubMed

    Brasse-Lagnel, Carole; Lavoinne, Alain; Husson, Annie

    2009-04-01

    Molecular data rapidly accumulating on the regulation of gene expression by amino acids in mammalian cells highlight the large variety of mechanisms that are involved. Transcription factors, such as the basic-leucine zipper factors, activating transcription factors and CCAAT/enhancer-binding protein, as well as specific regulatory sequences, such as amino acid response element and nutrient-sensing response element, have been shown to mediate the inhibitory effect of some amino acids. Moreover, amino acids exert a wide range of effects via the activation of different signalling pathways and various transcription factors, and a number of cis elements distinct from amino acid response element/nutrient-sensing response element sequences were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine, the most abundant amino acid, which at appropriate concentrations enhances a great number of cell functions via the activation of various transcription factors. The glutamine-responsive genes and the transcription factors involved correspond tightly to the specific effects of the amino acid in the inflammatory response, cell proliferation, differentiation and survival, and metabolic functions. Indeed, in addition to the major role played by nuclear factor-kappaB in the anti-inflammatory action of glutamine, the stimulatory role of activating protein-1 and the inhibitory role of C/EBP homology binding protein in growth-promotion, and the role of c-myc in cell survival, many other transcription factors are also involved in the action of glutamine to regulate apoptosis and intermediary metabolism in different cell types and tissues. The signalling pathways leading to the activation of transcription factors suggest that several kinases are involved, particularly mitogen-activated protein kinases. In most cases, however, the precise pathways from the entrance of the amino acid into the cell to the activation of gene

  12. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    PubMed

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-04

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

  13. Identification of nitrogen-fixing genes and gene clusters from metagenomic library of acid mine drainage.

    PubMed

    Dai, Zhimin; Guo, Xue; Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

    2014-01-01

    Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

  14. Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

    PubMed Central

    Yin, Huaqun; Liang, Yili; Cong, Jing; Liu, Xueduan

    2014-01-01

    Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of nif gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate nif genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel nif gene clusters. Metagenomic analyses revealed that 742 sequences were identified as nif genes including structural subunit genes nifH, nifD, nifK and various additional genes. The AMD community is massively dominated by the genus Acidithiobacillus. However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring nif, fix and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most nif genes in this cluster are most similar to those of Herbaspirillum seropedicae, but the organization of the nif gene cluster had significant differences from H. seropedicae. Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of nif genes exist in this gene cluster. nifQ gene falls into the same transcription unit with fixABCX genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community. PMID:24498417

  15. Cadmium Induces Retinoic Acid Signaling by Regulating Retinoic Acid Metabolic Gene Expression*

    PubMed Central

    Cui, Yuxia; Freedman, Jonathan H.

    2009-01-01

    The transition metal cadmium is an environmental teratogen. In addition, cadmium and retinoic acid can act synergistically to induce forelimb malformations. The molecular mechanism underlying the teratogenicity of cadmium and the synergistic effect with retinoic acid has not been addressed. An evolutionarily conserved gene, β,β-carotene 15,15′-monooxygenase (BCMO), which is involved in retinoic acid biosynthesis, was studied in both Caenorhabditis elegans and murine Hepa 1–6 cells. In C. elegans, bcmo-1 was expressed in the intestine and was cadmium inducible. Similarly, in Hepa 1–6 cells, Bcmo1 was induced by cadmium. Retinoic acid-mediated signaling increased after 24-h exposures to 5 and 10 μm cadmium in Hepa 1–6 cells. Examination of gene expression demonstrated that the induction of retinoic acid signaling by cadmium may be mediated by overexpression of Bcmo1. Furthermore, cadmium inhibited the expression of Cyp26a1 and Cyp26b1, which are involved in retinoic acid degradation. These results indicate that cadmium-induced teratogenicity may be due to the ability of the metal to increase the levels of retinoic acid by disrupting the expression of retinoic acid-metabolizing genes. PMID:19556237

  16. Tomato ABSCISIC ACID STRESS RIPENING (ASR) gene family revisited.

    PubMed

    Golan, Ido; Dominguez, Pia Guadalupe; Konrad, Zvia; Shkolnik-Inbar, Doron; Carrari, Fernando; Bar-Zvi, Dudy

    2014-01-01

    Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.

  17. Repeats in transforming acidic coiled-coil (TACC) genes.

    PubMed

    Trivedi, Seema

    2013-06-01

    Transforming acidic coiled-coil proteins (TACC1, 2, and 3) are essential proteins associated with the assembly of spindle microtubules and maintenance of bipolarity. Dysregulation of TACCs is associated with tumorigenesis, but studies of microsatellite instability in TACC genes have not been extensive. Microsatellite or simple sequence repeat instability is known to cause many types of cancer. The present in silico analysis of SSRs in human TACC gene sequences shows the presence of mono- to hexa-nucleotide repeats, with the highest densities found for mono- and di-nucleotide repeats. Density of repeats is higher in introns than in exons. Some of the repeats are present in regulatory regions and retained introns. Human TACC genes show conservation of many repeat classes. Microsatellites in TACC genes could be valuable markers for monitoring numerical chromosomal aberrations and or cancer.

  18. Modification of Monolignol Biosynthetic Pathway in Jute: Different Gene, Different Consequence.

    PubMed

    Shafrin, Farhana; Ferdous, Ahlan Sabah; Sarkar, Suprovath Kumar; Ahmed, Rajib; Amin, Al-; Hossain, Kawsar; Sarker, Mrinmoy; Rencoret, Jorge; Gutiérrez, Ana; Del Rio, Jose C; Sanan-Mishra, Neeti; Khan, Haseena

    2017-01-04

    Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16-25% reduction in acid insoluble lignin for the whole stem and ~13-14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition.

  19. Modification of Monolignol Biosynthetic Pathway in Jute: Different Gene, Different Consequence

    PubMed Central

    Shafrin, Farhana; Ferdous, Ahlan Sabah; Sarkar, Suprovath Kumar; Ahmed, Rajib; Amin, Al-; Hossain, Kawsar; Sarker, Mrinmoy; Rencoret, Jorge; Gutiérrez, Ana; del Rio, Jose C.; Sanan-Mishra, Neeti; Khan, Haseena

    2017-01-01

    Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16–25% reduction in acid insoluble lignin for the whole stem and ~13–14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition. PMID:28051165

  20. A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes.

    PubMed

    Casal, Enriqueta; Palomo, Laura; Cabrera, Diana; Falcon-Perez, Juan M

    2016-01-01

    There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of

  1. Catechol-O-Methyltransferase Val158Met Polymorphism and Clinical Response to Antipsychotic Treatment in Schizophrenia and Schizo-Affective Disorder Patients: a Meta-Analysis

    PubMed Central

    Huang, Eric; Zai, Clement C.; Lisoway, Amanda; Maciukiewicz, Malgorzata; Felsky, Daniel; Tiwari, Arun K.; Bishop, Jeffrey R.; Ikeda, Masashi; Molero, Patricio; Ortuno, Felipe; Porcelli, Stefano; Samochowiec, Jerzy; Mierzejewski, Pawel; Gao, Shugui; Crespo-Facorro, Benedicto; Pelayo-Terán, José M; Kaur, Harpreet; Kukreti, Ritushree; Meltzer, Herbert Y.; Lieberman, Jeffrey A.; Potkin, Steven G.; Müller, Daniel J.

    2016-01-01

    Background: The catechol-O-methyltransferase (COMT) enzyme plays a crucial role in dopamine degradation, and the COMT Val158Met polymorphism (rs4680) is associated with significant differences in enzymatic activity and consequently dopamine concentrations in the prefrontal cortex. Multiple studies have analyzed the COMT Val158Met variant in relation to antipsychotic response. Here, we conducted a meta-analysis examining the relationship between COMT Val158Met and antipsychotic response. Methods: Searches using PubMed, Web of Science, and PsycInfo databases (03/01/2015) yielded 23 studies investigating COMT Val158Met variation and antipsychotic response in schizophrenia and schizo-affective disorder. Responders/nonresponders were defined using each study’s original criteria. If no binary response definition was used, authors were asked to define response according to at least 30% Positive and Negative Syndrome Scale score reduction (or equivalent in other scales). Analysis was conducted under a fixed-effects model. Results: Ten studies met inclusion criteria for the meta-analysis. Five additional antipsychotic-treated samples were analyzed for Val158Met and response and included in the meta-analysis (ntotal=1416). Met/Met individuals were significantly more likely to respond than Val-carriers (P=.039, ORMet/Met=1.37, 95% CI: 1.02–1.85). Met/Met patients also experienced significantly greater improvement in positive symptoms relative to Val-carriers (P=.030, SMD=0.24, 95% CI: 0.024–0.46). Posthoc analyses on patients treated with atypical antipsychotics (n=1207) showed that Met/Met patients were significantly more likely to respond relative to Val-carriers (P=.0098, ORMet/Met=1.54, 95% CI: 1.11–2.14), while no difference was observed for typical-antipsychotic-treated patients (n=155) (P=.65). Conclusions: Our findings suggest that the COMT Val158Met polymorphism is associated with response to antipsychotics in schizophrenia and schizo-affective disorder

  2. Assessment of Cellular Estrogenic Activity Based on Estrogen Receptor-Mediated Reduction of Soluble-Form Catechol-O-Methyltransferase (COMT) Expression in an ELISA-Based System

    PubMed Central

    Ho, Philip Wing-Lok; Tse, Zero Ho-Man; Liu, Hui-Fang; Lu, Song; Ho, Jessica Wing-Man; Kung, Michelle Hiu-Wai; Ramsden, David Boyer; Ho, Shu-Leong

    2013-01-01

    Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10-9-10-7M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular

  3. Assessment of cellular estrogenic activity based on estrogen receptor-mediated reduction of soluble-form catechol-O-methyltransferase (COMT) expression in an ELISA-based system.

    PubMed

    Ho, Philip Wing-Lok; Tse, Zero Ho-Man; Liu, Hui-Fang; Lu, Song; Ho, Jessica Wing-Man; Kung, Michelle Hiu-Wai; Ramsden, David Boyer; Ho, Shu-Leong

    2013-01-01

    Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular

  4. A Novel Sensitive Method to Measure Catechol-O-Methyltransferase Activity Unravels the Presence of This Activity in Extracellular Vesicles Released by Rat Hepatocytes

    PubMed Central

    Casal, Enriqueta; Palomo, Laura; Cabrera, Diana; Falcon-Perez, Juan M.

    2016-01-01

    There is a clear need for drug treatments to be selected according to the characteristics of an individual patient, in order to improve efficacy and reduce the number and severity of adverse drug reactions. One of the main enzymes to take into account in pharmacogenomics is catechol O-methyltransferase (COMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine to catechols and catecholamines, like the neurotransmitters dopamine, epinephrine, and norepinephrine. Although, most of this enzyme is associated to intracellular vesicles, recently it has also been detected in extracellular vesicles secreted by hepatocytes and in serum circulating vesicles. COMT has implications in many neurological and psychiatric disorders like Parkinson's disease, chronic fatigue, pain response, schizophrenia, and bipolar disorders. Remarkably, genetic variations of COMT affect its activity and are associated to various human disorders from psychiatric diseases to estrogen-induced cancers. Consequently, the establishment of new methods to evaluate COMT activity is an important aspect to investigate the biology of this drug-metabolizing enzyme. Herein, we have developed a sensitive and selective method to determine COMT activity. We first optimized the activity in rat liver incubated with two different substrates; norepinephrine and dopamine. The enzymatically formed products (normetanephrine and 3-methoxytyramine, respectively) were extracted by solid-phase extraction using weak cation exchange cartridges, chromatographically separated, and detected and quantified using a mass spectrometer. The range of quantitation for both products was from 0.005 to 25 μg/mL. This methodology offers acceptable recovery for both enzymatic products (≥75%) and good accuracy and precision (≤15%). The lower limit of quantifications were 0.01 and 0.005 μM for 3-methoxytyramine and normetanephrine, respectively. Importantly, this sensitive assay was able to detect the presence of

  5. Detoxication of structurally diverse polycyclic aromatic hydrocarbon (PAH) o-quinones by human recombinant catechol-O-methyltransferase (COMT) via O-methylation of PAH catechols.

    PubMed

    Zhang, Li; Jin, Yi; Chen, Mo; Huang, Meng; Harvey, Ronald G; Blair, Ian A; Penning, Trevor M

    2011-07-22

    Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[³H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. ¹H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.

  6. Yeast genes involved in response to lactic acid and acetic acid: acidic conditions caused by the organic acids in Saccharomyces cerevisiae cultures induce expression of intracellular metal metabolism genes regulated by Aft1p.

    PubMed

    Kawahata, Miho; Masaki, Kazuo; Fujii, Tsutomu; Iefuji, Haruyuki

    2006-09-01

    Using two types of genome-wide analysis to investigate yeast genes involved in response to lactic acid and acetic acid, we found that the acidic condition affects metal metabolism. The first type is an expression analysis using DNA microarrays to investigate 'acid shock response' as the first step to adapt to an acidic condition, and 'acid adaptation' by maintaining integrity in the acidic condition. The other is a functional screening using the nonessential genes deletion collection of Saccharomyces cerevisiae. The expression analysis showed that genes involved in stress response, such as YGP1, TPS1 and HSP150, were induced under the acid shock response. Genes such as FIT2, ARN1 and ARN2, involved in metal metabolism regulated by Aft1p, were induced under the acid adaptation. AFT1 was induced under acid shock response and under acid adaptation with lactic acid. Moreover, green fluorescent protein-fused Aft1p was localized to the nucleus in cells grown in media containing lactic acid, acetic acid, or hydrochloric acid. Both analyses suggested that the acidic condition affects cell wall architecture. The depletion of cell-wall components encoded by SED1, DSE2, CTS1, EGT2, SCW11, SUN4 and YNL300W and histone acetyltransferase complex proteins encoded by YID21, EAF3, EAF5, EAF6 and YAF9 increased resistance to lactic acid. Depletion of the cell-wall mannoprotein Sed1p provided resistance to lactic acid, although the expression of SED1 was induced by exposure to lactic acid. Depletion of vacuolar membrane H+-ATPase and high-osmolarity glycerol mitogen-activated protein kinase proteins caused acid sensitivity. Moreover, our quantitative PCR showed that expression of PDR12 increased under acid shock response with lactic acid and decreased under acid adaptation with hydrochloric acid.

  7. Virus-induced silencing of Comt, pAmt and Kas genes results in a reduction of capsaicinoid accumulation in chili pepper fruits.

    PubMed

    del Rosario Abraham-Juárez, Ma; del Carmen Rocha-Granados, Ma; López, Mercedes G; Rivera-Bustamante, Rafael Francisco; Ochoa-Alejo, Neftalí

    2008-02-01

    Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways. Fragments of Comt (encoding a caffeic acid O-methyltransferase), pAmt (a putative aminotransferase), and Kas (a beta-keto-acyl-[acyl-carrier-protein] synthase) genes, that are differentially expressed in placenta tissue of pungent chili pepper, were individually inserted into a Pepper huasteco yellow veins virus (PHYVV)-derived vector to determine, by virus-induced gene silencing, irrespective of whether these genes are involved in the biosynthesis of capsaicinoids. Reduction of the respective mRNA levels as well as the presence of related siRNAs confirmed the silencing of these three genes. Morphological alterations were evident in plants inoculated with PHYVV::Comt and PHYVV::Kas constructs; however, plants inoculated with PHYVV::pAmt showed no evident alterations. On the other hand, fruit setting was normal in all cases. Biochemical analysis of placenta tissues showed that, indeed, independent silencing of all three genes led to a dramatic reduction in capsaicinoid content in the fruits demonstrating the participation of these genes in capsaicinoid biosynthesis. Using this approach it was possible to generate non-pungent chili peppers at high efficiency.

  8. Characterization and expression of caffeoyl-coenzyme A 3-O-methyltransferase proposed for the induced resistance response of Vitis vinifera L.

    PubMed Central

    Busam, G; Junghanns, K T; Kneusel, R E; Kassemeyer, H H; Matern, U

    1997-01-01

    Cell-suspension cultures of Vitis vinifera L. cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h. CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species. The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl- and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification. Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid. Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine. The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown. Treatment of the cultured V. vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective. The data imply for the first time (to our knowledge) that the expression of phenyl-propanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR. PMID:9390437

  9. Single amino acid polymorphism in aldehyde dehydrogenase gene superfamily.

    PubMed

    Priyadharshini Christy, J; George Priya Doss, C

    2015-01-01

    The aldehyde dehydrogenase gene superfamily comprises of 19 genes and 3 pseudogenes. These superfamily genes play a vital role in the formation of molecules that are involved in life processes, and detoxification of endogenous and exogenous aldehydes. ALDH superfamily genes associated mutations are implicated in various diseases, such as pyridoxine-dependent seizures, gamma-hydroxybutyric aciduria, type II Hyperprolinemia, Sjogren-Larsson syndrome including cancer and Alzheimer's disease. Accumulation of large DNA variations data especially Single Amino acid Polymorphisms (SAPs) in public databases related to ALDH superfamily genes insisted us to conduct a survey on the disease associated mutations and predict their function impact on protein structure and function. Overall this study provides an update and highlights the importance of pathogenic mutations in associated diseases. Using KD4v and Project HOPE a computational based platform, we summarized all the deleterious properties of SAPs in ALDH superfamily genes by the providing valuable insight into structural alteration rendered due to mutation. We hope this review might provide a way to define the deleteriousness of a SAP and helps to understand the molecular basis of the associated disease and also permits precise diagnosis and treatment in the near future.

  10. Differential Gene Expression of Longan Under Simulated Acid Rain Stress.

    PubMed

    Zheng, Shan; Pan, Tengfei; Ma, Cuilan; Qiu, Dongliang

    2017-03-16

    Differential gene expression profile was studied in Dimocarpus longan Lour. in response to treatments of simulated acid rain with pH 2.5, 3.5, and a control (pH 5.6) using differential display reverse transcription polymerase chain reaction (DDRT-PCR). Results showed that mRNA differential display conditions were optimized to find an expressed sequence tag (EST) related with acid rain stress. The potential encoding products had 80% similarity with a transcription initiation factor IIF of Gossypium raimondii and 81% similarity with a protein product of Theobroma cacao. This fragment is the transcription factor activated by second messenger substances in longan leaves after signal perception of acid rain.

  11. Identification of genes regulated by UV/salicylic acid.

    SciTech Connect

    Paunesku, T.; Chang-Liu, C.-M.; Shearin-Jones, P.; Watson, C.; Milton, J.; Oryhon, J.; Salbego, D.; Milosavljevic, A.; Woloschak, G. E.; CuraGen Corp.

    2000-02-01

    Purpose : Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells. The presumed effector molecule responsible for this modulation is NF-kappaB. In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid. Materials and methods : Differential-display RT-PCR was used to identify differentially expressed genes. Results : Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156). Conclusions : Sequences of four of these genes contained NF-kappaB DNA binding sites of the type that may attract transrepressor p55/p55 NF-kappaB homodimers. Down-regulation of these genes upon UV irradiation may contribute to increased cell survival via suppression of p53 independent apoptosis.

