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Sample records for putative o-methyltransferase accumulating

  1. 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.

  2. 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

  3. 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

  4. 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 ...

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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).

  19. 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.

  20. Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

    PubMed Central

    Molitoris, B A; Wilson, P D; Schrier, R W; Simon, F R

    1985-01-01

    and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores. Images PMID:3001141

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  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. 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

  8. 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

  9. Tonoplast Sugar Transporters (SbTSTs) putatively control sucrose accumulation in sweet sorghum stems.

    PubMed

    Bihmidine, Saadia; Julius, Benjamin T; Dweikat, Ismail; Braun, David M

    2016-01-01

    Carbohydrates are differentially partitioned in sweet versus grain sorghums. While the latter preferentially accumulate starch in the grain, the former primarily store large amounts of sucrose in the stem. Previous work determined that neither sucrose metabolizing enzymes nor changes in Sucrose transporter (SUT) gene expression accounted for the carbohydrate partitioning differences. Recently, 2 additional classes of sucrose transport proteins, Tonoplast Sugar Transporters (TSTs) and SWEETs, were identified; thus, we examined whether their expression tracked sucrose accumulation in sweet sorghum stems. We determined 2 TSTs were differentially expressed in sweet vs. grain sorghum stems, likely underlying the massive difference in sucrose accumulation. A model illustrating potential roles for different classes of sugar transport proteins in sorghum sugar partitioning is discussed.

  10. Tonoplast Sugar Transporters (SbTSTs) putatively control sucrose accumulation in sweet sorghum stems

    PubMed Central

    Bihmidine, Saadia; Julius, Benjamin T; Dweikat, Ismail; Braun, David M

    2016-01-01

    ABSTRACT Carbohydrates are differentially partitioned in sweet versus grain sorghums. While the latter preferentially accumulate starch in the grain, the former primarily store large amounts of sucrose in the stem. Previous work determined that neither sucrose metabolizing enzymes nor changes in Sucrose transporter (SUT) gene expression accounted for the carbohydrate partitioning differences. Recently, 2 additional classes of sucrose transport proteins, Tonoplast Sugar Transporters (TSTs) and SWEETs, were identified; thus, we examined whether their expression tracked sucrose accumulation in sweet sorghum stems. We determined 2 TSTs were differentially expressed in sweet vs. grain sorghum stems, likely underlying the massive difference in sucrose accumulation. A model illustrating potential roles for different classes of sugar transport proteins in sorghum sugar partitioning is discussed. PMID:26619184

  11. Identification of putative silicon transporters in the low accumulators N. tabacum and S. lycopersicum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Silicon (Si) has long been excluded from the list of essential elements based on a definition developed over 60 years ago. However, how can one deem an element “non-essential” if through millennia of adaptation a fundamental pathway of perception and accumulation leading to molecular changes within...

  12. 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...

  13. 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...

  14. 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…

  15. 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.

  16. Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing.

    PubMed

    Meyer, Rikke Louise; Saunders, Aaron Marc; Blackall, Linda Louise

    2006-02-01

    Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90 % identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic-aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6.6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33 % of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55 % of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

  17. 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°.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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 ...

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Comparative study of putative 9-cis-epoxycarotenoid dioxygenase and abscisic acid accumulation in the responses of Sunki mandarin and Rangpur lime to water deficit.

    PubMed

    Neves, D M; Filho, M A Coelho; Bellete, B S; Silva, M F G F; Souza, D T; Dos S Soares Filho, W; Costa, M G C; Gesteira, A S

    2013-09-01

    Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5 days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. Accumulator

    NASA Technical Reports Server (NTRS)

    Fenwick, J. R.; Karigan, G. H. (Inventor)

    1977-01-01

    An accumulator particularly adapted for use in controlling the pressure of a stream of fluid in its liquid phase utilizing the fluid in its gaseous phase was designed. The accumulator is characterized by a shell defining a pressure chamber having an entry throat for a liquid and adapted to be connected in contiguous relation with a selected conduit having a stream of fluid flowing through the conduit in its liquid phase. A pressure and volume stabilization tube, including an array of pressure relief perforations is projected into the chamber with the perforations disposed adjacent to the entry throat for accommodating a discharge of the fluid in either gaseous or liquid phases, while a gas inlet and liquid to gas conversion system is provided, the chamber is connected with a source of the fluid for continuously pressuring the chamber for controlling the pressure of the stream of liquid.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  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. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii

    PubMed Central

    Shtaida, Nastassia; Khozin-Goldberg, Inna; Solovchenko, Alexei; Chekanov, Konstantin; Didi-Cohen, Shoshana; Leu, Stefan; Cohen, Zvi; Boussiba, Sammy

    2014-01-01

    The chloroplast pyruvate dehydrogenase complex (cpPDC) catalyses the oxidative decarboxylation of pyruvate forming acetyl-CoA, an immediate primer for the initial reactions of de novo fatty acid (FA) synthesis. Little is known about the source of acetyl-CoA in the chloroplasts of photosynthetic microalgae, which are capable of producing high amounts of the storage lipid triacylglycerol (TAG) under conditions of nutrient stresses. We generated Chlamydomonas reinhardtii CC-1618 mutants with decreased expression of the PDC2_E1α gene, encoding the putative chloroplast pyruvate dehydrogenase subunit E1α, using artificial microRNA. A comparative study on the effects of PDC2_E1α silencing on FAs and TAG production in C. reinhardtii, grown photoautotrophically and mixotrophically, with and without a nitrogen source in the nutrient medium, was carried out. Reduced expression of PDC2 _E1α led to a severely hampered photoautotrophic growth phenotype with drastic impairment in TAG accumulation under nitrogen deprivation. In the presence of acetate, downregulation of PDC2_E1α exerted little to no effect on TAG production and photosynthetic activity. In contrast, under photoautotrophic conditions, especially in the absence of a nitrogen source, a dramatic decline in photosynthetic oxygen evolution and photosystem II quantum yield against a background of the apparent over-reduction of the photosynthetic electron chain was recorded. Our results suggest an essential role of cpPDC in the supply of carbon precursors for de novo FA synthesis in microalgae under conditions of photoautotrophy. A shortage of this supply is detrimental to the nitrogen-starvation-induced synthesis of storage TAG, an important carbon and energy sink in stressed Chlamydomonas cells, thereby impairing the acclimation ability of the microalga. PMID:25210079