  12. Nucleic acid modifications in regulation of gene expression

    PubMed Central

    Chen, Kai; Zhao, Boxuan Simen; He, Chuan

    2016-01-01

    Nucleic acids carry a wide range of different chemical modifications. In contrast to previous views that these modifications are static and only play fine-tuning functions, recent research advances paint a much more dynamic picture. Nucleic acids carry diverse modifications and employ these chemical marks to exert essential or critical influences in a variety of cellular processes in eukaryotic organisms. This review covers several nucleic acid modifications that play important regulatory roles in biological systems, especially in regulation of gene expression: 5-methylcytosine (5mC) and its oxidative derivatives, and N6 -methyladenine (6mA) in DNA; N6 -methyladenosine (m6A), pseudouridine (), and 5-methylcytosine (m5C) in messenger RNA and long non-coding RNA. Modifications in other non-coding RNAs, such as tRNA, miRNA, and snRNA, are also briefly summarized. We provide brief historical perspective of the field, and highlight recent progress in identifying diverse nucleic acid modifications and exploring their functions in different organisms. Overall, we believe that work in this field will yield additional layers of both chemical and biological complexity as we continue to uncover functional consequences of known nucleic acid modifications and discover new ones. PMID:26933737

  13. Unraveling new genes associated with seed development and metabolism in Bixa orellana L. by expressed sequence tag (EST) analysis.

    PubMed

    Soares, Virgínia L F; Rodrigues, Simone M; de Oliveira, Tahise M; de Queiroz, Talisson O; Lima, Lívia S; Hora-Júnior, Braz T; Gramacho, Karina P; Micheli, Fabienne; Cascardo, Júlio C M; Otoni, Wagner C; Gesteira, Abelmon S; Costa, Marcio G C

    2011-02-01

    The tropical tree Bixa orellana L. produces a range of secondary metabolites which biochemical and molecular biosynthesis basis are not well understood. In this work we have characterized a set of ESTs from a non-normalized cDNA library of B. orellana seeds to obtain information about the main developmental and metabolic processes taking place in developing seeds and their associated genes. After sequencing a set of randomly selected clones, most of the sequences were assigned with putative functions based on similarity, GO annotations and protein domains. The most abundant transcripts encoded proteins associated with cell wall (prolyl 4-hydroxylase), fatty acid (acyl carrier protein), and hormone/flavonoid (2OG-Fe oxygenase) synthesis, germination (MADS FLC-like protein) and embryo development (AP2/ERF transcription factor) regulation, photosynthesis (chlorophyll a-b binding protein), cell elongation (MAP65-1a), and stress responses (metallothionein- and thaumatin-like proteins). Enzymes were assigned to 16 different metabolic pathways related to both primary and secondary metabolisms. Characterization of two candidate genes of the bixin biosynthetic pathway, BoCCD and BoOMT, showed that they belong, respectively, to the carotenoid-cleavage dioxygenase 4 (CCD4) and caffeic acid O-methyltransferase (COMT) families, and are up-regulated during seed development. It indicates their involvement in the synthesis of this commercially important carotenoid pigment in seeds of B. orellana. Most of the genes identified here are the first representatives of their gene families in B. orellana.

  14. Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus Liberibacter asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in juvenile hormone biosynthesis...

  15. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  16. The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection[W][OPEN

    PubMed Central

    Breakspear, Andrew; Liu, Chengwu; Roy, Sonali; Stacey, Nicola; Rogers, Christian; Trick, Martin; Morieri, Giulia; Mysore, Kirankumar S.; Wen, Jiangqi; Oldroyd, Giles E.D.; Downie, J. Allan

    2014-01-01

    Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads. PMID:25527707

  17. Cationic liposome–nucleic acid complexes for gene delivery and gene silencing

    PubMed Central

    Ewert, Kai K.; Majzoub, Ramsey N.; Leal, Cecília

    2014-01-01

    Cationic liposomes (CLs) are studied worldwide as carriers of DNA and short interfering RNA (siRNA) for gene delivery and gene silencing, and related clinical trials are ongoing. Optimization of transfection efficiency and silencing efficiency by cationic liposome carriers requires a comprehensive understanding of the structures of CL–nucleic acid complexes and the nature of their interactions with cell membranes as well as events leading to release of active nucleic acids within the cytoplasm. Synchrotron x-ray scattering has revealed that CL–nucleic acid complexes spontaneously assemble into distinct liquid crystalline phases including the lamellar, inverse hexagonal, hexagonal, and gyroid cubic phases, and fluorescence microscopy has revealed CL–DNA pathways and interactions with cells. The combining of custom synthesis with characterization techniques and gene expression and silencing assays has begun to unveil structure–function relations in vitro. As a recent example, this review will briefly describe experiments with surface-functionalized PEGylated CL–DNA nanoparticles. The functionalization, which is achieved through custom synthesis, is intended to address and overcome cell targeting and endosomal escape barriers to nucleic acid delivery faced by PEGylated nanoparticles designed for in vivo applications. PMID:25587216

  18. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

    PubMed

    Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

    2015-12-01

    In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'.

  19. A gene network engineering platform for lactic acid bacteria.

    PubMed

    Kong, Wentao; Kapuganti, Venkata S; Lu, Ting

    2016-02-29

    Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.

  20. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  1. Iron Supply Affects Anthocyanin Content and Related Gene Expression in Berries of Vitis vinifera cv. Cabernet Sauvignon.

    PubMed

    Shi, Pengbao; Li, Bing; Chen, Haiju; Song, Changzheng; Meng, Jiangfei; Xi, Zhumei; Zhang, Zhenwen

    2017-02-14

    Anthocyanins are important compounds for red grape and red wine quality, and can be influenced by supply of nutrients such as nitrogen, phosphorus, potassium, zinc, and iron. The present work aims to gain a better understanding of the effect of iron supply on anthocyanins concentration in grape berries. To this end, own-rooted four-year-old Cabernet Sauvignon grapevines (Vitis vinifera) were fertigated every three days with 0, 23, 46, 92, and 184 μM iron (Fe) from ferric ethylenediamine di (o-hydroxyphenylacetic) acid (Fe-EDDHA) in a complete nutrient solution. Fe deficiency or excess generally led to higher concentrations of titratable acidity and skin/berry ratio, and to lower reducing sugar content, sugar/acid ratio, pH, berry weight, and concentration of anthocyanins. Most of the individual anthocyanins detected in this study, except cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, and cyanidin-3-O-(6-O-coumaryl)-glucoside, in moderate Fe treatment (46 μM) grapes were significantly higher than those of other treatments. Genes encoding chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), leucoanthocyanidin dioxygenase (LDOX), and anthocyanin O-methyltransferase (AOMT) exhibited higher transcript levels in berries from plants cultivated with 46 μM Fe compared to the ones cultivated with other Fe concentrations. We suggest that grape sugar content, anthocyanins content, and transcriptions of genes involved in anthocyanin biosynthesis were correlated with Fe supply concentrations.

  2. Amino acid regulation of mammalian gene expression in the intestine.

    PubMed

    Brasse-Lagnel, Carole G; Lavoinne, Alain M; Husson, Annie S

    2010-07-01

    Some amino acids exert a wide range of regulatory effects on gene expression via the activation of different signalling pathways and transcription factors, and a number of cis elements were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine and arginine, which modulate a number of cell functions through the activation of various pathways in different tissues. In the intestine, appropriate concentrations of both arginine and/or glutamine contribute to facilitate cell proliferation, to limit the inflammatory response and apoptosis, and to modulate intermediary metabolism through specific transcription factors. Particularly, besides its role as a major fuel for enterocytes, the regulatory effects of glutamine have been extensively studied and the molecular mechanisms involved appear diversified and complex. Indeed, in addition to a major role of NF-kappaB in its anti-inflammatory action and a stimulatory role of AP-1 in its growth-promoting action and cell survival, the involvement of some other transcription factors, such as PPAR-gamma or HSF-1, was shown to maintain intestinal cell integrity. The signalling pathways leading to the activation of transcription factors imply several kinases, particularly MAP kinases in the effect of glutamine and p70 S6 kinase for those of arginine, but in most cases the precise pathways from the entrance of the aminoacid into the cell to the activation of gene transcription has remained elusive.

  3. [Gene cloning and bioinformatics analysis of new gene for chlorogenic acid biosynthesis of Lonicera hypoglauca].

    PubMed

    Yu, Shu-lin; Huang, Lu-qi; Yuan, Yuan; Qi, Lin-jie; Liu, Da-hui

    2015-03-01

    To obtain the key genes for chlorogenic acid biosynthesis of Lonicera hypoglauca, four new genes ware obtained from the our dataset of L. hypoglauca. And we also predicted the structure and function of LHPAL4, LHHCT1 , LHHCT2 and LHHCT3 proteins. The phylogenetic tree showed that LHPAL4 was closely related with LHPAL1, LHHCT1 was closely related with LHHCT3, LHHCT2 clustered into a single group. By Real-time PCR to detect the gene expressed level in different organs of L. hypoglauca, we found that the transcripted level of LHPAL4, LHHCT1 and LHHCT3 was the highest in defeat flowers, and the transcripted level of LHHCT2 was the highest in leaves. These result provided a basis to further analysis the mechanism of active ingredients in different organs, as well as the element for in vitro biosynthesis of active ingredients.

  4. Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA.

    PubMed

    Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki; Kirimura, Kohtaro

    2014-05-01

    The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.

  5. Identification of differentially expressed genes in a spontaneous altered leaf shape mutant of the navel orange [Citrus sinensis (L.) Osbeck].

    PubMed

    Da, Xinlei; Yu, Keqin; Shen, Shihui; Zhang, Yajian; Wu, Juxun; Yi, Hualin

    2012-07-01

    Most of the economically important citrus cultivars have originated from bud mutations. Leaf shape and structure are important factors that impact plant photosynthesis. We found a spontaneous bud mutant exhibiting a narrow leaf phenotype in navel orange [Citrus sinensis (L.) Osbeck]. To identify and characterize the genes involved in the formation of this trait, we performed suppression subtractive hybridization (SSH) and macroarray analysis. A total of 221 non-redundant differentially expressed transcripts were obtained. These transcripts included cell wall- and microtubule-related genes and two transcription factor-encoding genes, yabby and wox, which are crucial for leaf morphogenesis. Many highly redundant transcripts were associated with stress responses, while others, encoding caffeic acid 3-O-methyltransferase (EC 2.1.1.68) and a myb-like transcription factor, might be involved in the lignin pathway, which produces a component of secondary walls. Furthermore, real-time quantitative RT-PCR was performed for selected genes to validate the quality of the expressed sequence tags (ESTs) from the SSH libraries. This study represents an attempt to investigate the molecular mechanism associated with a leaf shape mutation, and its results provide new clues for understanding leaf shape mutations in citrus.

  6. Increased Production of Fatty Acids and Triglycerides in Aspergillus oryzae by Enhancing Expressions of Fatty Acid Synthesis-Related Genes

    SciTech Connect

    Tamano, Koichi; Bruno, Kenneth S.; Karagiosis, Sue A.; Culley, David E.; Deng, Shuang; Collett, James R.; Umemura, Myco; Koike, Hideaki; Baker, Scott E.; Machida, Masa

    2013-01-01

    Microbial production of fats and oils is being developedas a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillusoryzae. Examination of the A.oryzaegenome demonstrates that it contains twofatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhancedthe expressionof fatty acid synthesis-related genes by replacing their promoters with thepromoter fromthe constitutively highly expressedgene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthasegenes we successfullyincreasedtheproduction of fatty acids and triglyceridesby more than two fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesteraseincreasedproductivity to a lesser extent.Increasing expression ofacetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored usingquantitative real-time RT-PCR. Our data demonstrates that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

  7. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

  8. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  9. Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase.

    PubMed Central

    Takeuchi, H; Shibano, Y; Morihara, K; Fukushima, J; Inami, S; Keil, B; Gilles, A M; Kawamoto, S; Okuda, K

    1992-01-01

    The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases. Images Fig. 2. PMID:1311172

  10. The PH gene determines fruit acidity and contributes to the evolution of sweet melons.

    PubMed

    Cohen, Shahar; Itkin, Maxim; Yeselson, Yelena; Tzuri, Galil; Portnoy, Vitaly; Harel-Baja, Rotem; Lev, Shery; Sa'ar, Uzi; Davidovitz-Rikanati, Rachel; Baranes, Nadine; Bar, Einat; Wolf, Dalia; Petreikov, Marina; Shen, Shmuel; Ben-Dor, Shifra; Rogachev, Ilana; Aharoni, Asaph; Ast, Tslil; Schuldiner, Maya; Belausov, Eduard; Eshed, Ravit; Ophir, Ron; Sherman, Amir; Frei, Benedikt; Neuhaus, H Ekkehard; Xu, Yimin; Fei, Zhangjun; Giovannoni, Jim; Lewinsohn, Efraim; Tadmor, Yaakov; Paris, Harry S; Katzir, Nurit; Burger, Yosef; Schaffer, Arthur A

    2014-06-05

    Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago.

  11. Tolerance to acetic acid is improved by mutations of the TATA-binding protein gene.

    PubMed

    An, Jieun; Kwon, Hyeji; Kim, Eunjung; Lee, Young Mi; Ko, Hyeok Jin; Park, Hongjae; Choi, In-Geol; Kim, Sooah; Kim, Kyoung Heon; Kim, Wankee; Choi, Wonja

    2015-03-01

    Screening a library of overexpressing mutant alleles of the TATA-binding gene SPT15 yielded two Saccharomyces cerevisiae strains (MRRC 3252 and 3253) with enhanced tolerance to acetic acid. They were also tolerant to propionic acid and hydrogen peroxide. Transcriptome profile analysis identified 58 upregulated genes and 106 downregulated genes in MRRC 3252. Stress- and protein synthesis-related transcription factors were predominantly enriched in the upregulated and downregulated genes respectively. Eight deletion mutants for some of the highly downregulated genes were acetic acid-tolerant. The level of intracellular reactive oxygen species was considerably lessened in MRRC 3252 and 3253 upon exposure to acetic acid. Metabolome profile analysis revealed that intracellular concentrations of 5 and 102 metabolites were increased and decreased, respectively, in MRRC 3252, featuring a large increase of urea and a significant decrease of amino acids. The dur1/2Δmutant, in which the urea degradation gene DUR1/2 is deleted, displayed enhanced tolerance to acetic acid. Enhanced tolerance to acetic acid was also observed on the medium containing a low concentration of amino acids. Taken together, this study identified two SPT15 alleles, nine gene deletions and low concentration of amino acids in the medium that confer enhanced tolerance to acetic acid.

  12. Identification of Genes Associated with Reproduction in the Mud Crab (Scylla olivacea) and Their Differential Expression following Serotonin Stimulation

    PubMed Central

    Kornthong, Napamanee; Cummins, Scott F.; Chotwiwatthanakun, Charoonroj; Khornchatri, Kanjana; Engsusophon, Attakorn; Hanna, Peter J.; Sobhon, Prasert

    2014-01-01

    The central nervous system (CNS) is often intimately involved in reproduction control and is therefore a target organ for transcriptomic investigations to identify reproduction-associated genes. In this study, 454 transcriptome sequencing was performed on pooled brain and ventral nerve cord of the female mud crab (Scylla olivacea) following serotonin injection (5 µg/g BW). A total of 197,468 sequence reads was obtained with an average length of 828 bp. Approximately 38.7% of 2,183 isotigs matched with significant similarity (E value < 1e−4) to sequences within the Genbank non-redundant (nr) database, with most significant matches being to crustacean and insect sequences. Approximately 32 putative neuropeptide genes were identified from nonmatching blast sequences. In addition, we identified full-length transcripts for crustacean reproductive-related genes, namely farnesoic acid o-methyltransferase (FAMeT), estrogen sulfotransferase (ESULT) and prostaglandin F synthase (PGFS). Following serotonin injection, which would normally initiate reproductive processes, we found up-regulation of FAMeT, ESULT and PGFS expression in the female CNS and ovary. Our data here provides an invaluable new resource for understanding the molecular role of the CNS on reproduction in S. olivacea. PMID:25542017

  13. The rice OsLpa1 gene encodse a novel protein involved in phytic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rice low phytic acid 1 (OsLpa1) gene was originally identified using a forward genetics approach. Mutation of this gene resulted in a 45% reduction in rice seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, the rice lpa1 mutant does not appear to differ significa...

  14. Molecular evolution of monotreme and marsupial whey acidic protein genes.

    PubMed

    Sharp, Julie A; Lefèvre, Christophe; Nicholas, Kevin R

    2007-01-01

    Whey acidic protein (WAP), a major whey protein present in milk of a number of mammalian species has characteristic cysteine-rich domains known as four-disulfide cores (4-DSC). Eutherian WAP, expressed in the mammary gland throughout lactation, has two 4-DSC domains, (DI-DII) whereas marsupial WAP, expressed only during mid-late lactation, contains an additional 4-DSC (DIII), and has a DIII-D1-DII configuration. We report the expression and evolution of echidna (Tachyglossus aculeatus) and platypus (Onithorhynchus anatinus) WAP cDNAs. Predicted translation of monotreme cDNAs showed echidna WAP contains two 4-DSC domains corresponding to DIII-DII, whereas platypus WAP contains an additional domain at the C-terminus with homology to DII and has the configuration DIII-DII-DII. Both monotreme WAPs represent new WAP protein configurations. We propose models for evolution of the WAP gene in the mammalian lineage either through exon loss from an ancient ancestor or by rapid evolution via the process of exon shuffling. This evolutionary outcome may reflect differences in lactation strategy between marsupials, monotremes, and eutherians, and give insight to biological function of the gene products. WAP four-disulfide core domain 2 (WFDC2) proteins were also identified in echidna, platypus and tammar wallaby (Macropus eugenii) lactating mammary cells. WFDC2 proteins are secreted proteins not previously associated with lactation. Mammary gland expression of tammar WFDC2 during the course of lactation showed WFDC2 was elevated during pregnancy, reduced in early lactation and absent in mid-late lactation.

  15. Hyaluronic acid enhances gene delivery into the cochlea.

    PubMed

    Shibata, Seiji B; Cortez, Sarah R; Wiler, James A; Swiderski, Donald L; Raphael, Yehoash

    2012-03-01

    Cochlear gene therapy can be a new avenue for the treatment of severe hearing loss by inducing regeneration or phenotypic rescue. One necessary step to establish this therapy is the development of a safe and feasible inoculation surgery, ideally without drilling the bony cochlear wall. The round window membrane (RWM) is accessible in the middle-ear space, but viral vectors placed on this membrane do not readily cross the membrane to the cochlear tissues. In an attempt to enhance permeability of the RWM, we applied hyaluronic acid (HA), a nontoxic and biodegradable reagent, onto the RWM of guinea pigs, prior to delivering an adenovirus carrying enhanced green fluorescent protein (eGFP) reporter gene (Ad-eGFP) at the same site. We examined distribution of eGFP in the cochlea 1 week after treatment, comparing delivery of the vector via the RWM, with or without HA, to delivery by a cochleostomy into the perilymph. We found that cochlear tissue treated with HA-assisted delivery of Ad-eGFP demonstrated wider expression of transgenes in cochlear cells than did tissue treated by cochleostomy injection. HA-assisted vector delivery facilitated expression in cells lining the scala media, which are less accessible and not transduced after perilymphatic injection. We assessed auditory function by measuring auditory brainstem responses and determined that thresholds were significantly better in the ears treated with HA-assisted Ad-eGFP placement on the RWM as compared with cochleostomy. Together, these data demonstrate that HA-assisted delivery of viral vectors provides an atraumatic and clinically feasible method to introduce transgenes into cochlear cells, thereby enhancing both research methods and future clinical application.

  16. Identification of a 12-gene fusaric acid biosynthetic gene cluster in Fusarium species through comparative and functional genomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a ...

  17. Molecular characterization of the acid-inducible asr gene of Escherichia coli and its role in acid stress response.

    PubMed

    Seputiene, Vaida; Motiejūnas, Domantas; Suziedelis, Kestutis; Tomenius, Henrik; Normark, Staffan; Melefors, Ojar; Suziedeliene, Edita

    2003-04-01

    Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli

  18. The PH gene determines fruit acidity and contributes to the evolution of sweet melons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acids are one of the three major components of fleshy fruit taste, together with sugars and volatile flavor compounds. However, the molecular-genetic control of acid accumulation in fruit is poorly understood and, to date, no genes responsible for acid accumulation in fleshy fruit have been function...

  19. Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene

    PubMed Central

    Mihálik, Daniel; Klčová, Lenka; Ondreičková, Katarína; Hudcovicová, Martina; Gubišová, Marcela; Klempová, Tatiana; Čertík, Milan; Pauk, János; Kraic, Ján

    2015-01-01

    The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%–0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%–1.40% (v/v) and 0%–1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat. PMID:26694368

  20. Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition[C][W

    PubMed Central

    Chantreau, Maxime; Portelette, Antoine; Dauwe, Rebecca; Kiyoto, Shingo; Crônier, David; Morreel, Kris; Arribat, Sandrine; Neutelings, Godfrey; Chabi, Malika; Boerjan, Wout; Yoshinaga, Arata; Mesnard, François; Grec, Sebastien; Chabbert, Brigitte; Hawkins, Simon

    2014-01-01

    Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply. PMID:25381351

  1. Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

    PubMed

    Chantreau, Maxime; Portelette, Antoine; Dauwe, Rebecca; Kiyoto, Shingo; Crônier, David; Morreel, Kris; Arribat, Sandrine; Neutelings, Godfrey; Chabi, Malika; Boerjan, Wout; Yoshinaga, Arata; Mesnard, François; Grec, Sebastien; Chabbert, Brigitte; Hawkins, Simon

    2014-11-01

    Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

  2. Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

    PubMed

    O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

    2015-08-03

    The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms.

  3. Executive function performance and change in aging is predicted by apolipoprotein E, intensified by catechol-O-methyltransferase and brain-derived neurotrophic factor, and moderated by age and lifestyle.

    PubMed

    Sapkota, Shraddha; Bäckman, Lars; Dixon, Roger A

    2017-01-03

    Recent studies have reported several genetic, health, and aging interaction effects in predicting cognitive performance and change. We used an accelerated longitudinal design to examine interactions among genetic, lifestyle, and aging for executive function (EF) in non-demented older adults (n = 634; age range = 53-95 years). The polymorphisms were apolipoprotein E (APOE), catechol-O-methyltransferase (COMT), and brain-derived neurotrophic factor (BDNF). We tested (1) independent and additive effects of APOE, COMT, and BDNF and (2) APOE effect modification for COMT + BDNF, on EF performance and 9-year change as separated by age and lifestyle activities. First, APOE ε4+ carriers had poorer EF performance and steeper 9-year decline. Second, APOE ε4+ carriers with (1) BDNF Met/Met genotype and (2) increasing allelic risk in the COMT + BDNF risk panel had poorer EF performance; these effects were moderated by lifestyle activities (composite of everyday social, physical, and cognitive activities). Examining APOE effect modification for COMT + BDNF risk panel effects with other moderating factors may help identify complex neurobiological and genetic underpinnings of polygenic phenotypes such as EF in aging.

  4. (1)H, (15)N, (13)C backbone resonance assignments of human soluble catechol O-methyltransferase in complex with S-adenosyl-L-methionine and 3,5-dinitrocatechol.

    PubMed

    Czarnota, Sylwia; Baxter, Nicola J; Cliff, Matthew J; Waltho, Jonathan P; Scrutton, Nigel S; Hay, Sam

    2017-04-01

    Catechol O-methyltransferase (COMT) is an enzyme that plays a major role in catechol neurotransmitter deactivation. Inhibition of COMT can increase neurotransmitter levels, which provides a means of treatment for Parkinson's disease, schizophrenia and depression. COMT exists as two isozymes: a soluble cytoplasmic form (S-COMT), expressed in the liver and kidneys and a membrane-bound form (MB-COMT), found mostly in the brain. Here we report the backbone (1)H, (15)N and (13)C chemical shift assignments of S-COMT in complex with S-adenosyl-L-methionine, 3,5-dinitrocatechol and Mg(2+). Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97 % of all backbone resonances were assigned in the complex, with 205 out of a possible 215 residues assigned in the (1)H-(15)N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS+ webserver is in good agreement with published X-ray crystal structures.

  5. Genome mining of mycosporine-like amino acid (MAA) synthesizing and non-synthesizing cyanobacteria: A bioinformatics study.

    PubMed

    Singh, Shailendra P; Klisch, Manfred; Sinha, Rajeshwar P; Häder, Donat-P

    2010-02-01

    Mycosporine-like amino acids (MAAs) are a family of more than 20 compounds having absorption maxima between 310 and 362 nm. These compounds are well known for their UV-absorbing/screening role in various organisms and seem to have evolutionary significance. In the present investigation we tested four cyanobacteria, e.g., Anabaena variabilis PCC 7937, Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301, for their ability to synthesize MAA and conducted genomic and phylogenetic analysis to identify the possible set of genes that might be involved in the biosynthesis of these compounds. Out of the four investigated species, only A. variabilis PCC 7937 was able to synthesize MAA. Genome mining identified a combination of genes, YP_324358 (predicted DHQ synthase) and YP_324357 (O-methyltransferase), which were present only in A. variabilis PCC 7937 and missing in the other studied cyanobacteria. Phylogenetic analysis revealed that these two genes are transferred from a cyanobacterial donor to dinoflagellates and finally to metazoa by a lateral gene transfer event. All other cyanobacteria, which have these two genes, also had another copy of the DHQ synthase gene. The predicted protein structure for YP_324358 also suggested that this product is different from the chemically characterized DHQ synthase of Aspergillus nidulans contrary to the YP_324879, which was predicted to be similar to the DHQ synthase. The present study provides a first insight into the genes of cyanobacteria involved in MAA biosynthesis and thus widens the field of research for molecular, bioinformatics and phylogenetic analysis of these evolutionary and industrially important compounds. Based on the results we propose that YP_324358 and YP_324357 gene products are involved in the biosynthesis of the common core (deoxygadusol) of all MAAs.

  6. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  7. Cloning of L-amino acid deaminase gene from Proteus vulgaris.

    PubMed

    Takahashi, E; Ito, K; Yoshimoto, T

    1999-12-01

    The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned and the nucleotide sequence of the enzyme gene was clarified. An open reading frame of 1,413 bp starting at an ATG methionine codon was found, which encodes a protein of 471 amino acid residues, the calculated molecular weight of which is 51,518. The amino acid sequence of P. vulgaris was 58.6% identical with the L-amino acid deaminase of P. mirabilis. A significantly conserved sequence was found around the FAD-binding sequence of flavo-proteins. The partially purified wild and recombinant enzymes had the same substrate specificity for L-amino acids to form the respective keto-acids, however not for D-amino acids.

  8. Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts.

    PubMed

    Zafar, S; Coates, D E; Cullinan, M P; Drummond, B K; Milne, T; Seymour, G J

    2014-10-01

    The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

  9. Nucleic acid binding property of the gene products of rice stripe virus.

    PubMed

    Liang, Delin; Ma, Xiangqiang; Qu, Zhicai; Hull, Roger

    2005-10-01

    GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function.

  10. Oleic acid attenuates trans-10,cis-12 conjugated linoleic acid-mediated inflammatory gene expression in human adipocytes.

    PubMed

    Reardon, Meaghan; Gobern, Semone; Martinez, Kristina; Shen, Wan; Reid, Tanya; McIntosh, Michael

    2012-11-01

    The weight loss supplement conjugated linoleic acid (CLA) consists of an equal mixture of trans-10,cis-12 (10,12) and cis-9,trans-11 (9,11) isomers. However, high levels of mixed CLA isomers, or the 10,12 isomer, causes chronic inflammation, lipodystrophy, or insulin resistance. We previously demonstrated that 10,12 CLA decreases de novo lipid synthesis along with the abundance and activity of stearoyl-CoA desaturase (SCD)-1, a δ-9 desaturase essential for the synthesis of monounsaturated fatty acids (MUFA). Thus, we hypothesized that the 10,12 CLA-mediated decrease in SCD-1, with the subsequent decrease in MUFA, was responsible for the observed effects. To test this hypothesis, 10,12 CLA-treated human adipocytes were supplemented with oleic acid for 12 h to 7 days, and inflammatory gene expression, insulin-stimulated glucose uptake, and lipid content were measured. Oleic acid reduced inflammatory gene expression in a dose-dependent manner, and restored the lipid content of 10,12 CLA-treated adipocytes without improving insulin-stimulated glucose uptake. In contrast, supplementation with stearic acid, a substrate for SCD-1, or 9,11 CLA did not prevent inflammatory gene expression by 10,12 CLA. Notably, 10,12 CLA impacted the expression of several G-protein coupled receptors that was attenuated by oleic acid. Collectively, these data show that oleic acid attenuates 10,12 CLA-induced inflammatory gene expression and lipid content, possibly by alleviating cell stress caused by the inhibition of MUFA needed for phospholipid and neutral lipid synthesis.

  11. Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens.

    PubMed Central

    Decker, H; Motamedi, H; Hutchinson, C R

    1993-01-01

    The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence of tcmP is not significantly similar to those of any known proteins in the available data bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression of tcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis. Images PMID:8509339

  12. EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHOLORACETC ACID

    EPA Science Inventory

    EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHLOROACETIC ACID

    Dichloroacetic acid COCA) is a major by-product ofwater disinfection by cWorination. Several
    studies have shown that DCA induces liver tumors in rodents when administered in drinkmg wate...

  13. recA gene product is responsible for inhibition of deoxyribonucleic acid synthesis after ultraviolet irradiation.

    PubMed Central

    Trgovcević, Z; Petranović, D; Petranović, M; Salaj-Smic, E

    1980-01-01

    Deoxyribonucleic acid synthesis after ultraviolet irradiation was studied in wild-type, uvrA, recB, recA recB, and recA Escherichia coli strains. Inhibition of deoxyribonucleic acid synthesis, which occurs almost immediately after exposing the cells to ultraviolet radiation, depends on the functional gene recA. PMID:6997276

  14. MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    EPA Science Inventory


    MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

  15. EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHLOROACETIC ACID

    EPA Science Inventory

    EARLY GENE EXPRESSION CHANGES IN THE LIVERS OF MICE EXPOSED TO DICHLOROACETIC ACID

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have shown that DCA induces liver tumors in rodents when administered in drinking wate...

  16. Regulation of the expression of key genes involved in HDL metabolism by unsaturated fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine the effects, and possible mechanisms of action, of unsaturated fatty acids on the expression of genes involved in HDL metabolism in HepG2 cells. The mRNA concentration of target genes was assessed by real time PCR. Protein concentrations were determined by wes...

  17. Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

    PubMed

    Desriac, N; Postollec, F; Coroller, L; Sohier, D; Abee, T; den Besten, H M W

    2013-10-01

    Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour.

  18. Regulation of hepatic gene expression by saturated fatty acids.

    PubMed

    Vallim, T; Salter, A M

    2010-01-01

    Diets rich in saturated fatty acids have long been associated with increased plasma cholesterol concentrations and hence increased risk of cardiovascular disease. More recently, they have also been suggested to promote the development of non-alcoholic fatty liver disease. While there is now considerable evidence to suggest that polyunsaturated fatty acids exert many of their effects through regulating the activity of transcription factors, including peroxisome proliferator activated receptors, sterol regulatory binding proteins (SREBPs) and liver X receptor, our understanding of how saturated fatty acids act is still limited. Here we review the potential mechanisms whereby saturated fatty acids modulate hepatic lipid metabolism thereby impacting on the synthesis, storage and secretion of lipids. Evidence is presented that their effects are, at least partly, mediated through modulation of the activity of the SREBP family of transcription factors.

  19. Properties of bacteriophage T4 mutants defective in gene 30 (deoxyribonucleic acid ligase) and the rII gene.

    PubMed

    Karam, J D; Barker, B

    1971-02-01

    In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.

  20. Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis.

    PubMed Central

    Rajgarhia, V B; Strohl, W R

    1997-01-01

    The structure of the Streptomyces sp. strain C5 daunorubicin type II polyketide synthase (PKS) gene region is different from that of other known type II PKS gene clusters. Directly downstream of the genes encoding ketoacylsynthase alpha and beta (KS alpha, KS beta) are two genes (dpsC, dpsD) encoding proteins of unproven function, both absent from other type II PKS gene clusters. Also in contrast to other type II PKS clusters, the gene encoding the acyl carrier protein (ACP), dpsG, is located about 6.8 kbp upstream of the genes encoding the daunorubicin KS alpha and KS beta. In this work, we demonstrate that the minimal genes required to produce aklanonic acid in heterologous hosts are dpsG (ACP), dauI (regulatory activator), dpsA (KS alpha), dpsB (KS beta), dpsF (aromatase), dpsE (polyketide reductase), and dauG (putative deoxyaklanonic acid oxygenase). The two unusual open reading frames, dpsC (KASIII homolog lacking a known active site) and dpsD (acyltransferase homolog), are not required to synthesize aklanonic acid. Additionally, replacement of dpsD or dpsCD in Streptomyces sp. strain C5 with a neomycin resistance gene (aphI) results in mutant strains that still produced anthracyclines. PMID:9098068

  1. Gene Expression Analysis of Alfalfa Seedlings Response to Acid-Aluminum

    PubMed Central

    Lv, Aimin; Wang, Shengyin; Huang, Bingru

    2016-01-01

    Acid-Aluminum (Al) is toxic to plants and greatly affects crop production worldwide. To understand the responses of plants to acid soils and Aluminum toxicity, we examined global gene expression using microarray data in alfalfa seedlings with the treatment of acid-Aluminum. 3,926 genes that were identified significantly up- or downregulated in response to Al3+ ions with pH 4.5 treatment, 66.33% of which were found in roots. Their functional categories were mainly involved with phytohormone regulation, reactive oxygen species, and transporters. Both gene ontology (GO) enrichment and KEGG analysis indicated that phenylpropanoid biosynthesis, phenylalanine metabolism, and flavonoid biosynthesis played a critical role on defense to Aluminum stress in alfalfa. In addition, we found that transcription factors such as the MYB and WRKY family proteins may be also involved in the regulation of reactive oxygen species reactions and flavonoid biosynthesis. Thus, the finding of global gene expression profile provided insights into the mechanisms of plant defense to acid-Al stress in alfalfa. Understanding the key regulatory genes and pathways would be advantageous for improving crop production not only in alfalfa but also in other crops under acid-Aluminum stress. PMID:28074175

  2. Folic acid rivals methylenetetrahydrofolate reductase (MTHFR) gene-silencing effect on MEPM cell proliferation and apoptosis.

    PubMed

    Xiao, Wen-Lin; Wu, Min; Shi, Bing

    2006-11-01

    It's clear that environmental factors play a role in the aetiology of orofacial clefting (OFC) and an important area of future research will be to unravel interactions that occur between candidate genes and environmental factors during early development of the embryo. Periconceptional folic acid supplementation may reduce the risk of OFC. Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene reduce availability of 5-methylenetetrahydrofolate, the predominant circulating form of folic acid. To determine the effect of MTHFR gene mutation on murine embryonic palatal mesenchymal (MEPM) cells and the interaction with folic acid supplement, we used RNAi study in the primary cultures of MEPM cells. The cells of MTHFR gene silencing grew slower and the apoptosis cell number was more than the cells of control. Supplement with 20 microg/ml folic acid was the best to preventing teratogenic effect of MTHFR gene silencing. By flow cytometry analysis of cell cycle, results were shown that the MEPM cells were retarded in G(0)/G(1) after MTHFR gene silencing. While using 20 microg/ml folic acid supplements could make cell transit the G(1)/S restriction point and the cells growth was close to normal level.

  3. The Role of a Catechol-O-Methyltransferase (COMT) Val158Met Genetic Polymorphism in Schizophrenia: A Systematic Review and Updated Meta-analysis on 32,816 Subjects.

    PubMed

    González-Castro, Thelma Beatriz; Hernández-Díaz, Yazmin; Juárez-Rojop, Isela Esther; López-Narváez, María Lilia; Tovilla-Zárate, Carlos Alfonso; Fresan, Ana

    2016-06-01

    An association between a catechol-O-methyltransferase (COMT) Val156Met (rs4680) polymorphism and schizophrenia has been reported in the literature, although no conclusive outcomes have been attained. The aim of this study was to evaluate the association of the COMT Val108/158Met polymorphism with schizophrenia in a systematic review and meta-analysis. We performed a keyword search on PubMed and EBSCO databases. All English language case-control studies published up to April 2015 were selected. A total of 67 studies were selected for inclusion. The genotype distribution of subjects with schizophrenia was compared with healthy control subjects, using allelic, additive, dominant and recessive models. The pooled results from the meta-analysis (15,565 cases and 17,251 healthy subjects) after the elimination of heterogeneity showed an association between COMT Val108/158Met and schizophrenia [recessive model: OR 1.08 CI 95 % (1.01-1.15)]. We conducted subgroup analyses according to ethnicity. An association was observed in our Caucasian population in the additive model [OR 1.21 CI 95 % (1.06-1.37)] and in the recessive model [OR 1.21 CI 95 % (1.11-1.32)], but not in the allelic or dominant models. However, when we analysed our Asian population after the elimination of heterogeneity, no evidence of a significant association was found in any of the genetic models. Our analyses indicate that there is an association between COMT Val108/158Met and schizophrenia in the general population. Furthermore, in Caucasian populations, this risk could be increased.

  4. Entacapone, a catechol-O-methyltransferase inhibitor, improves the motor activity and dopamine content of basal ganglia in a rat model of Parkinson's disease induced by Japanese encephalitis virus.