  19. The Maize Viviparous8 Locus, Encoding a Putative ALTERED MERISTEM PROGRAM1-Like Peptidase, Regulates Abscisic Acid Accumulation and Coordinates Embryo and Endosperm Development1[W

    PubMed Central

    Suzuki, Masaharu; Latshaw, Susan; Sato, Yutaka; Settles, A. Mark; Koch, Karen E.; Hannah, L. Curtis; Kojima, Mikiko; Sakakibara, Hitoshi; McCarty, Donald R.

    2008-01-01

    We describe a mutant of Zea mays isolated from a W22 inbred transposon population, widow's peak mutant1 (wpk1), with an altered pattern of anthocyanin synthesis and aleurone cell differentiation in endosperm. In addition, a failure of the developing mutant embryo to form leaf initials is associated with decreased expression of a subset of meristem regulatory genes that includes Abphyl1 and Td1. We show that the viviparous8 (vp8) mutant has a similar pleiotropic phenotype in the W22 inbred background in contrast to the viviparous embryo phenotype exhibited in the standard genetic background, and we confirmed that wpk1 is allelic to vp8. Further genetic analysis revealed that the standard vp8 stock contains an unlinked, partially dominant suppressor of the vp8 mutation that is not present in W22. Consistent with the early-onset viviparous phenotype of vp8, expression of several embryonic regulators, including LEC1/B3 domain transcription factors, was reduced in the mutant embryo. Moreover, reduced abscisic acid (ABA) content of vp8/wpk1 embryos was correlated with altered regulation of ABA biosynthesis, as well as ABA catabolic pathways. The ABA biosynthetic gene Vp14 was down-regulated in the nonsuppressed background, whereas the ZmABA8′oxA1a ABA 8′-hydroxylase gene was strongly up-regulated in both genetic backgrounds. Molecular analysis revealed that Vp8 encodes a putative peptidase closely related to Arabidopsis thaliana ALTERED MERISTEM PROGRAM1. Because the Vp8 regulates meristem development as well as seed maturation processes, including ABA accumulation, we propose that VP8 is required for synthesis of an unidentified signal that integrates meristem and embryo formation in seeds. PMID:18203869

  20. The Maize Viviparous8 locus, encoding a putative ALTERED MERISTEM PROGRAM1-like peptidase, regulates abscisic acid accumulation and coordinates embryo and endosperm development.

    PubMed

    Suzuki, Masaharu; Latshaw, Susan; Sato, Yutaka; Settles, A Mark; Koch, Karen E; Hannah, L Curtis; Kojima, Mikiko; Sakakibara, Hitoshi; McCarty, Donald R

    2008-03-01

    We describe a mutant of Zea mays isolated from a W22 inbred transposon population, widow's peak mutant1 (wpk1), with an altered pattern of anthocyanin synthesis and aleurone cell differentiation in endosperm. In addition, a failure of the developing mutant embryo to form leaf initials is associated with decreased expression of a subset of meristem regulatory genes that includes Abphyl1 and Td1. We show that the viviparous8 (vp8) mutant has a similar pleiotropic phenotype in the W22 inbred background in contrast to the viviparous embryo phenotype exhibited in the standard genetic background, and we confirmed that wpk1 is allelic to vp8. Further genetic analysis revealed that the standard vp8 stock contains an unlinked, partially dominant suppressor of the vp8 mutation that is not present in W22. Consistent with the early-onset viviparous phenotype of vp8, expression of several embryonic regulators, including LEC1/B3 domain transcription factors, was reduced in the mutant embryo. Moreover, reduced abscisic acid (ABA) content of vp8/wpk1 embryos was correlated with altered regulation of ABA biosynthesis, as well as ABA catabolic pathways. The ABA biosynthetic gene Vp14 was down-regulated in the nonsuppressed background, whereas the ZmABA8'oxA1a ABA 8'-hydroxylase gene was strongly up-regulated in both genetic backgrounds. Molecular analysis revealed that Vp8 encodes a putative peptidase closely related to Arabidopsis thaliana ALTERED MERISTEM PROGRAM1. Because the Vp8 regulates meristem development as well as seed maturation processes, including ABA accumulation, we propose that VP8 is required for synthesis of an unidentified signal that integrates meristem and embryo formation in seeds.

  1. 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.

  2. 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.

  3. 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...

  4. 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…

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  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. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  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. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  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. 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

  8. 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.

  9. 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

  10. 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

  11. The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination, is not readily released, and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots.

    PubMed

    Fossdal, Carl Gunnar; Nagy, Nina Elisabeth; Sharma, Praveen; Lönneborg, Anders

    2003-05-01

    The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.

  12. 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…

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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...

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  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. 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.

  10. Catechol-O-Methyltransferase Val158Met Polymorphism Associates with Individual Differences in Sleep Physiologic Responses to Chronic Sleep Loss

    PubMed Central

    Goel, Namni; Banks, Siobhan; Lin, Ling; Mignot, Emmanuel; Dinges, David F.