    PubMed

    Hamaue, Naoya; Ogata, Akihiko; Terado, Mutsuko; Tsuchida, Shirou; Yabe, Ichiro; Sasaki, Hidenao; Hirafuji, Masahiko; Togashi, Hiroko; Aoki, Takashi

    2010-01-14

    Levodopa is the main medication used for the treatment of Parkinson's disease. However, dyskinesia and wearing-off appear after the administration of high-dose levodopa for a long period. To combat the dyskinesia and wearing-off, levodopa is used together with a dopamine (DA) receptor agonist, and the amount of levodopa is decreased. We have reported the monoamine oxidase (MAO)-B inhibitor selegiline to be effective for treating motor dysfunction in Parkinson's disease model rats. We analyzed the improvement in motor functions and catecholamine contents in various brain regions induced by a combination of the catechol-O-methyltransferase (COMT) inhibitor entacapone and a levodopa/dopadecarboxylase inhibitor (DDCI) in Japanese encephalitis virus (JEV) induced Parkinson's disease model rats. Entacapone (10 mg/kg) was administered via a single oral administration with levodopa/DDCI (10 mg/kg). The motor functions of the JEV model rats were significantly worsened, compared with those of the healthy control rats. The motor functions in the Parkinson's disease model rats were significantly recovered to the same levels as the healthy control rats by the combined administration of entacapone and levodopa/DDCI. A significant improvement in motor function was not demonstrated in the case of the administration of levodopa/DDCI alone. The striatal DA concentrations in the model rat brains were significantly increased by using levodopa/DDCI together with entacapone. Motor function was recovered by raising the striatum DA density in the model rats. Thus, COMT inhibitors are useful for decreasing the amount of levodopa administered to Parkinson's disease patients.

  5. Gene expression levels are correlated with synonymous codon usage, amino acid composition, and gene architecture in the red flour beetle, Tribolium castaneum.

    PubMed

    Williford, Anna; Demuth, Jeffery P

    2012-12-01

    Gene expression levels correlate with multiple aspects of gene sequence and gene structure in phylogenetically diverse taxa, suggesting an important role of gene expression levels in the evolution of protein-coding genes. Here we present results of a genome-wide study of the influence of gene expression on synonymous codon usage, amino acid composition, and gene structure in the red flour beetle, Tribolium castaneum. Consistent with the action of translational selection, we find that synonymous codon usage bias increases with gene expression. However, the correspondence between tRNA gene copy number and optimal codons is weak. At the amino acid level, translational selection is suggested by the positive correlation between tRNA gene numbers and amino acid usage, which is stronger for highly expressed genes. In addition, there is a clear trend for increased use of metabolically cheaper, less complex amino acids as gene expression increases. tRNA gene numbers also correlate negatively with amino acid size/complexity (S/C) score indicating the coupling between translational selection and selection to minimize the use of large/complex amino acids. Interestingly, the analysis of 10 additional genomes suggests that the correlation between tRNA gene numbers and amino acid S/C score is widespread and might be explained by selection against negative consequences of protein misfolding. At the level of gene structure, three major trends are detected: 1) complete coding region length increases across low and intermediate expression levels but decreases in highly expressed genes; 2) the average intron size shows the opposite trend, first decreasing with expression, followed by a slight increase in highly expressed genes; and 3) intron density remains nearly constant across all expression levels. These changes in gene architecture are only in partial agreement with selection favoring reduced cost of biosynthesis.

  6. Regulatory Genes Controlling Fatty Acid Catabolism and Peroxisomal Functions in the Filamentous Fungus Aspergillus nidulans†

    PubMed Central

    Hynes, Michael J.; Murray, Sandra L.; Duncan, Anna; Khew, Gillian S.; Davis, Meryl A.

    2006-01-01

    The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5′ region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae. PMID:16682457

  7. Linoleic acid isomerase gene FgLAI12 affects sensitivity to salicylic acid, mycelial growth and virulence of Fusarium graminearum

    PubMed Central

    Zhang, Ya-Zhou; Wei, Zhen-Zhen; Liu, Cai-Hong; Chen, Qing; Xu, Bin-Jie; Guo, Zhen-Ru; Cao, Yong-Li; Wang, Yan; Han, Ya-Nan; Chen, Chen; Feng, Xiang; Qiao, Yuan-Yuan; Zong, Lu-Juan; Zheng, Ting; Deng, Mei; Jiang, Qian-Tao; Li, Wei; Zheng, You-Liang; Wei, Yu-Ming; Qi, Peng-Fei

    2017-01-01

    Fusarium graminearum is the major causal agent of fusarium head blight in wheat, a serious disease worldwide. Linoleic acid isomerase (LAI) catalyses the transformation of linoleic acid (LA) to conjugated linoleic acid (CLA), which is beneficial for human health. We characterised a cis-12 LAI gene of F. graminearum (FGSG_02668; FgLAI12), which was downregulated by salicylic acid (SA), a plant defence hormone. Disruption of FgLAI12 in F. graminearum resulted in decreased accumulation of cis-9,trans-11 CLA, enhanced sensitivity to SA, and increased accumulation of LA and SA in wheat spikes during infection. In addition, mycelial growth, accumulation of deoxynivalenol, and pathogenicity in wheat spikes were reduced. Re-introduction of a functional FgLAI12 gene into ΔFgLAI12 recovered the wild-type phenotype. Fluorescent microscopic analysis showed that FgLAI12 protein was usually expressed in the septa zone of conidia and the vacuole of hyphae, but was expressed in the cell membrane of hyphae in response to exogenous LA, which may be an element of LA metabolism during infection by F. graminearum. The cis-12 LAI enzyme encoded by FgLAI12 is critical for fungal response to SA, mycelial growth and virulence in wheat. The gene FgLAI12 is potentially valuable for biotechnological synthesis of cis-9,trans-11 CLA. PMID:28387243

  8. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production.

    PubMed

    Michelucci, Alessandro; Cordes, Thekla; Ghelfi, Jenny; Pailot, Arnaud; Reiling, Norbert; Goldmann, Oliver; Binz, Tina; Wegner, André; Tallam, Aravind; Rausell, Antonio; Buttini, Manuel; Linster, Carole L; Medina, Eva; Balling, Rudi; Hiller, Karsten

    2013-05-07

    Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.

  9. ALTERED GENE EXPRESSION IN MOUSE LIVERS AFTER DICHLOROACETIC ACID EXPOSURE

    EPA Science Inventory

    Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated that DCA exhibits hepatocarcinogenic effects in rodents when administered in drinking water. The mechanism(s) involved in DCA induction of cancer are not clear...

  10. Interfering ribonucleic acids that suppress expression of multiple unrelated genes

    PubMed Central

    Passioura, Toby; Gozar, Mary M; Goodchild, Amber; King, Andrew; Arndt, Greg M; Poidinger, Michael; Birkett, Donald J; Rivory, Laurent P

    2009-01-01

    Background Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance. We have investigated the possibility of using a single short synthetic duplex RNA to suppress the expression of VEGF-A and ICAM-1; genes implicated in the progression of ocular neovascular diseases such as diabetic retinopathy. Results Duplex RNA were designed to have incomplete complementarity with the 3'UTR sequences of both target genes. One such duplex, CODEMIR-1, was found to suppress VEGF and ICAM-1 by 90 and 60%, respectively in ARPE-19 cells at a transfected concentration of 40 ng/mL. Use of a cyan fusion reporter with target sites constructed in its 3'UTR demonstrated that the repression of VEGF and ICAM-1 by CODEMIR-1 was indeed due to interaction with the target sequence. An exhaustive analysis of sequence variants of CODEMIR-1 demonstrated a clear positive correlation between activity against VEGF (but not ICAM-1) and the length of the contiguous complementary region (from the 5' end of the guide strand). Various strategies, including the use of inosine bases at the sites of divergence of the target sequences were investigated. Conclusion Our work demonstrates the possibility of designing multitargeting dsRNA to suppress more than one disease-altering gene. This warrants further investigation as a possible therapeutic approach. PMID:19531249

  11. Utilization of lactic acid bacterial genes in Synechocystis sp. PCC 6803 in the production of lactic acid.

    PubMed

    Joseph, Ancy; Aikawa, Shimpei; Sasaki, Kengo; Tsuge, Yota; Matsuda, Fumio; Tanaka, Tsutomu; Kondo, Akihiko

    2013-01-01

    Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO2 has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plantarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Co-expression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 ± 0.02 or 0.14 ± 0.02 mM after 18 d of cultivation.

  12. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis

    PubMed Central

    Nair, Ramesh B.; Bastress, Kristen L.; Ruegger, Max O.; Denault, Jeff W.; Chapple, Clint

    2004-01-01

    Recent research has significantly advanced our understanding of the phenylpropanoid pathway but has left in doubt the pathway by which sinapic acid is synthesized in plants. The reduced epidermal fluorescence1 (ref1) mutant of Arabidopsis thaliana accumulates only 10 to 30% of the sinapate esters found in wild-type plants. Positional cloning of the REF1 gene revealed that it encodes an aldehyde dehydrogenase, a member of a large class of NADP+-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Consistent with this finding, extracts of ref1 leaves exhibit low sinapaldehyde dehydrogenase activity. These data indicate that REF1 encodes a sinapaldehyde dehydrogenase required for sinapic acid and sinapate ester biosynthesis. When expressed in Escherichia coli, REF1 was found to exhibit both sinapaldehyde and coniferaldehyde dehydrogenase activity, and further phenotypic analysis of ref1 mutant plants showed that they contain less cell wall–esterified ferulic acid. These findings suggest that both ferulic acid and sinapic acid are derived, at least in part, through oxidation of coniferaldehyde and sinapaldehyde. This route is directly opposite to the traditional representation of phenylpropanoid metabolism in which hydroxycinnamic acids are instead precursors of their corresponding aldehydes. PMID:14729911

  13. Tissue Specific Expression Levels of Apoptosis Involved Genes Have Correlations with Codon and Amino Acid Usage

    PubMed Central

    Sadeghi, Iman; Salavaty, Abbas; Nasiri, Habib

    2016-01-01

    Different mechanisms, including transcriptional and post transcriptional processes, regulate tissue specific expression of genes. In this study, we report differences in gene/protein compositional features between apoptosis involved genes selectively expressed in human tissues. We found some correlations between codon/amino acid usage and tissue specific expression level of genes. The findings can be significant for understanding the translational selection on these features. The selection may play an important role in the differentiation of human tissues and can be considered for future studies in diagnosis of some diseases such as cancer. PMID:28154517

  14. Multiplexed analysis of genes using nucleic acid-stabilized silver-nanocluster quantum dots.

    PubMed

    Enkin, Natalie; Wang, Fuan; Sharon, Etery; Albada, H Bauke; Willner, Itamar

    2014-11-25

    Luminescent nucleic acid-stabilized Ag nanoclusters (Ag NCs) are applied for the optical detection of DNA and for the multiplexed analysis of genes. Two different sensing modules including Ag NCs as luminescence labels are described. One sensing module involves the assembly of a three-component sensing module composed of a nucleic acid-stabilized Ag NC and a quencher-modified nucleic acid hybridized with a nucleic acid scaffold that is complementary to the target DNA. The luminescence of the Ag NCs is quenched in the sensing module nanostructure. The strand displacement of the scaffold by the target DNA separates the nucleic acid-functionalized Ag NCs, leading to the turned-on luminescence of the NCs and to the optical readout of the sensing process. By implementing two different-sized Ag NC-modified sensing modules, the parallel multiplexed analysis of two genes (the Werner Syndrome gene and the HIV, human immunodeficiency, gene), using 615 and 560 nm luminescent Ag NCs, is demonstrated. The second sensing module includes the nucleic acid functionalized Ag NCs and the quencher-modified nucleic acid hybridized with a hairpin DNA scaffold. The luminescence of the Ag NCs is quenched in the sensing module. Opening of the hairpin by the target DNA triggers the luminescence of the Ag NCs, due to the spatial separation of the Ag NCs/quencher units. The system is applied for the optical detection of the BRAC1 gene. In addition, by implementing two-sized Ag NCs, the multiplexed analysis of two genes by the hairpin sensing module approach is demonstrated.

  15. The expansion of amino-acid repeats is not associated to adaptive evolution in mammalian genes

    PubMed Central

    2009-01-01

    Background The expansion of amino acid repeats is determined by a high mutation rate and can be increased or limited by selection. It has been suggested that recent expansions could be associated with the potential of adaptation to new environments. In this work, we quantify the strength of this association, as well as the contribution of potential confounding factors. Results Mammalian positively selected genes have accumulated more recent amino acid repeats than other mammalian genes. However, we found little support for an accelerated evolutionary rate as the main driver for the expansion of amino acid repeats. The most significant predictors of amino acid repeats are gene function and GC content. There is no correlation with expression level. Conclusions Our analyses show that amino acid repeat expansions are causally independent from protein adaptive evolution in mammalian genomes. Relaxed purifying selection or positive selection do not associate with more or more recent amino acid repeats. Their occurrence is slightly favoured by the sequence context but mainly determined by the molecular function of the gene. PMID:20021652

  16. Analysis of the aspartic acid metabolic pathway using mutant genes.

    PubMed

    Azevedo, R A

    2002-01-01

    Amino acid metabolism is a fundamental process for plant growth and development. Although a considerable amount of information is available, little is known about the genetic control of enzymatic steps or regulation of several pathways. Much of the information about biochemical pathways has arisen from the use of mutants lacking key enzymes. Although mutants were largely used already in the 60's, by bacterial and fungal geneticists, it took plant research a long time to catch up. The advance in this area was rapid in the 80's, which was followed in the 90's by the development of techniques of plant transformation. In this review we present an overview of the aspartic acid metabolic pathway, the key regulatory enzymes and the mutants and transgenic plants produced for lysine and threonine metabolism. We also discuss and propose a new study of high-lysine mutants.

  17. Foreign gene recruitment to the fatty acid biosynthesis pathway in diatoms.

    PubMed

    Chan, Cheong Xin; Baglivi, Francesca L; Jenkins, Christina E; Bhattacharya, Debashish

    2013-09-01

    Diatoms are highly successful marine and freshwater algae that contribute up to 20% of global carbon fixation. These species are leading candidates for biofuel production owing to ease of culturing and high fatty acid content. To assist in strain improvement and downstream applications for potential use as a biofuel, it is important to understand the evolution of lipid biosynthesis in diatoms. The evolutionary history of diatoms is however complicated by likely multiple endosymbioses involving the capture of foreign cells and horizontal gene transfer into the host genome. Using a phylogenomic approach, we assessed the evolutionary history of 12 diatom genes putatively encoding functions related to lipid biosynthesis. We found evidence of gene transfer likely from a green algal source for seven of these genes, with the remaining showing either vertical inheritance or evolutionary histories too complicated to interpret given current genome data. The functions of horizontally transferred genes encompass all aspects of lipid biosynthesis (initiation, biosynthesis, and desaturation of fatty acids) as well as fatty acid elongation, and are not restricted to plastid-targeted proteins. Our findings demonstrate that the transfer, duplication, and subfunctionalization of genes were key steps in the evolution of lipid biosynthesis in diatoms and other photosynthetic eukaryotes. This target pathway for biofuel research is highly chimeric and surprisingly, our results suggest that research done on related genes in green algae may have application to diatom models.

  18. Mutations of the Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel homolog, induce L-glutamic acid production.

    PubMed

    Nakamura, Jun; Hirano, Seiko; Ito, Hisao; Wachi, Masaaki

    2007-07-01

    Corynebacterium glutamicum is a biotin auxotroph that secretes L-glutamic acid in response to biotin limitation; this process is employed in industrial L-glutamic acid production. Fatty acid ester surfactants and penicillin also induce L-glutamic acid secretion, even in the presence of biotin. However, the mechanism of L-glutamic acid secretion remains unclear. It was recently reported that disruption of odhA, encoding a subunit of the 2-oxoglutarate dehydrogenase complex, resulted in L-glutamic acid secretion without induction. In this study, we analyzed odhA disruptants and found that those which exhibited constitutive L-glutamic acid secretion carried additional mutations in the NCgl1221 gene, which encodes a mechanosensitive channel homolog. These NCgl1221 gene mutations lead to constitutive L-glutamic acid secretion even in the absence of odhA disruption and also render cells resistant to an L-glutamic acid analog, 4-fluoroglutamic acid. Disruption of the NCgl1221 gene essentially abolishes L-glutamic acid secretion, causing an increase in the intracellular L-glutamic acid pool under biotin-limiting conditions, while amplification of the wild-type NCgl1221 gene increased L-glutamate secretion, although only in response to induction. These results suggest that the NCgl1221 gene encodes an L-glutamic acid exporter. We propose that treatments that induce L-glutamic acid secretion alter membrane tension and trigger a structural transformation of the NCgl1221 protein, enabling it to export L-glutamic acid.

  19. Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas.

    PubMed

    Kaur, Simran; Norkina, Oxana; Ziemer, Donna; Samuelson, Linda C; De Lisle, Robert C

    2004-08-01

    The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.

  20. Dynamic changes in prefrontal cortex gene expression following lysergic acid diethylamide administration.

    PubMed

    Nichols, Charles D; Garcia, Efrain E; Sanders-Bush, Elaine

    2003-03-17

    Lysergic acid diethylamide (LSD) is a psychoactive drug that transiently alters human perception, behavior, and mood at extremely low doses. Certain aspects of the behavior elicited by acute doses of LSD closely resemble symptoms of mental disorders such as schizophrenia. Characterizing gene expression profiles after LSD will be important for understanding how it alters behavior, and will lead to novel insights into disorders, such as schizophrenia, whose behavioral symptoms resemble the temporary effects of hallucinogenic drugs. We previously identified a small collection of genes within the rat prefrontal cortex that respond to LSD. Many of the products of these genes are involved in the process of synaptic plasticity. In the current report, we present a detailed analysis of the expression of these genes within the brain using RNase protection analysis. We find that the gene response to LSD is quite dynamic. The expression of some genes increases rapidly and decreases rapidly, while other genes change more gradually. Dose-response studies show two classes of expression; gene expression maximally stimulated at lower doses, versus gene expression that continues to rise at the higher doses. The role of the 5-HT(1A) and 5-HT(2A) receptor in mediating the increases in gene expression was examined in a series of experiments using receptor specific antagonists. Most expression increases were due to activation of the 5-HT(2A) receptor, however expression of two genes had neither a 5-HT(1A) nor a 5-HT(2A) receptor component.

  1. Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

    PubMed Central

    Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

    2016-01-01

    The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

  2. Heterogenous expression of poly-gamma-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3.

    PubMed

    Wang, N; Yang, G; Che, C; Liu, Y

    2011-01-01

    Bacillus licheniformis WBL-3, one of poly-gamma-glutamic acid (gamma-PGA) producers, depends on the existence of glutamate in the medium. In this paper, gamma-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgsBCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IF03336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced gamma-PGA extracellularly. The yield of gamma-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar gamma-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize gamma-PGA.

  3. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68.