    2011-01-01

    Background The COMT Val158Met polymorphism modulates cortical dopaminergic catabolism, and predicts individual differences in prefrontal executive functioning in healthy adults and schizophrenic patients, and associates with EEG differences during sleep loss. We assessed whether the COMT Val158Met polymorphism was a novel marker in healthy adults of differential vulnerability to chronic partial sleep deprivation (PSD), a condition distinct from total sleep loss and one experienced by millions on a daily and persistent basis. Methodology/Principal Findings 20 Met/Met, 64 Val/Met, and 45 Val/Val subjects participated in a protocol of two baseline 10h time in bed (TIB) nights followed by five consecutive 4 h TIB nights. Met/Met subjects showed differentially steeper declines in non-REM EEG slow-wave energy (SWE)—the putative homeostatic marker of sleep drive—during PSD, despite comparable baseline SWE declines. Val/Val subjects showed differentially smaller increases in slow-wave sleep and smaller reductions in stage 2 sleep during PSD, and had more stage 1 sleep across nights and a shorter baseline REM sleep latency. The genotypes, however, did not differ in performance across various executive function and cognitive tasks and showed comparable increases in subjective and physiological sleepiness in response to chronic sleep loss. Met/Met genotypic and Met allelic frequencies were higher in whites than African Americans. Conclusions/Significance The COMT Val158Met polymorphism may be a genetic biomarker for predicting individual differences in sleep physiology—but not in cognitive and executive functioning—resulting from sleep loss in a healthy, racially-diverse adult population of men and women. Beyond healthy sleepers, our results may also provide insight for predicting sleep loss responses in patients with schizophrenia and other psychiatric disorders, since these groups repeatedly experience chronically-curtailed sleep and demonstrate COMT

  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. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  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. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  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 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

  1. 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

  2. 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.

  3. Insect-Induced Conifer Defense. White Pine Weevil and Methyl Jasmonate Induce Traumatic Resinosis, de Novo Formed Volatile Emissions, and Accumulation of Terpenoid Synthase and Putative Octadecanoid Pathway Transcripts in Sitka Spruce1[w

    PubMed Central

    Miller, Barbara; Madilao, Lufiani L.; Ralph, Steven; Bohlmann, Jörg

    2005-01-01

    Stem-boring insects and methyl jasmonate (MeJA) are thought to induce similar complex chemical and anatomical defenses in conifers. To compare insect- and MeJA-induced terpenoid responses, we analyzed traumatic oleoresin mixtures, emissions of terpenoid volatiles, and expression of terpenoid synthase (TPS) genes in Sitka spruce (Picea sitchensis) following attack by white pine weevils (Pissodes strobi) or application of MeJA. Both insects and MeJA caused traumatic resin accumulation in stems, with more accumulation induced by the weevils. Weevil-induced terpenoid emission profiles were also more complex than emissions induced by MeJA. Weevil feeding caused a rapid release of a blend of monoterpene olefins, presumably by passive evaporation of resin compounds from stem feeding sites. These compounds were not found in MeJA-induced emissions. Both weevils and MeJA caused delayed, diurnal emissions of (−)-linalool, indicating induced de novo biosynthesis of this compound. TPS transcripts strongly increased in stems upon insect attack or MeJA treatment. Time courses and intensity of induced TPS transcripts were different for monoterpene synthases, sesquiterpene synthases, and diterpene synthases. Increased levels of weevil- and MeJA-induced TPS transcripts accompanied major changes in terpenoid accumulation in stems. Induced TPS expression profiles in needles were less complex than those in stems and matched induced de novo emissions of (−)-linalool. Overall, weevils and MeJA induced similar, but not identical, terpenoid defense responses in Sitka spruce. Findings of insect- and MeJA-induced accumulation of allene oxide synthase-like and allene oxide cyclase-like transcripts are discussed in the context of traumatic resinosis and induced volatile emissions in this gymnosperm system. PMID:15618433

  4. Superoxide serves as a putative signal molecule for plant cell division: overexpression of CaRLK1 promotes the plant cell cycle via accumulation of O2(-) and decrease in H2 O2.

    PubMed

    Lee, Dong Ju; Choi, Hyun Jun; Moon, Mid-Eum; Chi, Youn-Tae; Ji, Kon-Young; Choi, Doil

    2017-02-01

    Reactive oxygen species (ROS) exert both positive and negative effects on plant growth and development and therefore receive a great deal of attention in current research. A hot pepper, Capsicum annuum receptor-like kinase 1 (CaRLK1) was ectopically expressed in Nicotiana tabacum BY-2 cell and Nicotiana benthamiana plants. This ectopic expression of CaRLK1 enhanced cell division and proliferation in both heterologous systems. Apparently, CaRLK1 is involved in controlling the cell cycle, possibly by inducing expressions of cyclin B1, cyclin D3, cyclin-dependent protein kinase 3, condensin complex subunit 2 and anaphase-promoting complex subunit 11 genes. CaRLK1 overexpression also increased transcript accumulation of NADPH oxidase genes, generation of O2(-) and catalase (CAT) activity/protein levels. In parallel, it decreased cellular H2 O2 levels and cell size. Treatment with Tiron or diphenyleneiodonium (DPI) both decreased the cell division rate and O2(-) concentrations, but increased cellular H2 O2 levels. Tobacco BY-2 cells overexpressing CaRLK1 were more sensitive to amino-1,2,4-triazole (3-AT), a CAT inhibitor, than control cells, suggesting that the increased H2 O2 levels may not function as a signal for cell division and proliferation. Overexpression of CaRLK1 stimulated progression of the cell cycle from G0 /G1 phase into the S phase. It is concluded that the CaRLK1 protein plays a pivotal role in controlling the level of O2(-) as signaling molecule which promotes cell division, concomitant with a reduction in H2 O2 by the induction of CAT activity/protein.

  5. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  6. Accumulate-Repeat-Accumulate-Accumulate-Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Sam; Thorpe, Jeremy

    2004-01-01

    Inspired by recently proposed Accumulate-Repeat-Accumulate (ARA) codes [15], in this paper we propose a channel coding scheme called Accumulate-Repeat-Accumulate-Accumulate (ARAA) codes. These codes can be seen as serial turbo-like codes or as a subclass of Low Density Parity Check (LDPC) codes, and they have a projected graph or protograph representation; this allows for a high-speed iterative decoder implementation using belief propagation. An ARAA code can be viewed as a precoded Repeat-and-Accumulate (RA) code with puncturing in concatenation with another accumulator, where simply an accumulator is chosen as the precoder; thus ARAA codes have a very fast encoder structure. Using density evolution on their associated protographs, we find examples of rate-lJ2 ARAA codes with maximum variable node degree 4 for which a minimum bit-SNR as low as 0.21 dB from the channel capacity limit can be achieved as the block size goes to infinity. Such a low threshold cannot be achieved by RA or Irregular RA (IRA) or unstructured irregular LDPC codes with the same constraint on the maximum variable node degree. Furthermore by puncturing the accumulators we can construct families of higher rate ARAA codes with thresholds that stay close to their respective channel capacity thresholds uniformly. Iterative decoding simulation results show comparable performance with the best-known LDPC codes but with very low error floor even at moderate block sizes.

  7. 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.

  8. 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.

  9. (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.

  10. 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

  11. 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.

  12. 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.

  13. Accumulate-Repeat-Accumulate-Accumulate Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Samuel; Thorpe, Jeremy

    2007-01-01

    Accumulate-repeat-accumulate-accumulate (ARAA) codes have been proposed, inspired by the recently proposed accumulate-repeat-accumulate (ARA) codes. These are error-correcting codes suitable for use in a variety of wireless data-communication systems that include noisy channels. ARAA codes can be regarded as serial turbolike codes or as a subclass of low-density parity-check (LDPC) codes, and, like ARA codes they have projected graph or protograph representations; these characteristics make it possible to design high-speed iterative decoders that utilize belief-propagation algorithms. The objective in proposing ARAA codes as a subclass of ARA codes was to enhance the error-floor performance of ARA codes while maintaining simple encoding structures and low maximum variable node degree.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Putative porin of Bradyrhizobium sp. (Lupinus) bacteroids induced by glyphosate.

    PubMed

    de María, Nuria; Guevara, Angeles; Serra, M Teresa; García-Luque, Isabel; González-Sama, Alfonso; García de Lacoba, Mario; de Felipe, M Rosario; Fernández-Pascual, Mercedes

    2007-08-01

    Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed.

  4. Accumulate Repeat Accumulate Coded Modulation

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative coded modulation scheme called 'Accumulate Repeat Accumulate Coded Modulation' (ARA coded modulation). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes that are combined with high level modulation. Thus at the decoder belief propagation can be used for iterative decoding of ARA coded modulation on a graph, provided a demapper transforms the received in-phase and quadrature samples to reliability of the bits.

  5. The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy.

    PubMed

    Samanani, Nailish; Alcantara, Joenel; Bourgault, Richard; Zulak, Katherine G; Facchini, Peter J

    2006-08-01

    The benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)-norcoclaurine 6-O-methyltransferase (6OMT), (S)-coclaurine N-methyltransferase (CNMT), (S)-3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) and salutaridinol-7-O-acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co-localization of a sieve element-specific H(+)-ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4'OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4'OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type-specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.

  6. Putative Porin of Bradyrhizobium sp. (Lupinus) Bacteroids Induced by Glyphosate▿

    PubMed Central

    de María, Nuria; Guevara, Ángeles; Serra, M. Teresa; García-Luque, Isabel; González-Sama, Alfonso; de Lacoba, Mario García; de Felipe, M. Rosario; Fernández-Pascual, Mercedes

    2007-01-01

    Application of glyphosate (N-[phosphonomethyl] glycine) to Bradyrhizobium sp. (Lupinus)-nodulated lupin plants caused modifications in the protein pattern of bacteroids. The most significant change was the presence of a 44-kDa polypeptide in bacteroids from plants treated with the higher doses of glyphosate employed (5 and 10 mM). The polypeptide has been characterized by the amino acid sequencing of its N terminus and the isolation and nucleic acid sequencing of its encoding gene. It is putatively encoded by a single gene, and the protein has been identified as a putative porin. Protein modeling revealed the existence of several domains sharing similarity to different porins, such as a transmembrane beta-barrel. The protein has been designated BLpp, for Bradyrhizobium sp. (Lupinus) putative porin, and would be the first porin described in Bradyrhizobium sp. (Lupinus). In addition, a putative conserved domain of porins has been identified which consists of 87 amino acids, located in the BLpp sequence 30 amino acids downstream of the N-terminal region. In bacteroids, mRNA of the BLpp gene shows a basal constitutive expression that increases under glyphosate treatment, and the expression of the gene is seemingly regulated at the transcriptional level. By contrast, in free-living bacteria glyphosate treatment leads to an inhibition of BLpp mRNA accumulation, indicating a different effect of glyphosate on BLpp gene expression in bacteroids and free-living bacteria. The possible role of BLpp in a metabolite interchange between Bradyrhizobium and lupin is discussed. PMID:17557843

  7. Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

    PubMed

    Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj

    2016-07-01

    PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity.

  8. Protein repair L-isoaspartyl methyltransferase in plants. Phylogenetic distribution and the accumulation of substrate proteins in aged barley seeds.