    PubMed

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

  4. Cloning and phylogenetic analysis of a fatty acid elongase gene from Nannochloropsis oculata CS179

    NASA Astrophysics Data System (ADS)

    Pan, Kehou; Ma, Xiaolei; Yu, Jianzhong; Zhu, Baohua; Yang, Guanpin

    2009-12-01

    Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids (LCPUFAs). Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5' and 3' RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the cryptosporidium. But the NJ-tree revealed that the N. oculata CS179 elongase clustered with those of the microalgae Phaeodactylum tricornutum, Ostreococcus tauri and Thalassiosira pseudonana.

  5. Serum homocysteine, vitamin B12, folic acid levels and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in vitiligo.

    PubMed

    Yasar, Ali; Gunduz, Kamer; Onur, Ece; Calkan, Mehmet

    2012-01-01

    The aim of this study was to determine serum vitamin B12, folic acid and homocysteine (Hcy) levels as well as MTHFR (C677, A1298C) gene polymorphisms in patients with vitiligo, and to compare the results with healthy controls. Forty patients with vitiligo and 40 age and sex matched healthy subjects were studied. Serum vitamin B12 and folate levels were determined by enzyme-linked immunosorbent assay. Plasma Hcy levels and MTHFR polymorphisms were determined by chemiluminescence and real time PCR methods, respectively. Mean serum vitamin B12 and Hcy levels were not significantly different while folic acid levels were significantly lower in the control group. There was no significant relationship between disease activity and vitamin B12, folic acid and homocystein levels. No significant difference in C677T gene polymorphism was detected. Heterozygote A1298C gene polymorphism in the patient group was statistically higher than the control group. There was no significant relationship between MTHFR gene polymorphisms and vitamin B12, folic acid and homocysteine levels. In conclusion, vitamin B12, folate and Hcy levels are not altered in vitiligo and MTHFR gene mutations (C677T and A1298C) do not seem to create susceptibility for vitiligo.

  6. Salicylic acid and gentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens.

    PubMed

    Campos, Laura; Granell, Pablo; Tárraga, Susana; López-Gresa, Pilar; Conejero, Vicente; Bellés, José María; Rodrigo, Ismael; Lisón, Purificación

    2014-04-01

    We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SA-induced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA.

  7. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

    PubMed

    Lin, Jingyu; Mazarei, Mitra; Zhao, Nan; Zhu, Junwei J; Zhuang, Xiaofeng; Liu, Wusheng; Pantalone, Vincent R; Arelli, Prakash R; Stewart, Charles N; Chen, Feng

    2013-12-01

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

  8. Effects of Oils Rich in Linoleic and α-Linolenic Acids on Fatty Acid Profile and Gene Expression in Goat Meat

    PubMed Central

    Ebrahimi, Mahdi; Rajion, Mohamed Ali; Goh, Yong Meng

    2014-01-01

    Alteration of the lipid content and fatty acid (FA) composition of foods can result in a healthier product. The aim of this study was to determine the effect of flaxseed oil or sunflower oil in the goat diet on fatty acid composition of muscle and expression of lipogenic genes in the semitendinosus (ST) muscle. Twenty-one entire male Boer kid goats were fed diets containing different levels of linoleic acid (LA) and α-linolenic acid (LNA) for 100 days. Inclusion of flaxseed oil increased (p < 0.05) the α-linolenic acid (C18:3n-3) concentration in the ST muscle. The diet high in α-linolenic acid (p < 0.05) decreased the arachidonic acid (C20:4n-6) and conjugated linolenic acid (CLA) c-9 t-11 content in the ST muscle. There was a significant (p < 0.05) upregulation of PPARα and PPARγ gene expression and downregulation of stearoyl-CoA desaturase (SCD) gene in the ST muscle for the high α-linolenic acid group compared with the low α-linolenic acid group. The results of the present study show that flaxseed oil as a source of α-linolenic acid can be incorporated into the diets of goats to enrich goat meat with n-3 fatty acids, upregulate the PPARα and PPARγ, and downregulate the SCD gene expression. PMID:25255382

  9. Antitumor Molecular Mechanism of Chlorogenic Acid on Inducting Genes GSK-3β and APC and Inhibiting Gene β-Catenin

    PubMed Central

    Xu, Ruoshi; Kang, Qiumei; Ren, Jie; Li, Zukun; Xu, Xiaoping

    2013-01-01

    Objective. Inhibiting gene β-catenin and inducting genes GSK-3β and APC, promoting the tumor cell apoptosis in Wnt pathway, by chlorogenic acid were discussed (CGA). Method. The different genes were scanned by the 4∗44K mouse microarray chips. The effect of the three genes was confirmed by RT-PCR technique with CGA dosage of 5, 10, and 20 mg/kg. Result. The expression of GSK-3β and APC was upregulated in group of 20 mg/kg dosage (P < 0.05) and the expression of β-catenin was downregulated in the same dosage (P < 0.05). Conclusion. The results infer that the multimeric protein complex of β-catenin could be increased by CGA upregulated genes GSK-3β and APC, which could inhibit the free β-catenin into the nucleus to connect with TCF. So the transcriptional expression of the target genes will be cut to abnormal cell proliferation. It is probably one of the ways that can stop the tumor increase by CGA. PMID:23844319

  10. Unconjugated Bile Acids Influence Expression of Circadian Genes: A Potential Mechanism for Microbe-Host Crosstalk

    PubMed Central

    Govindarajan, Kalaimathi; MacSharry, John; Casey, Patrick G.; Shanahan, Fergus

    2016-01-01

    Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression. PMID:27907092

  11. Green Tea Extract and Catechol-O-Methyltransferase Genotype Modify Fasting Serum Insulin and Plasma Adiponectin Concentrations in a Randomized Controlled Trial of Overweight and Obese Postmenopausal Women1234

    PubMed Central

    Dostal, Allison M; Samavat, Hamed; Espejo, Luis; Arikawa, Andrea Y; Stendell-Hollis, Nicole R; Kurzer, Mindy S

    2016-01-01

    Background: Green tea consumption has been associated with favorable changes in body weight and obesity-related hormones, although it is not known whether these changes result from green tea polyphenols or caffeine. Objective: We examined the impact of decaffeinated green tea extract (GTE) containing 843 mg of (−)-epigallocatechin-3-gallate on anthropometric variables, obesity-associated hormones, and glucose homeostasis. Methods: The Minnesota Green Tea Trial was a 12-mo randomized, double-blind, placebo-controlled clinical trial of 937 healthy postmenopausal women assigned to either decaffeinated GTE (1315 mg total catechins/d) or a placebo, stratified by catechol-O-methyltransferase (COMT) genotype. This study was conducted in a subset of 237 overweight and obese participants [body mass index (BMI) ≥25 kg/m2]. Results: No changes in energy intake, body weight, BMI, or waist circumference (WC) were observed over 12 mo in women taking GTE (n = 117) or placebo (n = 120). No differences were seen in circulating leptin, ghrelin, adiponectin, or glucose concentrations at month 12. Participants randomly assigned to GTE with baseline insulin ≥10 μIU/mL (n = 23) had a decrease in fasting serum insulin from baseline to month 12 (−1.43 ± 0.59 μIU/mL), whereas those randomly assigned to placebo with baseline insulin ≥10 μIU/mL (n = 19) had an increase in insulin over 12 mo (0.55 ± 0.64 μIU/mL, P < 0.01). Participants with the homozygous high-activity (G/G) form of COMT had significantly lower adiponectin (5.97 ± 0.50 compared with 7.58 ± 0.53 μg/mL, P = 0.03) and greater insulin concentrations (7.63 ± 0.53 compared with 6.18 ± 0.36 μIU/mL, P = 0.02) at month 12 compared with those with the low-activity (A/A) genotype, regardless of treatment group. Conclusions: Decaffeinated GTE was not associated with reductions in body weight, BMI, or WC and did not alter energy intake or mean hormone concentrations in healthy postmenopausal women over 12 mo. GTE

  12. Use of catechol-O-methyltransferase inhibition to minimize L-3,4-dihydroxyphenylalanine-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque.

    PubMed

    Huot, Philippe; Johnston, Tom H; Snoeren, Tessa; Koprich, James B; Hill, Michael P; Fox, Susan H; Brotchie, Jonathan M

    2013-03-01

    L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia is a complication of dopaminergic treatment in Parkinson's disease. Lowering the L-DOPA dose reduces dyskinesia but also reduces the antiparkinsonian benefit. A therapy that could enhance the antiparkinsonian action of low-dose L-DOPA (LDl) without exacerbating dyskinesia would thus be of considerable therapeutic benefit. This study assessed whether catechol-O-methyltransferase (COMT) inhibition, as an add-on to LDl, might be a means to achieve this goal. Cynomolgus macaques were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Dyskinesia was established by chronic treatment with L-DOPA. Two doses of L-DOPA were identified - high-dose L-DOPA (LDh), which provided good antiparkinsonian benefit but was compromised by disabling dyskinesia, and LDl, which was sub-threshold for providing significant antiparkinsonian benefit, without dyskinesia. LDh and LDl were administered in acute challenges in combination with vehicle and, for LDl, with the COMT inhibitor entacapone (5, 15 and 45 mg/kg). The duration of antiparkinsonian benefit (ON-time), parkinsonism and dyskinesia were determined. The ON-time after LDh was ∼170 min and the ON-time after LDl alone (∼98 min) was not significantly different to vehicle (∼37 min). In combination with LDl, entacapone significantly increased the ON-time (5, 15 and 45 mg/kg being ∼123, ∼148 and ∼180 min, respectively). The ON-time after LDl/entacapone 45 mg/kg was not different to that after LDh. However, whereas the percentage ON-time that was compromised by disabling dyskinesia was ∼56% with LDh, it was only ∼31% with LDl/entacapone 45 mg/kg. In addition to the well-recognized action of COMT inhibition to reduce wearing-OFF, the data presented suggest that COMT inhibition in combination with low doses of L-DOPA has potential as a strategy to alleviate dyskinesia.

  13. Gene expression profiles of murine fatty liver induced by the administration of valproic acid

    SciTech Connect

    Lee, Min-Ho; Hong, Il; Kim, Mingoo; Lee, Byung Hoon; Kim, Ju-Han; Kang, Kyung-Sun; Kim, Hyung-Lae; Yoon, Byung-Il; Chung, Heekyoung; Kong, Gu; Lee, Mi-Ock . E-mail: molee@snu.ac.kr

    2007-04-01

    Valproic acid (VPA) has been used as anticonvulsants, however, it induces hepatotoxicity such as microvesicular steatosis and necrosis in the liver. To explore the mechanisms of VPA-induced steatosis, we profiled the gene expression patterns of the mouse liver that were altered by treatment with VPA using microarray analysis. VPA was orally administered as a single dose of 100 mg/kg (low-dose) or 1000 mg/kg (high-dose) to ICR mice and the animals were killed at 6, 24, or 72 h after treatment. Serum alanine aminotransferase and aspartate aminotransferase levels were not significantly altered in the experimental animals. However, symptoms of steatosis were observed at 72 h with low-dose and at 24 h and 72 h with high-dose. After microarray data analysis, 1910 genes were selected by two-way ANOVA (P < 0.05) as VPA-responsive genes. Hierarchical clustering revealed that gene expression changes depended on the time rather than the dose of VPA treatment. Gene profiling data showed striking changes in the expression of genes associated with lipid, fatty acid, and steroid metabolism, oncogenesis, signal transduction, and development. Functional categorization of 1156 characteristically up- and down-regulated genes (cutoff > 1.5-fold) revealed that 60 genes were involved in lipid metabolism that was interconnected with biological pathways for biosynthesis of triglyceride and cholesterol, catabolism of fatty acid, and lipid transport. This gene expression profile may be associated with the known steatogenic hepatotoxicity of VPA and it may provide useful information for prediction of hepatotoxicity of unknown chemicals or new drug candidates through pattern recognition.

  14. Human-specific amino acid changes found in 103 protein-coding genes.

    PubMed

    Kitano, Takashi; Liu, Yu-Hua; Ueda, Shintaroh; Saitou, Naruya

    2004-05-01

    We humans have many characteristics that are different from those of the great apes. These human-specific characters must have arisen through mutations accumulated in the genome of our direct ancestor after the divergence of the last common ancestor with chimpanzee. Gene trees of human and great apes are necessary for extracting these human-specific genetic changes. We conducted a systematic analysis of 103 protein-coding genes for human, chimpanzee, gorilla, and orangutan. Nucleotide sequences for 18 genes were newly determined for this study, and those for the remaining genes were retrieved from the DDBJ/EMBL/GenBank database. The total number of amino acid changes in the human lineage was 147 for 26,199 codons (0.56%). The total number of amino acid changes in the human genome was, thus, estimated to be about 80,000. We applied the acceleration index test and Fisher's synonymous/nonsynonymous exact test for each gene tree to detect any human-specific enhancement of amino acid changes compared with ape branches. Six and two genes were shown to have significantly higher nonsynonymous changes at the human lineage from the acceleration index and exact tests, respectively. We also compared the distribution of the differences of the nonsynonymous substitutions on the human lineage and those on the great ape lineage. Two genes were more conserved in the ape lineage, whereas one gene was more conserved in the human lineage. These results suggest that a small proportion of protein-coding genes started to evolve differently in the human lineage after it diverged from the ape lineage.

  15. A Systems Genetics Approach Identifies Gene Regulatory Networks Associated with Fatty Acid Composition in Brassica rapa Seed1

    PubMed Central

    Xiao, Dong; Bucher, Johan; Jin, Mina; Boyle, Kerry; Fobert, Pierre; Maliepaard, Chris

    2016-01-01

    Fatty acids in seeds affect seed germination and seedling vigor, and fatty acid composition determines the quality of seed oil. In this study, quantitative trait locus (QTL) mapping of fatty acid and transcript abundance was integrated with gene network analysis to unravel the genetic regulation of seed fatty acid composition in a Brassica rapa doubled haploid population from a cross between a yellow sarson oil type and a black-seeded pak choi. The distribution of major QTLs for fatty acids showed a relationship with the fatty acid types: linkage group A03 for monounsaturated fatty acids, A04 for saturated fatty acids, and A05 for polyunsaturated fatty acids. Using a genetical genomics approach, expression quantitative trait locus (eQTL) hotspots were found at major fatty acid QTLs on linkage groups A03, A04, A05, and A09. An eQTL-guided gene coexpression network of lipid metabolism-related genes showed major hubs at the genes BrPLA2-ALPHA, BrWD-40, a number of seed storage protein genes, and the transcription factor BrMD-2, suggesting essential roles for these genes in lipid metabolism. Three subnetworks were extracted for the economically important and most abundant fatty acids erucic, oleic, linoleic, and linolenic acids. Network analysis, combined with comparison of the genome positions of cis- or trans-eQTLs with fatty acid QTLs, allowed the identification of candidate genes for genetic regulation of these fatty acids. The generated insights in the genetic architecture of fatty acid composition and the underlying complex gene regulatory networks in B. rapa seeds are discussed. PMID:26518343

  16. The "putative" role of transcription factors from HlWRKY family in the regulation of the final steps of prenylflavonid and bitter acids biosynthesis in hop (Humulus lupulus L.).

    PubMed

    Matoušek, Jaroslav; Kocábek, Tomáš; Patzak, Josef; Bříza, Jindřich; Siglová, Kristýna; Mishra, Ajay Kumar; Duraisamy, Ganesh Selvaraj; Týcová, Anna; Ono, Eiichiro; Krofta, Karel

    2016-10-01

    Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.

  17. Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2002-10-15

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  18. Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

    DOEpatents

    Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

    2003-03-04

    The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

  19. Molecular cloning and functional characterization of a Δ6-fatty acid desaturase gene from Rhizopus oryzae.

    PubMed

    Zhu, Yu; Zhang, Bi-Bo

    2013-09-01

    The objective was to screen for and isolate a novel enzyme with the specific activity of a Δ6-fatty acid desaturase from Rhizopus oryzae. In this study, R. oryzae was identified as a novel fungal species that produces large amounts of γ-linolenic acid. A full-length cDNA, designated here as RoD6D, with high homology to fungal Δ6-fatty acid desaturase genes was isolated from R. oryzae by using the rapid amplification of cDNA ends method. It had an open reading frame of 1176 bp encoding a deduced polypeptide of 391 amino acids. Bioinformatics analysis characterized the putative RoD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, a hydropathy profile, and a cytochrome b5 -like domain in the N terminus. When the coding sequence was expressed in the Saccharomyces cerevisiae strain INVScl, the encoded product of RoD6D exhibited Δ6-fatty acid desaturase activity that led to the accumulation of γ-linolenic acid. The corresponding genomic sequence of RoD6D was 1565 bp in length, with five introns. This is the first report on the characterization and gene cloning of a Δ6-fatty acid desaturase of R. oryzae from Douchi.

  20. Monitoring Gene Expression In Vivo with Nucleic Acid Molecular Switches

    SciTech Connect

    David C. Ward; Patricia Bray-Ward

    2005-01-26

    The overall objectives of this project were (1) to develop allosteric ribozymes capable of acting as molecular switches for monitoring the levels of both wild-type and mutant mRNA species in living cells and whole animals and (2) to develop highly efficient reagents to deliver nucleic acid molecular switches into living cells, tissues and animals with the ultimate goal of expression profiling specific mRNAs of diagnostic or prognostic value within tumors in animals. During the past year, we have moved our laboratory to Nevada and in the moving process we have lost electronic and paper copies of prior progress reports concerning the construction and biological properties of the molecular switches. Since there was minimal progress during the last year on molecular switches, we are relying on past project reports to provide a summary of our data on this facet of the grant. Here we are summarizing the work done on the delivery reagents and their application to inducing mutations in living cells, which will include work done during the no cost extension.

  1. Bugs, genes, fatty acids, and serotonin: Unraveling inflammatory bowel disease?

    PubMed Central

    Kaunitz, Jonathan; Nayyar, Piyush

    2015-01-01

    The annual incidence of the inflammatory bowel diseases (IBDs) ulcerative colitis and Crohn’s disease has increased at an alarming rate. Although the specific pathophysiology underlying IBD continues to be elusive, it is hypothesized that IBD results from an aberrant and persistent immune response directed against microbes or their products in the gut, facilitated by the genetic susceptibility of the host and intrinsic alterations in mucosal barrier function. In this review, we will describe advances in the understanding of how the interaction of host genetics and the intestinal microbiome contribute to the pathogenesis of IBD, with a focus on bacterial metabolites such as short chain fatty acids (SCFAs) as possible key signaling molecules.  In particular, we will describe alterations of the intestinal microbiota in IBD, focusing on how genetic loci affect the gut microbial phylogenetic distribution and the production of their major microbial metabolic product, SCFAs. We then describe how enteroendocrine cells and myenteric nerves express SCFA receptors that integrate networks such as the cholinergic and serotonergic neural systems and the glucagon-like peptide hormonal pathway, to modulate gut inflammation, permeability, and growth as part of an integrated model of IBD pathogenesis.  Through this integrative approach, we hope that novel hypotheses will emerge that will be tested in reductionist, hypothesis-driven studies in order to examine the interrelationship of these systems in the hope of better understanding IBD pathogenesis and to inform novel therapies. PMID:27508055

  2. Disruption of multiple genes whose deletion causes lactic-acid resistance improves lactic-acid resistance and productivity in Saccharomyces cerevisiae.

    PubMed

    Suzuki, Toshihiro; Sakamoto, Takatoshi; Sugiyama, Minetaka; Ishida, Nobuhiro; Kambe, Hiromi; Obata, Shusei; Kaneko, Yoshinobu; Takahashi, Haruo; Harashima, Satoshi

    2013-05-01

    To create strains that have high productivity of lactic acid without neutralization, a genome-wide screening for strains showing hyper-resistance to 6% l-lactic acid (pH 2.6) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 94 genes whose disruption led to resistance to 6% lactic acid in rich medium. We also found that multiple combinations of Δdse2, Δscw11, Δeaf3, and/or Δsed1 disruption led to enhanced resistance to lactic acid depending upon their combinations. In particular, the quadruple disruptant Δdse2Δscw11Δeaf3Δsed1 grew well in 6% lactic acid with the shortest lag phase. We then introduced an exogenous lactate dehydrogenase gene (LDH) into those single and multiple disruptants to evaluate their productivity of lactic acid. It was found that the quadruple disruptant displaying highest lactic-acid resistance showed a 27% increase of lactic-acid productivity as compared with the LDH-harboring wild-type strain. These observations suggest that disruption of multiple genes whose deletion leads to lactic-acid resistance is an effective way to enhance resistance to lactic acid, leading to high lactic-acid productivity without neutralization.

  3. Enzymatic Kolbe-Schmitt reaction to form salicylic acid from phenol: enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase.

    PubMed

    Kirimura, Kohtaro; Gunji, Hiroaki; Wakayama, Rumiko; Hattori, Takasumi; Ishii, Yoshitaka

    2010-04-02

    Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol; it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 degrees C for 9h.

  4. Expressing yeast SAMdc gene confers broad changes in gene expression and alters fatty acid composition in tomato fruit.

    PubMed

    Kolotilin, Igor; Koltai, Hinanit; Bar-Or, Carmiya; Chen, Lea; Nahon, Sahadia; Shlomo, Haviva; Levin, Ilan; Reuveni, Moshe

    2011-07-01

    Tomato (Solanum lycopersicum) fruits expressing a yeast S-adenosyl methionine decarboxylase (ySAMdc) gene under control of a ripening-induced promoter show altered phytonutrient content and broad changes in gene expression. Genome-wide transcriptional alterations in pericarp tissues of the ySAMdc-expressing fruits are shown. Consistent with the ySAMdc expression pattern from the ripening-induced promoter, very minor transcriptional alterations were detected at the mature green developmental stage. At the breaker and red stages, altered levels of numerous transcripts were observed with a general tendency toward upregulation in the transgenic fruits. Ontological analysis of up- and downregulated transcript groups revealed various affected metabolic processes, mainly carbohydrate and amino acid metabolism, and protein synthesis, which appeared to be intensified in the ripening transgenic fruits. Other functional ontological categories of altered transcripts represented signal transduction, transcription regulation, RNA processing, molecular transport and stress response, as well as metabolism of lipids, glycans, xenobiotics, energy, cofactors and vitamins. In addition, transcript levels of genes encoding structural enzymes for several biosynthetic pathways showed strong correlations to levels of specific metabolites that displayed altered levels in transgenic fruits. Increased transcript levels of fatty acid biosynthesis enzymes were accompanied by a change in the fatty acid profile of transgenic fruits, most notably increasing ω-3 fatty acids at the expense of other lipids. Thus, SAMdc is a prime target in manipulating the nutritional value of tomato fruits. Combined with analyses of selected metabolites in the overripe fruits, a model of enhanced homeostasis of the pericarp tissue in the polyamine-accumulating tomatoes is proposed.

  5. Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furfural (2-furaldehyde) is a furan formed by dehydration of pentose sugars. Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid and Coenzyme A intermediates. To identify genes involved in furan metabolism, two P. putida transposo...

  6. GENE EXPRESSION PATTERNS OF CD-1 DAY-8 EMBRYO CULTURES EXPOSED TO BROMOCHLORO ACETIC ACID

    EPA Science Inventory

    Gene expression patterns of CD-1 day-8 embryo cultures exposed to bromochloro acetic acid

    Edward D. Karoly?*, Judith E. Schmid* and E. Sidney Hunter III*
    ?Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina and *Reproductiv...

  7. Differential influence of distinct fatty acids on cardiomyocyte metabolic gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diabetes mellitus increases risk for cardiovascular disease, and exposes the heart to high plasma fatty acid (FA) levels, which induce genes promoting FA oxidation (e.g., malonyl-CoA decarboxylase; mcd), as well as those suppressing carbohydrate oxidation (e.g., pyruvate dehydrogenase kinase 4; pdk4...

  8. Characterization of the Fatty Acid Desaturase Genes in Cucumber: Structure, Phylogeny, and Expression Patterns

    PubMed Central

    Dong, Chun-Juan; Cao, Ning; Zhang, Zhi-Gang; Shang, Qing-Mao

    2016-01-01

    Fatty acid desaturases (FADs) introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids, and therefore play a critical role in plant development and acclimation to environmental stresses. In this study, 23 full-length FAD genes in cucumber (Cucumis sativus L.) were identified through database searches, including three CsFAB2 genes, two CsFAD2 genes, fourteen CsFAD5 genes, and one gene each for CsFAD3, CsFAD4, CsFAD6 and CsFAD7. These cucumber FAD genes were distributed on all seven chromosomes and two additional scaffolds. Based on a phylogenetic analysis, the cucumber FAD proteins were clustered into five subfamilies with their counterparts from other plants. Gene structures and protein sequences were considerably conserved in each subfamily. All three CsFAB2 proteins shared conserved structure with the known plant soluble FAD proteins. The other cucumber FADs belonged to the membrane-bound FADs and contained three highly conserved histidine boxes. Additionally, the putative endoplasmic reticulum retention signal was found at the C-termini of the CsFAD2 and CsFAD3 proteins, while the N-termini of CsFAD4, CsFAD5, CsFAD6, CsFAD7 and three CsFAB2s contained a predicted chloroplast signal peptide, which was consistent with their associated metabolic pathways. Furthermore, a gene expression analysis showed that CsFAD2 and CsFAD3 were universally expressed in all tested tissues, whereas the other cucumber FAD genes were preferentially expressed in the cotyledons or leaves. The tissue-specific expression patterns of cucumber FAD genes were correlated well with the differences in the fatty acid compositions ofroots and leaves. Finally, the cucumber FAD genes showed a cold-induced and heat-repressed expression pattern, although with distinct regulatory time courses among the different CsFAD members, which indicates the potential roles of the FADs in temperature stress resistance in cucumber. PMID:26938877

  9. Gene cloning of an efficiency oleate hydratase from Stenotrophomonas nitritireducens for polyunsaturated fatty acids and its application in the conversion of plant oils to 10-hydroxy fatty acids.

    PubMed

    Kang, Woo-Ri; Seo, Min-Ju; Shin, Kyung-Chul; Park, Jin-Byung; Oh, Deok-Kun

    2017-01-01

    Hydroxy fatty acids are used as precursors of lactones and dicarboxylic acids, as starting materials of polymers, and as additives in coatings and paintings. Stenotrophomonas nitritireducens efficiently converts cis-9 polyunsaturated fatty acids (PUFAs) to 10-hydroxy fatty acids. However, gene encoding enzyme involved in this conversion has not been identified to date. We purified a putative fatty acid double-bond hydratase from S. nitritireducens by ultrafiltration and HiPrep DEAE FF and Resource Q ion exchange chromatographies. Peptide sequences of the purified enzyme were obtained by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Sequence of the partial gene encoding this putative fatty acid double-bond hydratase was determined by degenerate polymerase chain reaction (PCR) based on the peptide sequences. The remaining gene sequence was identified by rapid amplification of cDNA ends using cDNA of S. nitritireducens as a template, and the full-length gene was cloned subsequently. The expressed enzyme was identified as an oleate hydratase by determining its kinetic parameters toward unsaturated fatty acids. S. nitritireducens oleate hydratase showed higher activity toward PUFAs compared with other available oleate hydratases. This suggested that the enzyme could be used effectively to convert plant oils to 10-hydroxy fatty acids because these oils contained unsaturated fatty acids such as oleic acid (OA) and linoleic acid (LA) and PUFAs such as α-linolenic acid and/or γ-linolenic acid. The enzyme converted soybean oil and perilla seed oil hydrolyzates containing 10 mM total unsaturated fatty acids, including OA, LA, and ALA, to 8.87 and 8.70 mM total 10-hydroxy fatty acids, respectively, in 240 min. To our knowledge, this is the first study on the biotechnological conversion of PUFA-containing oils to hydroxy fatty acids. Biotechnol. Bioeng. 2017;114: 74-82. © 2016 Wiley Periodicals, Inc.

  10. Exploring the diversity of arsenic resistance genes from acid mine drainage microorganisms.

    PubMed

    Morgante, Verónica; Mirete, Salvador; de Figueras, Carolina G; Postigo Cacho, Marina; González-Pastor, José E

    2015-06-01

    The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance.

  11. Gene organization around the phenylalanyl-transfer ribonucleic acid synthetase locus in Escherichia coli.

    PubMed Central

    Comer, M M

    1981-01-01

    The organization of seven genes located at about 38 min on the genetic map of Escherichia coli was examined; these genes included pheS and pheT, which code for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid synthetase, and thrS, the structural gene for threonyl-transfer ribonucleic acid synthetase. Deletion mutants were isolated from an F-prime-containing merodiploid strain and were characterized genetically. Seventeen different kinds of deletions extending into pheS of pheT were identified. These deletions unambiguously defined the gene order as aroD pps himA pheT pheS thrS pfkB. Mutants with deletions covering either pheS or pheT, but not both, were analyzed further by assay of phenylalanyl-transfer ribonucleic acid synthetase. The phenotype of the mutants with a deletion from pfkB through pheS was anomalous; although the pheT gene was apparently still present, its product, the beta subunit, was much reduced in activity. PMID:7012115

  12. Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase.

    PubMed

    Casqueiro, J; Gutiérrez, S; Bañuelos, O; Fierro, F; Velasco, J; Martín, J F

    1998-09-01

    A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

  13. Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance.

    PubMed

    Lee, Yeji; Nasution, Olviyani; Choi, Eunyong; Choi, In-Geol; Kim, Wankee; Choi, Wonja

    2015-08-01

    Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.

  14. Transcriptional Analysis of Essential Genes of the Escherichia coli Fatty Acid Biosynthesis Gene Cluster by Functional Replacement with the Analogous Salmonella typhimurium Gene Cluster

    PubMed Central

    Zhang, Yan; Cronan, John E.

    1998-01-01

    The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome. A difficulty in analysis of the fab cluster by the polar allele duplication approach (Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:3614–3620, 1996) is that several of these genes are essential for the growth of E. coli. We overcame this complication by use of the fab gene cluster of Salmonella typhimurium, a close relative of E. coli, to provide functions necessary for growth. The S. typhimurium fab cluster was isolated by complementation of an E. coli fabD mutant and was found to encode proteins with >94% homology to those of E. coli. However, the S. typhimurium sequences cannot recombine with the E. coli sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the plsX transcripts initiate at promoters located far upstream and include the upstream rpmF ribosomal protein gene, a promoter located upstream of the plsX coding sequence (probably within the upstream gene, rpmF) is sufficient for normal growth. We have also found that the fabG gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (Ω-Cm cassette) between the fabD and fabG genes of the E. coli chromosome abolished fabG transcription and blocked cell growth, thus providing the first indication that fabG is an essential gene. Insertion of the Ω-Cm cassette between fabH and fabD caused greatly decreased transcription of the fabD and fabG genes and slower cellular growth, indicating that fabD has only a weak promoter(s). PMID:9642179

  15. Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

    PubMed Central

    Li, Mimi; Peng, Bin; Guo, Haisong; Yan, Qinqin; Hang, Yueyu

    2013-01-01

    The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants. PMID:24358289

  16. The Effect of Multiple Single Nucleotide Polymorphisms in the Folic Acid Pathway Genes on Homocysteine Metabolism

    PubMed Central

    Liang, Shuang; Zhou, Yuanpeng; Wang, Huijun; Qian, Yanyan; Ma, Duan; Tian, Weidong; Persaud-Sharma, Vishwani; Yu, Chen; Ren, Yunyun; Zhou, Shufeng; Li, Xiaotian

    2014-01-01

    Objective. To investigate the joint effects of the single nucleotide polymorphisms (SNPs) of genes in the folic acid pathway on homocysteine (Hcy) metabolism. Methods. Four hundred women with normal pregnancies were enrolled in this study. SNPs were identified by MassARRAY. Serum folic acid and Hcy concentration were measured. Analysis of variance (ANOVA) and support vector machine (SVM) regressions were used to analyze the joint effects of SNPs on the Hcy level. Results. SNPs of MTHFR (rs1801133 and rs3733965) were significantly associated with maternal serum Hcy level. In the different genotypes of MTHFR (rs1801133), SNPs of RFC1 (rs1051266), TCN2 (rs9606756), BHMT (rs3733890), and CBS (rs234713 and rs2851391) were linked with the Hcy level adjusted for folic acid concentration. The integrated SNPs scores were significantly associated with the residual Hcy concentration (RHC) (r = 0.247). The Hcy level was significantly higher in the group with high SNP scores than that in other groups with SNP scores of less than 0.2 (P = 0.000). Moreover, this difference was even more significant in moderate and high levels of folic acid. Conclusion. SNPs of genes in the folic acid pathway possibly affect the Hcy metabolism in the presence of moderate and high levels of folic acid. PMID:24524080

  17. Docosahexaenoic acid regulates gene expression in HUVEC cells treated with polycyclic aromatic hydrocarbons.

    PubMed

    Gdula-Argasińska, Joanna; Czepiel, Jacek; Totoń-Żurańska, Justyna; Jurczyszyn, Artur; Perucki, William; Wołkow, Paweł

    2015-07-16

    The molecular mechanism of inflammation and carcinogenesis induced by exposure of polycyclic aromatic hydrocarbons (PAHs) is not clearly understood. Our study was undertaken due to the strong pro-carcinogenic potential and reactivity of PAH-metabolites, as well as the susceptibility of polyunsaturated fatty acids to oxidation. The aim of this study was to evaluate the pro- or anti-inflammatory impact of n-3 docosahexaenoic acid on human primary umbilical vein endothelial cells (HUVEC) exposed to polycyclic aromatic hydrocarbons. We analysed the influence of docosahexaenoic acid (DHA) and/or PAHs supplementation on the fatty acid profile of cell membranes, on cyclooxygenase-2 (COX-2), aryl hydrocarbon receptor (AHR), and glutathione S transferase Mu1 (GSTM1) protein expression as well as on the prostaglandin synthase 2 (PTGS2), AHR, GSTM1, PLA2G4A, and cytochrome P450 CYP1A1 gene expression. We observed that COX-2 and AHR protein expression was increased while GSTM1 expression was decreased in cells exposed to DHA and PAHs. Docosahexaenoic acid down-regulated CYP1A1 and up-regulated the AHR and PTGS2 genes. Our findings suggested that DHA contributes significantly to alleviate the harmful effects caused by PAHs in endothelial cells. Moreover, these results suggest that a diet rich in n-3 fatty acids is helpful to reduce the harmful effects of PAHs exposure on human living in heavily polluted areas.

  18. An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene.

    PubMed Central

    Slock, J; Stahly, D P; Han, C Y; Six, E W; Crawford, I P

    1990-01-01

    McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site. PMID:2123867

  19. Enhanced gene expression in epithelial cells transfected with amino acid-substituted gemini nanoparticles.

    PubMed

    Yang, Peng; Singh, Jagbir; Wettig, Shawn; Foldvari, Marianna; Verrall, Ronald E; Badea, Ildiko

    2010-08-01

    Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. Through modification of the alkyl tail length and the chemical nature of the spacer, new compounds can be generated with the potential to improve the efficiency of gene delivery. Amino acid (glycine and lysine) and dipeptide (glycyl-lysine and lysyl-lysine) substituted spacers of gemini surfactants were synthesized, and their efficiency of gene delivery was assessed in epithelial cells for topical cutaneous and mucosal applications. Three different epithelial cell lines, COS-7, PAM212 and Sf 1Ep cells, were transfected with plasmid DNA encoding for interferon gamma and green fluorescent protein complexed with the amino acid-substituted gemini compounds in the presence of 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine as a helper lipid. Gene expression was quantified by ELISA. Size, zeta potential and circular dichroism measurements were used to characterize the plasmid-gemini (PG) and plasmid-gemini surfactant-helper lipid (PGL) complexes. Gene expression was found to increase up to 72h and then declined by the 7th day. In general, the glycine-substituted surfactant showed consistently high gene expression in all three cell lines. Results of physicochemical and spectroscopic studies of the complexes indicate that substitution of the gemini spacer does not interfere with compaction of the DNA. The superior performance of these spacer-substituted gemini surfactants might be attributed to their better biocompatibility compared to the surfactants possessing unsubstituted spacers.

  20. Fatty acid regulates gene expression and growth of human prostate cancer PC-3 cells

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Chen, Y.; Tjandrawinata, R. R.

    2001-01-01

    It has been proposed that the omega-6 fatty acids increase the rate of tumor growth. Here we test that hypothesis in the PC-3 human prostate tumor. We found that the essential fatty acids, linoleic acid (LA) and arachidonic acid (AA), and the AA metabolite PGE(2) stimulate tumor growth while oleic acid (OA) and the omega-3 fatty acid, eicosapentaenoic acid (EPA) inhibited growth. In examining the role of AA in growth response, we extended our studies to analyze changes in early gene expression induced by AA. We demonstrate that c-fos expression is increased within minutes of addition in a dose-dependent manner. Moreover, the immediate early gene cox-2 is also increased in the presence of AA in a dose-dependent manner, while the constitutive cox-1 message was not increased. Three hours after exposure to AA, the synthesis of PGE(2) via COX-2 was also increased. Previous studies have demonstrated that AA was primarily delivered by low density lipoprotein (LDL) via its receptor (LDLr). Since it is known that hepatomas, acute myelogenous leukemia and colorectal tumors lack normal cholesterol feedback, we examined the role of the LDLr in growth regulation of the PC-3 prostate cancer cells. Analysis of ldlr mRNA expression and LDLr function demonstrated that human PC-3 prostate cancer cells lack normal feedback regulation. While exogenous LDL caused a significant stimulation of cell growth and PGE(2) synthesis, no change was seen in regulation of the LDLr by LDL. Taken together, these data show that normal cholesterol feedback of ldlr message and protein is lost in prostate cancer. These data suggest that unregulated over-expression of LDLr in tumor cells would permit increased availability of AA, which induces immediate early genes c-fos and cox-2 within minutes of uptake.