    PubMed Central

    Mudgett, M B; Lowenson, J D; Clarke, S

    1997-01-01

    Protein L-isoaspartate (D-aspartate) O-methyltransferases (MTs; EC 2.1.1.77) can initiate the conversion of detrimental L-isoaspartyl residues in spontaneously damaged proteins to normal L-aspartyl residues. We detected this enzyme in 45 species from 23 families representing most of the divisions of the plant kingdom. MT activity is often localized in seeds, suggesting that it has a role in their maturation, quiescence, and germination. The relationship among MT activity, the accumulation of abnormal protein L-isoaspartyl residues, and seed viability was explored in barley (Hordeum vulgare cultivar Himalaya) seeds, which contain high levels of MT. Natural aging of barley seeds for 17 years resulted in a significant reduction in MT activity and in seed viability, coupled with increased levels of "unrepaired" L-isoaspartyl residues. In seeds heated to accelerate aging, we found no reduction of MT activity, but we did observe decreased seed viability and the accumulation of isoaspartyl residues. Among populations of accelerated aged seed, those possessing the highest levels of L-isoaspartyl-containing proteins had the lowest germination percentages. These results suggest that the MT present in seeds cannot efficiently repair all spontaneously damaged proteins containing altered aspartyl residues, and their accumulation during aging may contribute to the loss of seed viability. PMID:9414558

  9. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  10. 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.

  11. 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.

  12. Ten Putative Contributors to the Obesity Epidemic

    PubMed Central

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  13. 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.

  14. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    PubMed

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  15. Biogenic Origin for Earth's Oldest Putative Microfossils

    SciTech Connect

    De Gregorio, B.; Sharp, T; Flynn, G; Wirick, S; Hervig, R

    2009-01-01

    Carbonaceous microbe-like features preserved within a local chert unit of the 3.5 Ga old Apex Basalt in Western Australia may represent some of the oldest evidence of life on Earth. However, the biogenicity of these putative microfossils has been called into question, primarily because the sample collection locality is a black, carbon-rich, brecciated chert dike representing an Archean submarine hydrothermal spring, suggesting a formation via an abiotic organic synthesis mechanism. Here we describe the macromolecular hydrocarbon structure, carbon bonding, functional group chemistry, and biotic element abundance of carbonaceous matter associated with these filamentous features. These characteristics are similar to those of biogenic kerogen from the ca. 1.9 Ga old Gunflint Formation. Although an abiotic origin cannot be entirely ruled out, it is unlikely that known abiotic synthesis mechanisms could recreate both the structural and compositional complexity of this ancient carbonaceous matter. Thus, we find that a biogenic origin for this material is more likely, implying that the Apex microbe-like features represent authentic biogenic organic matter.

  16. Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins.

    PubMed

    Del Valle, M E; Cobo, T; Cobo, J L; Vega, J A

    2012-08-01

    The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited.

  17. Putative Bronchopulmonary Flagellated Protozoa in Immunosuppressed Patients

    PubMed Central

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Çelik, Pınar; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be “flagellated protozoa” have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells. PMID:24804259

  18. The Biogeography of Putative Microbial Antibiotic Production.

    PubMed

    Morlon, Hélène; O'Connor, Timothy K; Bryant, Jessica A; Charkoudian, Louise K; Docherty, Kathryn M; Jones, Evan; Kembel, Steven W; Green, Jessica L; Bohannan, Brendan J M

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics.

  19. The Biogeography of Putative Microbial Antibiotic Production

    PubMed Central

    Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  20. PUTATIVE ADVERSE OUTCOME PATHWAY FOR INHIBITON ...

    EPA Pesticide Factsheets

    The adverse outcome pathway (AOP) provides a framework for organizing knowledge to define links between a molecular initiating event (MIE) and an adverse outcome (AO) occurring at a higher level of biological organization, such as the individual or population. The AOP framework proceeds from a general (e.g., not chemical specific) molecular mode of action, designated as a MIE, through stepwise changes in biological status, defined as key events (KEs), to a final AO that can be used in risk assessment. Because aromatase-inhibiting pharmaceuticals are widely used to treat breast cancer patients, we explored the unintended consequences that might occur in fish exposed to these chemicals through wastewater discharge into the aquatic environment. Unlike mammals, fish have two isoforms of aromatase, one that predominates in the ovary (cyp19a1a) and a second (cyp19a1b) that prevails in the brain. Aromatase activity in fish brain can be 100 to 1000 times that in mammals and is associated with reproduction. We have developed a putative AOP for inhibition of brain aromatase in fish leading to reproductive dysfunction based on review of relevant literature and reproductive experiments with the marine fish cunner (Tautogolabrus adspersus) exposed to aromatase-inhibiting pharmaceuticals in the laboratory. The first KE in this AOP is a decrease in brain aromatase activity due to exposure to an aromatase inhibitor. KEs then progress through subsequent steps including decreas

  1. The Progress and Prospects of Putative Biomarkers for Liver Cancer Stem Cells in Hepatocellular Carcinoma

    PubMed Central

    Yang, Ting

    2016-01-01

    Accumulating evidence suggests that hepatocellular carcinoma (HCC) is organized by liver cancer stem cells (LCSCs), which are a subset of cells with “stem-like” characteristics. Identification of the LCSCs is a fundamental and important problem in HCC research. LCSCs have been investigated by various stem cell biomarkers. There is still lack of consensus regarding the existence of a “global” marker for LCSCs in HCC. In this review article, we summarize the progress and prospects of putative biomarkers for LCSCs in the past decades, which is essential to develop future therapies targeting CSCs and to predict prognosis and curative effect of these therapies. PMID:27610139

  2. 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-...

  3. Chimpanzee accumulative stone throwing.

    PubMed

    Kühl, Hjalmar S; Kalan, Ammie K; Arandjelovic, Mimi; Aubert, Floris; D'Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M; Zuberbuehler, Klaus; Boesch, Christophe

    2016-02-29

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites.

  4. Accumulation of the planets

    NASA Technical Reports Server (NTRS)

    Wetherill, G. W.

    1987-01-01

    In modeling the accumulation of planetesimals into planets, it is appropriate to distinguish between two stages: an early stage, during which approximately 10 km diameter planetesimals accumulate locally to form bodies approximate 10 to the 25th g in mass; and a later stage in which the approximately 10 to the 25th g planetesimals accumulate into the final planets. In the terrestrial planet region, an initial planetesimal swarm corresponding to the critical mass of dust layer gravitational instabilities is considered. In order to better understand the accumulation history of Mercury-sized bodies, 19 Monte-Carlo simulations of terrestrial planet growth were calculated. A Monte Carlo technique was used to investigate the orbital evolution of asteroidal collision debris produced interior to 2.6 AU. It was found that there are two regions primarily responsible for production of Earth-crossing meteoritic material and Apollo objects. The same techniques were extended to include the origin of Earth-approaching asteroidal bodies. It is found that these same two resonant mechanisms predict a steady-state number of Apollo-Amor about 1/2 that estimated based on astronomical observations.

  5. Chimpanzee accumulative stone throwing

    PubMed Central

    Kühl, Hjalmar S.; Kalan, Ammie K.; Arandjelovic, Mimi; Aubert, Floris; D’Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E.; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J.; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M.; Zuberbuehler, Klaus; Boesch, Christophe

    2016-01-01

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites. PMID:26923684

  6. Proteomic Characterization of Differential Abundant Proteins Accumulated between Lower and Upper Epidermises of Fleshy Scales in Onion (Allium cepa L.) Bulbs

    PubMed Central

    Wu, Xiaolin

    2016-01-01

    The onion (Allium cepa L.) is widely planted worldwide as a valuable vegetable crop. The scales of an onion bulb are a modified type of leaf. The one-layer-cell epidermis of onion scales is commonly used as a model experimental material in botany and molecular biology. The lower epidermis (LE) and upper epidermis (UE) of onion scales display obvious differences in microscopic structure, cell differentiation and pigment synthesis; however, associated proteomic differences are unclear. LE and UE can be easily sampled as single-layer-cell tissues for comparative proteomic analysis. In this study, a proteomic approach based on 2-DE and mass spectrometry (MS) was applied to compare LE and UE of fleshy scales from yellow and red onions. We identified 47 differential abundant protein spots (representing 31 unique proteins) between LE and UE in red and yellow onions. These proteins are mainly involved in pigment synthesis, stress response, and cell division. Particularly, the differentially accumulated chalcone-flavanone isomerase and flavone O-methyltransferase 1-like in LE may result in the differences in the onion scale color between red and yellow onions. Moreover, stress-related proteins abundantly accumulated in both LE and UE. In addition, the differential accumulation of UDP-arabinopyranose mutase 1-like protein and β-1,3-glucanase in the LE may be related to the different cell sizes between LE and UE of the two types of onion. The data derived from this study provides new insight into the differences in differentiation and developmental processes between onion epidermises. This study may also make a contribution to onion breeding, such as improving resistances and changing colors. PMID:28036352

  7. Proteomic Characterization of Differential Abundant Proteins Accumulated between Lower and Upper Epidermises of Fleshy Scales in Onion (Allium cepa L.) Bulbs.

    PubMed

    Wu, Si; Ning, Fen; Wu, Xiaolin; Wang, Wei

    2016-01-01

    The onion (Allium cepa L.) is widely planted worldwide as a valuable vegetable crop. The scales of an onion bulb are a modified type of leaf. The one-layer-cell epidermis of onion scales is commonly used as a model experimental material in botany and molecular biology. The lower epidermis (LE) and upper epidermis (UE) of onion scales display obvious differences in microscopic structure, cell differentiation and pigment synthesis; however, associated proteomic differences are unclear. LE and UE can be easily sampled as single-layer-cell tissues for comparative proteomic analysis. In this study, a proteomic approach based on 2-DE and mass spectrometry (MS) was applied to compare LE and UE of fleshy scales from yellow and red onions. We identified 47 differential abundant protein spots (representing 31 unique proteins) between LE and UE in red and yellow onions. These proteins are mainly involved in pigment synthesis, stress response, and cell division. Particularly, the differentially accumulated chalcone-flavanone isomerase and flavone O-methyltransferase 1-like in LE may result in the differences in the onion scale color between red and yellow onions. Moreover, stress-related proteins abundantly accumulated in both LE and UE. In addition, the differential accumulation of UDP-arabinopyranose mutase 1-like protein and β-1,3-glucanase in the LE may be related to the different cell sizes between LE and UE of the two types of onion. The data derived from this study provides new insight into the differences in differentiation and developmental processes between onion epidermises. This study may also make a contribution to onion breeding, such as improving resistances and changing colors.

  8. Choline metabolism in glycinebetaine accumulating and non-accumulating near-isogenic lines of Zea mays and Sorghum bicolor.