  1. Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids1[OPEN

    PubMed Central

    Deason, Nicholas; DellaPenna, Dean

    2017-01-01

    Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed’s nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and single-nucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants. PMID:27872244

  2. Gene Overexpression and Biochemical Characterization of the Biotechnologically Relevant Chlorogenic Acid Hydrolase from Aspergillus niger▿

    PubMed Central

    Benoit, Isabelle; Asther, Michèle; Bourne, Yves; Navarro, David; Canaan, Stéphane; Lesage-Meessen, Laurence; Herweijer, Marga; Coutinho, Pedro M.; Asther, Marcel; Record, Eric

    2007-01-01

    The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter−1, i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 × 106 M−1 s−1 toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme. PMID:17630312

  3. Subchronic effects of valproic acid on gene expression profiles for lipid metabolism in mouse liver

    SciTech Connect

    Lee, Min-Ho |; Kim, Mingoo |; Lee, Byung-Hoon |; Kim, Ju-Han |; Kang, Kyung-Sun |; Kim, Hyung-Lae |; Yoon, Byung-Il |; Chung, Heekyoung; Kong, Gu |; Lee, Mi-Ock ||

    2008-02-01

    Valproic acid (VPA) is used clinically to treat epilepsy, however it induces hepatotoxicity such as microvesicular steatosis. Acute hepatotoxicity of VPA has been well documented by biochemical studies and microarray analysis, but little is known about the chronic effects of VPA in the liver. In the present investigation, we profiled gene expression patterns in the mouse liver after subchronic treatment with VPA. VPA was administered orally at a dose of 100 mg/kg/day or 500 mg/kg/day to ICR mice, and the livers were obtained after 1, 2, or 4 weeks. The activities of serum liver enzymes did not change, whereas triglyceride concentration increased significantly. Microarray analysis revealed that 1325 genes of a set of 32,996 individual genes were VPA responsive when examined by two-way ANOVA (P < 0.05) and fold change (> 1.5). Consistent with our previous results obtained using an acute VPA exposure model (Lee et al., Toxicol Appl Pharmacol. 220:45-59, 2007), the most significantly over-represented biological terms for these genes included lipid, fatty acid, and steroid metabolism. Biological pathway analysis suggests that the genes responsible for increased biosynthesis of cholesterol and triglyceride, and for decreased fatty acid {beta}-oxidation contribute to the abnormalities in lipid metabolism induced by subchronic VPA treatment. A comparison of the VPA-responsive genes in the acute and subchronic models extracted 15 commonly altered genes, such as Cyp4a14 and Adpn, which may have predictive power to distinguish the mode of action of hepatotoxicants. Our data provide a better understanding of the molecular mechanisms of VPA-induced hepatotoxicity and useful information to predict steatogenic hepatotoxicity.

  4. Expanding Duplication of Free Fatty Acid Receptor-2 (GPR43) Genes in the Chicken Genome.

    PubMed

    Meslin, Camille; Desert, Colette; Callebaut, Isabelle; Djari, Anis; Klopp, Christophe; Pitel, Frédérique; Leroux, Sophie; Martin, Pascal; Froment, Pascal; Guilbert, Edith; Gondret, Florence; Lagarrigue, Sandrine; Monget, Philippe

    2015-04-24

    Free fatty acid receptors (FFAR) belong to a family of five G-protein coupled receptors that are involved in the regulation of lipid metabolism, so that their loss of function increases the risk of obesity. The aim of this study was to determine the expansion of genes encoding paralogs of FFAR2 in the chicken, considered as a model organism for developmental biology and biomedical research. By estimating the gene copy number using quantitative polymerase chain reaction, genomic DNA resequencing, and RNA sequencing data, we showed the existence of 23 ± 1.5 genes encoding FFAR2 paralogs in the chicken genome. The FFAR2 paralogs shared an identity from 87.2% up to 99%. Extensive gene conversion was responsible for this high degree of sequence similarities between these genes, and this concerned especially the four amino acids known to be critical for ligand binding. Moreover, elevated nonsynonymous/synonymous substitution ratios on some amino acids within or in close-vicinity of the ligand-binding groove suggest that positive selection may have reduced the effective rate of gene conversion in this region, thus contributing to diversify the function of some FFAR2 paralogs. All the FFAR2 paralogs were located on a microchromosome in a same linkage group. FFAR2 genes were expressed in different tissues and cells such as spleen, peripheral blood mononuclear cells, abdominal adipose tissue, intestine, and lung, with the highest rate of expression in testis. Further investigations are needed to determine whether these chicken-specific events along evolution are the consequence of domestication and may play a role in regulating lipid metabolism in this species.

  5. Nutrient uptake by marine invertebrates: cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus).

    PubMed

    Meyer, Eli; Manahan, Donal T

    2009-08-01

    Transport of amino acids from low concentrations in seawater by marine invertebrates has been extensively studied, but few of the genes involved in this physiological process have been identified. We have characterized three amino acid transporter genes cloned from embryos of the sea urchin Strongylocentrotus purpuratus. These genes show phylogenetic proximity to classical amino acid transport systems, including Gly and B0+, and the inebriated gene (INE). Heterologous expression of these genes in frog oocytes induced a 40-fold increase in alanine transport above endogenous levels, demonstrating that these genes mediate alanine transport. Antibodies specific to one of these genes (Sp-AT1) inhibited alanine transport, confirming the physiological activity of this gene in larvae. Whole-mount antibody staining of larvae revealed expression of Sp-AT1 in the ectodermal tissues associated with amino acid transport, as independently demonstrated by autoradiographic localization of radioactive alanine. Maximum rates of alanine transport increased 6-fold during early development, from embryonic to larval stages. Analysis of gene expression during this developmental period revealed that Sp-AT1 transcript abundance remained nearly constant, while that of another transporter gene (Sp-AT2) increased 11-fold. The functional characterization of these genes establishes a molecular biological basis for amino acid transport by developmental stages of marine invertebrates.

  6. Up-regulation of the expression of the gene for liver fatty acid-binding protein by long-chain fatty acids.

    PubMed Central

    Meunier-Durmort, C; Poirier, H; Niot, I; Forest, C; Besnard, P

    1996-01-01

    The role of fatty acids in the expression of the gene for liver fatty acid-binding protein (L-FABP) was investigated in the well-differentiated FAO rat hepatoma cell line. Cells were maintained in serum-free medium containing 40 microM BSA/320 microM oleate. Western blot analysis showed that oleate triggered an approx. 4-fold increase in the cytosolic L-FABP level in 16 h. Oleate specifically stimulated L-FABP mRNA in time-dependent and dose-dependent manners with a maximum 7-fold increase at 16 h in FAO cells. Preincubation of FAO cells with cycloheximide prevented the oleate-mediated induction of L-FABP mRNA, showing that protein synthesis was required for the action of fatty acids. Run-on transcription assays demonstrated that the control of L-FABP gene expression by oleate was, at least in part, transcriptional. Palmitic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid were similarly potent whereas octanoic acid was inefficient. This regulation was also found in normal hepatocytes. Therefore long-chain fatty acids are strong inducers of L-FABP gene expression. FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action. PMID:8912685

  7. The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana.

    PubMed

    Zhang, Gaoyang; Zhang, Yujia; Xu, Jiantang; Niu, Xiaoping; Qi, Jianmin; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, LiHui; Su, Jianguang

    2014-08-10

    The Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene from jute using homology clone (primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named "CcCCoAOMT1". Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1 gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene's function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1 in these transgenic A.thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44-21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1 gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1 gene inhibition.

  8. Controllably local gene delivery mediated by polyelectrolyte multilayer films assembled from gene-loaded nanopolymersomes and hyaluronic acid

    PubMed Central

    Teng, Wei; Wang, Qinmei; Chen, Ying; Huang, Hongzhang

    2014-01-01

    To explore a spatiotemporally controllable gene delivery system with high efficiency and safety, polyelectrolyte multilayer (PEM) films were constructed on titanium or quartz substrates via layer-by-layer self-assembly technique by using plasmid deoxyribonucleic acid-loaded lipopolysaccharide–amine nanopolymersomes (pNPs) as polycations and hyaluronic acid (HA) as polyanions. pNPs were chosen because they have high transfection efficiency (>95%) in mesenchymal stem cells (MSCs) and induce significant angiogenesis in zebrafish in conventional bolus transfection. The assembly process of PEM films was confirmed by analyses of quartz crystal microbalance with dissipation, X-ray photoelectron spectroscopy, infrared, contact angle, and zeta potential along with atomic force microscopy observation. Quartz crystal microbalance with dissipation analysis reveals that this film grows in an exponential mode, pNPs are the main contributor to the film mass, and the film mass can be modulated in a relatively wide range (1.0–29 μg/cm2) by adjusting the deposition layer number. Atomic force microscopy observation shows that the assembly leads to the formation of a patterned film with three-dimensional tree-like nanostructure, where the branches are composed of beaded chains (pNP beads are strung on HA molecular chains), and the incorporated pNPs keep structure intact. In vitro release experiment shows that plasmid deoxyribonucleic acid can be gradually released from films over 14 days, and the released plasmid deoxyribonucleic acid exists in a complex form. In vitro cell experiments demonstrate that PEM films can enhance the adhesion and proliferation of MSCs and efficiently transfect MSCs in situ in vitro for at least 4 days. Our results suggest that a (pNPs/HA)n system can mediate efficient transfection in stem cells in a spatially and temporally controllable pattern, highlighting its huge potential in local gene therapy. PMID:25378927

  9. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    PubMed

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P < 0.05). We identified one SNP in the genomic sequence of the gene and found that this SNP was associated significantly with body length (P < 0.05), but not with resistance to S. agalactiae. The results of this study suggest that the LAO gene plays an important role in innate immune responses to the bacterial pathogen in tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

  10. Transcriptome analysis and identification of genes associated with omega-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Perilla (Perilla frutescens (L.) var frutescens) produces high levels of a-linolenic acid (ALA), an omega-3 fatty acid important to health and development. To uncover key genes involved in fatty acid (FA) and triacylglycerol (TAG) synthesis in perilla, we conducted deep sequencing of cD...

  11. Retinoic acid regulates several genes in bile acid and lipid metabolism via upregulation of small heterodimer partner in hepatocytes.

    PubMed

    Mamoon, Abulkhair; Subauste, Angela; Subauste, Maria C; Subauste, Jose

    2014-10-25

    Retinoic acid (RA) affects multiple aspects of development, embryogenesis and cell differentiation processes. The liver is a major organ that stores RA suggesting that retinoids play an important role in the function of hepatocytes. In our previous studies, we have demonstrated the involvement of small heterodimer partner (SHP) in RA-induced signaling in a non-transformed hepatic cell line AML 12. In the present study, we have identified several critical genes in lipid homeostasis (Apoa1, Apoa2 and ApoF) that are repressed by RA-treatment in a SHP dependent manner, in vitro and also in vivo with the use of the SHP null mice. In a similar manner, RA also represses several critical genes involved in bile acid metabolism (Cyp7a1, Cyp8b1, Mdr2, Bsep, Baat and Ntcp) via upregulation of SHP. Collectively our data suggest that SHP plays a major role in RA-induced potential changes in pathophysiology of metabolic disorders in the liver.

  12. Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

    PubMed

    Tagashira, Yusuke; Shimizu, Tomoe; Miyamoto, Masanobu; Nishida, Sho; Yoshida, Kaoru T

    2015-04-24

    The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP₆) biosynthesis-related genes, as InsP₆ is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP₆ biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP₆ and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP₆ biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP₆ biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

  13. A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides.

    PubMed

    Ment, Dana; Alkan, Noam; Luria, Neta; Bi, Fang-Cheng; Reuveni, Eli; Fluhr, Robert; Prusky, Dov

    2015-02-01

    Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

  14. Characterization of the bovine gene LIPE and possible influence on fatty acid composition of meat

    PubMed Central

    Goszczynski, Daniel Estanislao; Mazzucco, Juliana Papaleo; Ripoli, María Verónica; Villarreal, Edgardo Leopoldo; Rogberg-Muñoz, Andrés; Mezzadra, Carlos Alberto; Melucci, Lilia Magdalena; Giovambattista, Guillermo

    2014-01-01

    LIPE is an intracellular neutral lipase, which is capable of hydrolyzing a variety of esters and plays a key role in the mobilization of fatty acids from diacylglycerols. The objectives of this study were to characterize the genetic polymorphism of bovine LIPE gene and to evaluate the possible association between three SNPs in the coding regions of this gene with the fatty acid composition of meat in a cattle population. Forty-three unrelated animals from different cattle breeds were re-sequenced and 21 SNPs were detected over approximately 2600 bp, five of these SNPs were novel. Three SNPs were selected, on the basis of evolutionary conservation, to perform validation and association studies in a crossbred cattle population. Our results may suggest a possible association of SNP1 with contents of oleic acid and total monounsaturated fatty acids (p < 0.01), and SNP2 and SNP3 with Heneicosylic acid content (p < 0.01), may be helpful to improve the quality of meat and improve health. PMID:25606458

  15. Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification.

    PubMed Central

    Kranz, R G; Gabbert, K K; Madigan, M T

    1997-01-01

    The photosynthetic bacterium Rhodobacter capsulatus can grow with short- to long-chain fatty acids as the sole carbon source (R. G. Kranz, K. K. Gabbert, T. A. Locke, and M. T. Madigan, Appl. Environ. Microbiol. 63:3003-3009, 1997). Concomitant with growth on fatty acids is the production to high levels of the polyester storage compounds called polyhydroxyalkanoates (PHAs). Here, we describe colony screening and selection systems to analyze the production of PHAs in R. capsulatus. A screen with Nile red dissolved in acetone distinguishes between PHA producers and nonproducers. Unlike the wild type, an R. capsulatus PhaC- strain with the gene encoding PHA synthase deleted is unable to grow on solid media containing high concentrations of certain fatty acids. It is proposed that this deficiency is due to the inability of the PhaC- strain to detoxify the surrounding medium by consumption of fatty acids and their incorporation into PHAs. This fatty acid toxicity phenotype is used in selection for the cloning and characterization of heterologous phaC genes. PMID:9251190

  16. Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

    PubMed

    Simonson, Oscar E; Svahn, Mathias G; Törnquist, Elisabeth; Lundin, Karin E; Smith, C I E

    2005-01-01

    Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

  17. Differential expression of fatty acid synthase genes, Acl, Fat and Kas, in Capsicum fruit.

    PubMed

    Aluru, Maneesha R; Mazourek, Michael; Landry, Laurie G; Curry, Jeanne; Jahn, Molly; O'Connell, Mary A

    2003-07-01

    The biosynthesis of capsaicinoids in the placenta of chilli fruit is modelled to require components of the fatty acid synthase (FAS) complex. Three candidate genes for subunits in this complex, Kas, Acl, and Fat, isolated based on differential expression, were characterized. Transcription of these three genes was placental-specific and RNA abundance was positively correlated with degree of pungency. Kas and Acl were mapped to linkage group 1 and Fat to linkage group 6. None of the genes is linked to the pungency locus, C, on linkage group 2. KAS accumulation was positively correlated with pungency. Western blots of placental extracts and histological sections both demonstrated that the accumulation of this enzyme was correlated with fruit pungency and KAS was immunolocalized to the expected cell layer, the placental epidermis. Enzyme activity of the recombinant form of the placental-specific KAS was confirmed using crude cell extracts. These FAS components are fruit-specific members of their respective gene families. These genes are predicted to be associated with Capsicum fruit traits, for example, capsaicinoid biosynthesis or fatty acid biosynthesis necessary for placental development.

  18. Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response.

    PubMed

    Kurar, E; Splitter, G A

    1997-12-01

    Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. As a test of this new methodology, the immune response to the in vivo-expressed Brucella abortus ribosomal L7/12 gene in the muscle cells of mice was examined. To accomplish this goal the eukaryotic expression systems pcDNA3 and p6 were used. Single intramuscular injection of the L7/L12 gene driven by the human cytomegalovirus (CMV) promoter (pcDNA3) or bovine MHC 1 promoter (p6) resulted in intracellular expression of the B. abortus L7/L12 immunodominant protein encoded by this gene. This application facilitated directed antigen presentation to the immune system and established specific antibody and T-cell responses compared with vector only (pcDNA3) negative controls and B. abortus S19 injected positive controls. Although pcDNA3-encoded L7/L12 gene-inoculated mice possessed significant protection, p6-L7/L12 did not engender significant protection against B. abortus S2308 infection compared to positive control mice. These data suggest a promising antigen-specific response, and L7/L12 nucleic acid vaccination may be an initial step in the development of genetically engineered candidate vaccines against brucellosis. This study for the first time focuses on DNA immunization of a gene from B. abortus.

  19. Structure and expression of the Drosophila ubiquitin-80-amino-acid fusion-protein gene.

    PubMed Central

    Barrio, R; del Arco, A; Cabrera, H L; Arribas, C

    1994-01-01

    In the fruitfly Drosophila, as in all eukaryotes examined so far, some ubiquitin-coding sequences appear fused to unrelated open reading frames. Two of these fusion genes have been previously described (the homologues of UBI1-UBI2 and UBI4 in yeast), and we report here the organization and expression of a third one, the DUb80 gene (the homologue of UBI3 in yeast). This gene encodes a ubiquitin monomer fused to an 80-amino-acid extension which is homologous with the ribosomal protein encoded by the UB13 gene. The 5' regulatory region of DUb80 shares common features with another ubiquitin fusion gene, DUb52, and with the ribosomal protein genes of Drosophila, Xenopus and mouse. We also find helix-loop-helix protein-binding sequences (E-boxes). The DUb80 gene is transcribed to a 0.9 kb mRNA which is particularly abundant under conditions of high protein synthesis, such as in ovaries and exponentially growing cells. Images Figure 3 Figure 4 PMID:8068011

  20. The Saccharomyces Cerevisiae Spt7 Gene Encodes a Very Acidic Protein Important for Transcription in Vivo

    PubMed Central

    Gansheroff, L. J.; Dollard, C.; Tan, P.; Winston, F.

    1995-01-01

    Mutations in the SPT7 gene of Saccharomyces cerevisiae originally were identified as suppressors of Ty and {delta small} insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Other genes that have been identified in mutant hunts of this type have been shown to play a role in transcription. In this work we show that SPT7 is also important for proper transcription in vivo. We have cloned and sequenced the SPT7 gene and have shown that it encodes a large, acidic protein that is localized to the nucleus. The SPT7 protein contains a bromodomain sequence; a deletion that removes the bromodomain from the SPT7 protein causes no detectable mutant phenotype. Strains that contain an spt7 null mutation are viable but grow very slowly and have transcriptional defects at many loci including insertion mutations, Ty elements, the INO1 gene and the MFA1 gene. These transcriptional defects and other mutant phenotypes are similar to those caused by certain mutations in SPT15, which encodes the TATA binding protein (TBP). The similarity of the phenotypes of spt7 and spt15 mutants, including effects of spt7 mutations on the transcription start site of certain genes, suggests that SPT7 plays an important role in transcription initiation in vivo. PMID:7713415

  1. Effect of chronic valproic Acid treatment on hepatic gene expression profile in wfs1 knockout mouse.

    PubMed

    Punapart, Marite; Eltermaa, Mall; Oflijan, Julia; Sütt, Silva; Must, Anne; Kõks, Sulev; Schalkwyk, Leonard C; Fernandes, Catherine; Vasar, Eero; Soomets, Ursel; Terasmaa, Anton

    2014-01-01

    Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.