    PubMed

    Peel, Gregory J; Mickelbart, Michael V; Rhodes, David

    2010-03-01

    Glycinebetaine (GB) is a compatible solute that is accumulated by some plant species, especially under conditions leading to tissue osmotic stress. Genetic modification for accumulation of GB in an attempt to produce more stress tolerant plants has been a focus for several groups in recent years. However, attempts to increase tissue GB concentrations have been unsuccessful, with many transgenic lines accumulating far lower concentrations than naturally-occurring GB accumulators. A better understanding of the metabolic regulation of GB synthesis is necessary for successful molecular breeding and biotechnology. We utilized previously developed near-isogenic lines for GB accumulation to characterize the biochemical basis for GB deficiency in maize and sorghum. Salinity resulted in increased accumulation of choline in both accumulating and non-accumulating lines. When grown in the presence of NaCl, GB-non-accumulating lines had increased concentrations of choline and phosphocholine, but not GB. Decreased GB synthesis can be explained from the increased concentrations of phosphocholine in planta and the strong inhibition of N-phosphoethanolamine methyltransferase by phosphocholine observed in vitro. The lack of GB accumulation in GB-/- homozygous NILs was not due to the lack of the putative choline monooxygenase (the enzyme responsible for choline oxidation to betaine aldehyde) gene or protein that we describe. The previously identified bet1 locus does not appear to be choline monooxygenase. However, the lack of GB synthesis does affect the synthesis and turnover of choline moieties in GB non-accumulating lines, which may lead to alterations in overall 1-carbon metabolism in plants.

  9. Control of helium accumulation

    SciTech Connect

    Varadarajan, V.; Miley, G.H.

    1990-01-01

    The fishbone like oscillations in ignited tokamaks are addressed in an exploratory manner. The effects of the strong m = 1 oscillations and the weak high-frequency oscillations are examined in order to explore the feasibility of utilizing these oscillations for alpha accumulation control. The prospects of achieving small scale continuous alpha removal from the plasma center by mild fishbone-like oscillations are examined.

  10. Selenium accumulation by plants

    PubMed Central

    White, Philip J.

    2016-01-01

    Background Selenium (Se) is an essential mineral element for animals and humans, which they acquire largely from plants. The Se concentration in edible plants is determined by the Se phytoavailability in soils. Selenium is not an essential element for plants, but excessive Se can be toxic. Thus, soil Se phytoavailability determines the ecology of plants. Most plants cannot grow on seleniferous soils. Most plants that grow on seleniferous soils accumulate <100 mg Se kg–1 dry matter and cannot tolerate greater tissue Se concentrations. However, some plant species have evolved tolerance to Se, and commonly accumulate tissue Se concentrations >100 mg Se kg–1 dry matter. These plants are considered to be Se accumulators. Some species can even accumulate Se concentrations of 1000–15 000 mg Se kg–1 dry matter and are called Se hyperaccumulators. Scope This article provides an overview of Se uptake, translocation and metabolism in plants and highlights the possible genetic basis of differences in these between and within plant species. The review focuses initially on adaptations allowing plants to tolerate large Se concentrations in their tissues and the evolutionary origin of species that hyperaccumulate Se. It then describes the variation in tissue Se concentrations between and within angiosperm species and identifies genes encoding enzymes limiting the rates of incorporation of Se into organic compounds and chromosomal loci that might enable the development of crops with greater Se concentrations in their edible portions. Finally, it discusses transgenic approaches enabling plants to tolerate greater Se concentrations in the rhizosphere and in their tissues. Conclusions The trait of Se hyperaccumulation has evolved several times in separate angiosperm clades. The ability to tolerate large tissue Se concentrations is primarily related to the ability to divert Se away from the accumulation of selenocysteine and selenomethionine, which might be incorporated

  11. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    USGS Publications Warehouse

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  12. Overabundance of Putative Cancer Stem Cells in Human Skin Keratinocyte Cells Malignantly Transformed by Arsenic

    PubMed Central

    Sun, Yang; Tokar, Erik J.; Waalkes, Michael P.

    2012-01-01

    Arsenic is a human skin carcinogen. Cancer is probably a disease driven by stem cells (SCs), and SCs are likely a key target during arsenic oncogenesis. In utero arsenic exposure predisposes mice to skin cancers that overproduce cancer SCs (CSCs) and have distorted CSC signaling and population dynamics. Therefore, we hypothesized CSC accumulation may occur during arsenic-induced malignant transformation in vitro of human skin keratinocytes. Thus, the HaCaT cell line, malignantly transformed by arsenite (100nM, 30 weeks; termed As-TM cells) in prior work, was further studied for the quantity and nature of SCs after this transformation. SCs were isolated from passage-matched control and As-TM cells by a magnetic bead system that enriches for CD34-positive cells. There were 2.5 times more SCs isolated from As-TM cells than control. Holoclone production from As-TM putative CSCs was 2.5-fold higher by 1 week and 3.5-fold higher by 2 weeks than control SCs. Potential malignant phenotype was assessed in isolated SC/CSCs. Transcript level of SC/CSC markers were elevated in both isolated As-TM CSCs and control SCs compared with parental cells, but compared with control SCs, As-TM putative CSCs had elevated CD34, K5, K14, K15, and K19 transcripts and dramatically stronger staining for p63, Rac1, K5, Notch1, and K19. As-TM putative CSCs also showed markedly elevated MMP-9 secretion and colony formation, indicators of cancer phenotype, even compared with total population of As-TM cells. Thus, malignant phenotype is particularly pronounced in CSCs after arsenic-induced transformation of human skin cells and occurs concurrently with a potential overproduction of these cells. PMID:22011395

  13. Heat exchanger-accumulator

    DOEpatents

    Ecker, Amir L.

    1980-01-01

    What is disclosed is a heat exchanger-accumulator for vaporizing a refrigerant or the like, characterized by an upright pressure vessel having a top, bottom and side walls; an inlet conduit eccentrically and sealingly penetrating through the top; a tubular overflow chamber disposed within the vessel and sealingly connected with the bottom so as to define an annular outer volumetric chamber for receiving refrigerant; a heat transfer coil disposed in the outer volumetric chamber for vaporizing the liquid refrigerant that accumulates there; the heat transfer coil defining a passageway for circulating an externally supplied heat exchange fluid; transferring heat efficiently from the fluid; and freely allowing vaporized refrigerant to escape upwardly from the liquid refrigerant; and a refrigerant discharge conduit penetrating sealingly through the top and traversing substantially the length of the pressurized vessel downwardly and upwardly such that its inlet is near the top of the pressurized vessel so as to provide a means for transporting refrigerant vapor from the vessel. The refrigerant discharge conduit has metering orifices, or passageways, penetrating laterally through its walls near the bottom, communicating respectively interiorly and exteriorly of the overflow chamber for controllably carrying small amounts of liquid refrigerant and oil to the effluent stream of refrigerant gas.