  2. Identification and Functional Analysis of the Mycophenolic Acid Gene Cluster of Penicillium roqueforti.

    PubMed

    Del-Cid, Abdiel; Gil-Durán, Carlos; Vaca, Inmaculada; Rojas-Aedo, Juan F; García-Rico, Ramón O; Levicán, Gloria; Chávez, Renato

    2016-01-01

    The filamentous fungus Penicillium roqueforti is widely known as the ripening agent of blue-veined cheeses. Additionally, this fungus is able to produce several secondary metabolites, including the meroterpenoid compound mycophenolic acid (MPA). Cheeses ripened with P. roqueforti are usually contaminated with MPA. On the other hand, MPA is a commercially valuable immunosuppressant. However, to date the molecular basis of the production of MPA by P. roqueforti is still unknown. Using a bioinformatic approach, we have identified a genomic region of approximately 24.4 kbp containing a seven-gene cluster that may be involved in the MPA biosynthesis in P. roqueforti. Gene silencing of each of these seven genes (named mpaA, mpaB, mpaC, mpaDE, mpaF, mpaG and mpaH) resulted in dramatic reductions in MPA production, confirming that all of these genes are involved in the biosynthesis of the compound. Interestingly, the mpaF gene, originally described in P. brevicompactum as a MPA self-resistance gene, also exerts the same function in P. roqueforti, suggesting that this gene has a dual function in MPA metabolism. The knowledge of the biosynthetic pathway of MPA in P. roqueforti will be important for the future control of MPA contamination in cheeses and the improvement of MPA production for commercial purposes.

  3. Bioinformatics analysis of the oxidosqualene cyclase gene and the amino acid sequence in mangrove plants

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wati, R.

    2017-01-01

    This study described the bioinformatics methods to analyze seven oxidosqualene cyclase (OSC) genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, similarity, subcellular localization and phylogenetic. The physical and chemical properties of seven mangrove OSC showed variation among the genes. The percentage of the secondary structure of seven mangrove OSC genes followed the order of a helix > random coil > extended chain structure. The values of chloroplast or signal peptide were too low, indicated that no chloroplast transit peptide or signal peptide of secretion pathway in mangrove OSC genes. The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the mangrove OSC gene, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, Kandelia KcMS join with Bruguiera BgLUS, Rhizophora RsM1 was close to Bruguiera BgbAS, and Rhizophora RcCAS join with Kandelia KcCAS. The present study, therefore, supported the previous results that plant OSC genes form distinct clusters in the tree.

  4. Identification and Functional Analysis of the Mycophenolic Acid Gene Cluster of Penicillium roqueforti

    PubMed Central

    Del-Cid, Abdiel; Gil-Durán, Carlos; Vaca, Inmaculada; Rojas-Aedo, Juan F.; García-Rico, Ramón O.; Levicán, Gloria; Chávez, Renato

    2016-01-01

    The filamentous fungus Penicillium roqueforti is widely known as the ripening agent of blue-veined cheeses. Additionally, this fungus is able to produce several secondary metabolites, including the meroterpenoid compound mycophenolic acid (MPA). Cheeses ripened with P. roqueforti are usually contaminated with MPA. On the other hand, MPA is a commercially valuable immunosuppressant. However, to date the molecular basis of the production of MPA by P. roqueforti is still unknown. Using a bioinformatic approach, we have identified a genomic region of approximately 24.4 kbp containing a seven-gene cluster that may be involved in the MPA biosynthesis in P. roqueforti. Gene silencing of each of these seven genes (named mpaA, mpaB, mpaC, mpaDE, mpaF, mpaG and mpaH) resulted in dramatic reductions in MPA production, confirming that all of these genes are involved in the biosynthesis of the compound. Interestingly, the mpaF gene, originally described in P. brevicompactum as a MPA self-resistance gene, also exerts the same function in P. roqueforti, suggesting that this gene has a dual function in MPA metabolism. The knowledge of the biosynthetic pathway of MPA in P. roqueforti will be important for the future control of MPA contamination in cheeses and the improvement of MPA production for commercial purposes. PMID:26751579

  5. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

    PubMed Central

    Li, Chengchen; Gui, Shunhua; Yang, Tao; Walk, Thomas; Wang, Xiurong; Liao, Hong

    2012-01-01

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs. Methods BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis. Key Results In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions. Conclusions Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions. PMID:21948626

  6. Comparative Studies of Mammalian Acid Lipases: Evidence for a New Gene Family in Mouse and Rat (Lipo)

    PubMed Central

    Holmes, Roger S; Cox, Laura A; VandeBerg, John L

    2010-01-01

    At least six families of mammalian acid lipases (E.C. 3.1.1.-) catalyse the hydrolysis of triglycerides in the body, designated as LIPA (lysosomal), LIPF (gastric), LIPJ (testis) and LIPK, LIPM and LIPN (epidermal), which belong to the AB hydrolase superfamily. In this study, in silico methods were used to predict the amino acid sequences, secondary and tertiary structures, and gene locations for acid lipase genes and encoded proteins using data from several mammalian genome projects. Mammalian acid lipase genes were located within a gene cluster for each of the 8 mammalian genomes examined, including human (Homo sapiens), chimpanzee (Pons troglodytes), rhesus monkey (Macacca mulatta), mouse (Mus musculus), rat (Rattus norvegicus), cow (Bos taurus), horse (Equus caballus) and dog (Canis familaris), with each containing 9 coding exons. Human and mouse acid lipases shared 44-87% sequence identity and exhibited sequence alignments and identities for key amino acid residues and conservation of predicted secondary and tertiary structures with those previously reported for human gastric lipase (LIPF) (Roussel et al., 1999). Evidence for a new family of acid lipase genes is reported for mouse and rat genomes, designated as Lipo. Mouse acid lipase genes are subject to differential mRNA tissue expression, with Lipa showing wide tissue expression, while others have a more restricted tissue expression in the digestive tract (Lipf), salivary gland (Lipo) and epidermal tissues (Lipk, Lipm and Lipn). Phylogenetic analyses of the mammalian acid lipase gene families suggested that these genes are products of gene duplication events prior to eutherian mammalian evolution and derived from an ancestral vertebrate LIPA gene, which is present in the frog, Xenopus tropicalis. PMID:20598663

  7. Subtle gene-environment interactions driving paranoia in daily life.

    PubMed

    Simons, C J P; Wichers, M; Derom, C; Thiery, E; Myin-Germeys, I; Krabbendam, L; van Os, J

    2009-02-01

    It has been suggested that genes impact on the degree to which minor daily stressors cause variation in the intensity of subtle paranoid experiences. The objective of the present study was to test the hypothesis that catechol-O-methyltransferase (COMT) Val(158)Met and brain-derived neurotrophic factor (BDNF) Val(66)Met in part mediate genetic effects on paranoid reactivity to minor stressors. In a general population sample of 579 young adult female twins, on the one hand, appraisals of (1) event-related stress and (2) social stress and, on the other hand, feelings of paranoia in the flow of daily life were assessed using momentary assessment technology for five consecutive days. Multilevel regression analyses were used to examine moderation of daily life stress-induced paranoia by COMT Val(158)Met and BDNF Val(66)Met genotypes. Catechol-O-methyltransferase Val carriers displayed more feelings of paranoia in response to event stress compared with Met carriers. Brain-derived neurotrophic factor Met carriers showed more social-stress-induced paranoia than individuals with the Val/Val genotype. Thus, paranoia in the flow of daily life may be the result of gene-environment interactions that can be traced to different types of stress being moderated by different types of genetic variation.

  8. Analysis of gene expression in Homarus americanus larvae exposed to sublethal concentrations of endosulfan during metamorphosis.

    PubMed

    Bauer, Megan; Greenwood, Spencer J; Clark, K Fraser; Jackman, Paula; Fairchild, Wayne

    2013-12-01

    Agricultural pesticide runoff has been suspected as the cause of numerous fish kills in rivers throughout Prince Edward Island but the impact on the surrounding marine environment is unknown. Endosulfan, an organochlorine pesticide, is a potent neurotoxin and molt inhibitor used to combat the Colorado potato beetle however it has the potential to affect non-target organisms including the American lobster (Homarus americanus). Metamorphosis is a critical stage of development and the effects of contaminant exposure during this time are largely unknown in lobster. A 14day endosulfan exposure was performed to identify the effects on survival, development and gene expression in lobster larvae during metamorphosis; all of which were predicted to be negatively impacted. The higher endosulfan concentrations resulted in greater mortality and a significant increase in the number of days required to reach metamorphosis in surviving animals. A custom made H. americanus microarray was used for monitoring the changes in expression of 14,592 genes at the termination of the exposure. Genes with >1.5 fold change and identified as being significant at p<0.05 using one-way ANOVA were selected for further analysis. A total of 707 genes were identified as being significantly differentiated, however with only ~40% annotation of the array, the majority of these genes were unknown. Annotated genes of interest were involved in many processes: development, metabolism, immunity and oxidative stress response and gene regulation. Nine genes of interest (histone H1, farnesoic acid O-methyltransferase, cuticle protein, glutathione S-transferase, thioredoxin, NADH dehydrogenase, ecdysone nuclear receptor Fushi tarazu F1 (FTZ-F1), ferritin and ecdysone inducible protein E75 (EIP-E75)) were selected for RT-qPCR validation of the microarray results. The RT-qPCR method was more sensitive than the microarray yet detected similar expression patterns. The two highest endosulfan concentrations resulted

  9. Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium

    DOE PAGES

    Yurchenko, Olga P.; Park, Sunjung; Ilut, Daniel C.; ...

    2014-11-18

    The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterizemore » the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation. Results: Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings. Conclusions: The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior

  10. Identification of a retinoic acid-responsive neural enhancer in the Ciona intestinalis Hox1 gene.

    PubMed

    Kanda, Miyuki; Ikeda, Taku; Fujiwara, Shigeki

    2013-02-01

    The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci-Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci-Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid-response element (RARE). Mutation of the RARE in the Ci-Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci-Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA-dependent and one RA-independent.

  11. Position effect variegation of an acid phosphatase gene in Drosophila melanogaster.

    PubMed

    Frisardi, M C; MacIntyre, R J

    1984-01-01

    X-ray mutagenesis has produced a series of deficiencies in a duplication of part of the third chromosome containing the acid phosphatase gene (Acph-1) in Drosophila melanogaster. In one of these deficiencies, Acph-1 is shown to be undergoing position effect variegation. Naturally occurring electrophoretic variants of the enzyme were used to visualize and determine quantitatively the extent of variegation of the allele which is cis to the heterochromatic breakpoint. Alteration of genotypic background and temperature provided further evidence for position effect. Rocket immunoelectrophoresis was used to correlate the levels of acid phosphatase activity and protein in flies containing the deficiency. A novel result indicates that the variegation is not the consequence of an averaging of active and inactive cells, but rather due to a quantitative alteration of gene activity within at least some individual cells.

  12. A co-expression gene network associated with developmental regulation of apple fruit acidity.

    PubMed

    Bai, Yang; Dougherty, Laura; Cheng, Lailiang; Xu, Kenong

    2015-08-01

    Apple fruit acidity, which affects the fruit's overall taste and flavor to a large extent, is primarily determined by the concentration of malic acid. Previous studies demonstrated that the major QTL malic acid (Ma) on chromosome 16 is largely responsible for fruit acidity variations in apple. Recent advances suggested that a natural mutation that gives rise to a premature stop codon in one of the two aluminum-activated malate transporter (ALMT)-like genes (called Ma1) is the genetic causal element underlying Ma. However, the natural mutation does not explain the developmental changes of fruit malate levels in a given genotype. Using RNA-seq data from the fruit of 'Golden Delicious' taken at 14 developmental stages from 1 week after full-bloom (WAF01) to harvest (WAF20), we characterized their transcriptomes in groups of high (12.2 ± 1.6 mg/g fw, WAF03-WAF08), mid (7.4 ± 0.5 mg/g fw, WAF01-WAF02 and WAF10-WAF14) and low (5.4 ± 0.4 mg/g fw, WAF16-WAF20) malate concentrations. Detailed analyses showed that a set of 3,066 genes (including Ma1) were expressed not only differentially (P FDR < 0.05) between the high and low malate groups (or between the early and late developmental stages) but also in significant (P < 0.05) correlation with malate concentrations. The 3,066 genes fell in 648 MapMan (sub-) bins or functional classes, and 19 of them were significantly (P FDR < 0.05) co-enriched or co-suppressed in a malate dependent manner. Network inferring using the 363 genes encompassed in the 19 (sub-) bins, identified a major co-expression network of 239 genes. Since the 239 genes were also differentially expressed between the early (WAF03-WAF08) and late (WAF16-WAF20) developmental stages, the major network was considered to be associated with developmental regulation of apple fruit acidity in 'Golden Delicious'.

  13. Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

    PubMed

    Cohen, S; Tzuri, G; Harel-Beja, R; Itkin, M; Portnoy, V; Sa'ar, U; Lev, S; Yeselson, L; Petrikov, M; Rogachev, I; Aharoni, A; Ophir, R; Tadmor, Y; Lewinsohn, E; Burger, Y; Katzir, N; Schaffer, A A

    2012-07-01

    Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit

  14. Analysis of ldh genes in Lactobacillus casei BL23: role on lactic acid production.

    PubMed

    Rico, Juan; Yebra, María Jesús; Pérez-Martínez, Gaspar; Deutscher, Josef; Monedero, Vicente

    2008-06-01

    Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.

  15. Poly (ADP-ribose) glycohydrolase regulates retinoic acid receptor-mediated gene expression.

    PubMed

    Le May, Nicolas; Iltis, Izarn; Amé, Jean-Christophe; Zhovmer, Alexander; Biard, Denis; Egly, Jean-Marc; Schreiber, Valérie; Coin, Frédéric

    2012-12-14

    Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.

  16. [Analysis of critical genes expression of chlorogenic acid and luteolin biosyntheses in Lonicera confusa].

    PubMed

    Qin, Shuang-Shuang; Huang, Lu-Qi; Yuan, Yuan; Yu, Li-Ying

    2014-07-01

    This study analysed the tissue specific expression of critical genes involved in chlorogenic acid and luteolin biosynthesis, for exploiting the molecular mechanism of components biosynthesis in Lonicera confusa. Expression of PAL, 4CL, C4H, CHS, CHI, FNS and HQT gene families of chlorogenic acid and luteolin biosynthesis-related genes in buds and leaves of L. confusa were analyed by Real-time PCR. Expressions of PAL1, C4H1, 4CL1, CHS1, CHI3 and HQT2 in buds were lower than that in leaves, and expressions of PAL3, 4CL2, CHI2 and FNS2 in buds were higher than that in leaves. The results indicated that that PAL3 and 4CL2 may be associated with accumulation of chlorogenic acid, and the expression patterns of PAL1, CHS1, CHI3 and HQT2 in buds and leaves of L. confusa were different with L. japonica. This study provided some theoretical basis for the further research on genetic mechanism of active components differences in L. confusa and L. japonica.

  17. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering.

    PubMed

    Chen, Yingying; Stabryla, Lisa; Wei, Na

    2016-01-29

    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

  18. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering

    PubMed Central

    Chen, Yingying; Stabryla, Lisa

    2016-01-01

    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production. PMID:26826231

  19. Styrene lower catabolic pathway in Pseudomonas fluorescens ST: identification and characterization of genes for phenylacetic acid degradation.

    PubMed

    Di Gennaro, Patrizia; Ferrara, Silvia; Ronco, Ilaria; Galli, Enrica; Sello, Guido; Papacchini, Maddalena; Bestetti, Giuseppina

    2007-08-01

    Pseudomonas fluorescens ST is a styrene degrading microorganism that, by the sequential oxidation of the vinyl side chain, converts styrene to phenylacetic acid. The cluster of styrene upper pathway catabolic genes (sty genes) has been previously localized on a chromosomal region. This report describes the isolation, sequencing and analysis of a new chromosomal fragment deriving from the ST strain genomic bank that contains the styrene lower degradative pathway genes (paa genes), involved in the metabolism of phenylacetic acid. Analysis of the paa gene cluster led to the description of 14 putative genes: a gene encoding a phenylacetyl-CoA ligase (paaF), the enzyme required for the activation of phenylacetic acid; five ORFs encoding the subunits of a ring hydroxylation multienzymatic system (paaGHIJK); the gene paaW encoding a membrane protein of unknown function; five genes for a beta-oxidation-like system (paaABCDE), involved in the steps following the aromatic ring cleavage; a gene encoding a putative permease (paaL) and a gene (paaN) probably involved in the aromatic ring cleavage. The function of some of the isolated genes has been proved by means of biotransformation experiments.

  20. Glutamic acid promotes monacolin K production and monacolin K biosynthetic gene cluster expression in Monascus.

    PubMed

    Zhang, Chan; Liang, Jian; Yang, Le; Chai, Shiyuan; Zhang, Chenxi; Sun, Baoguo; Wang, Chengtao

    2017-12-01

    This study investigated the effects of glutamic acid on production of monacolin K and expression of the monacolin K biosynthetic gene cluster. When Monascus M1 was grown in glutamic medium instead of in the original medium, monacolin K production increased from 48.4 to 215.4 mg l(-1), monacolin K production increased by 3.5 times. Glutamic acid enhanced monacolin K production by upregulating the expression of mokB-mokI; on day 8, the expression level of mokA tended to decrease by Reverse Transcription-polymerase Chain Reaction. Our findings demonstrated that mokA was not a key gene responsible for the quantity of monacolin K production in the presence of glutamic acid. Observation of Monascus mycelium morphology using Scanning Electron Microscope showed glutamic acid significantly increased the content of Monascus mycelium, altered the permeability of Monascus mycelium, enhanced secretion of monacolin K from the cell, and reduced the monacolin K content in Monascus mycelium, thereby enhancing monacolin K production.

  1. Transcriptome Profiling of Shewanella oneidensis Gene Expressionfollowing Exposure to Acidic and Alkaline pH

    SciTech Connect

    Leaphart, Adam B.; Thompson, Dorothea K.; Huang, Katherine; Alm,Eric; Wan, Xiu-Feng; Arkin, Adam P.; Brown, Steven D.; Wu, Liyou; Yan,Tingfen; Liu, Xueduan; Wickham, Gene S.; Zhou, Jizhong

    2007-04-02

    The molecular response of Shewanella oneidensis MR-1 tovariations in extracellular pH was investigated based on genomewide geneexpression profiling. Microarray analysis revealed that cells elicitedboth general and specific transcriptome responses when challenged withenvironmental acid (pH 4) or base (pH 10) conditions over a 60