  14. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    PubMed Central

    Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

    2016-01-01

    Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

  15. Gastrokines: stomach-specific proteins with putative homeostatic and tumor suppressor roles.

    PubMed

    Menheniott, Trevelyan R; Kurklu, Bayzar; Giraud, Andrew S

    2013-01-15

    During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.

  16. Solids Accumulation Scouting Studies

    SciTech Connect

    Duignan, M. R.; Steeper, T. J.; Steimke, J. L.

    2012-09-26

    The objective of Solids Accumulation activities was to perform scaled testing to understand the behavior of remaining solids in a Double Shell Tank (DST), specifically AW-105, at Hanford during multiple fill, mix, and transfer operations. It is important to know if fissionable materials can concentrate when waste is transferred from staging tanks prior to feeding waste treatment plants. Specifically, there is a concern that large, dense particles containing plutonium could accumulate in poorly mixed regions of a blend tank heel for tanks that employ mixing jet pumps. At the request of the DOE Hanford Tank Operations Contractor, Washington River Protection Solutions, the Engineering Development Laboratory of the Savannah River National Laboratory performed a scouting study in a 1/22-scale model of a waste staging tank to investigate this concern and to develop measurement techniques that could be applied in a more extensive study at a larger scale. Simulated waste tank solids: Gibbsite, Zirconia, Sand, and Stainless Steel, with stainless steel particles representing the heavier particles, e.g., plutonium, and supernatant were charged to the test tank and rotating liquid jets were used to mix most of the solids while the simulant was pumped out. Subsequently, the volume and shape of the mounds of residual solids and the spatial concentration profiles for the surrogate for heavier particles were measured. Several techniques were developed and equipment designed to accomplish the measurements needed and they included: 1. Magnetic particle separator to remove simulant stainless steel solids. A device was designed and built to capture these solids, which represent the heavier solids during a waste transfer from a staging tank. 2. Photographic equipment to determine the volume of the solids mounds. The mounds were photographed as they were exposed at different tank waste levels to develop a composite of topographical areas. 3. Laser rangefinders to determine the volume of

  17. Spectral Evidence of Aqueous Activity in Two Putative Martian Paleolakes

    NASA Technical Reports Server (NTRS)

    Roush, Ted L.; Marzo, Giuseppe A.; Fonti, Sergio; Orofino, Vincenzo; Blanco, Armando

    2010-01-01

    CRISM observations of putative paleolakes in Cankuzo and Luqa craters exhibit spectral features consistent with the activity of water. The spatial distributions suggest different formation scenarios for each site. In Cankuzo the distribution suggests postimpact alteration whereas in Luqa there are hints of possible formation of a layer of phyllosilicate materials.

  18. Sulfur Isotope Composition of Putative Primary Troilite in Chondrules

    NASA Technical Reports Server (NTRS)

    Tachibana, Shogo; Huss, Gary R.

    2002-01-01

    Sulfur isotope compositions of putative primary troilites in chondrules from Bishunpur were measured by ion probe. These primary troilites have the same S isotope compositions as matrix troilites and thus appear to be isotopically unfractionated. Additional information is contained in the original extended abstract.

  19. Bartonella henselae AS A PUTATIVE CAUSE OF CONGENITAL CHOLESTASIS

    PubMed Central

    VELHO, Paulo Eduardo Neves Ferreira; BELLOMO-BRANDÃO, Maria Ângela; DRUMMOND, Marina Rovani; MAGALHÃES, Renata Ferreira; HESSEL, Gabriel; BARJAS-CASTRO, Maria de Lourdes; ESCANHOELA, Cecília Amélia Fazzio; NEGRO, Gilda Maria Barbaro DEL; OKAY, Thelma Suely

    2016-01-01

    SUMMARY Severe anemia and cholestatic hepatitis are associated with bartonella infections. A putative vertical Bartonella henselae infection was defined on the basis of ultrastructural and molecular analyses in a three-year-old child with anemia, jaundice and hepatosplenomegaly since birth. Physicians should consider bartonellosis in patients with anemia and hepatitis of unknown origin. PMID:27410916

  20. Developing putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content dataDeveloping putative AOPs from high content data

    EPA Science Inventory

    Developing putative AOPs from high content data Shannon M. Bell1,2, Stephen W. Edwards2 1 Oak Ridge Institute for Science and Education 2 Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,...

  1. pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.

    PubMed

    Gong, Xiaoyan; Hurtado, Oscar; Wang, Baohua; Wu, Congqing; Yi, Mihwa; Giraldo, Martha; Valent, Barbara; Goodin, Michael; Farman, Mark

    2015-02-01

    As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

  2. Detoxification of polycyclic aromatic hydrocarbons (PAHs) in Arabidopsis thaliana involves a putative flavonol synthase

    PubMed Central

    Hernández-Vega, Juan C.; Cady, Brian; Kayanja, Gilbert; Mauriello, Anthony; Cervantes, Natalie; Gillespie, Andrea; Lavia, Lisa; Trujillo, Joshua; Alkio, Merianne; Colón-Carmona, Adán

    2017-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants with cytotoxic, teratogenic and carcinogenic properties. Bioremediation studies with bacteria have led to the identification of dioxygenases (DOXs) in the first step to degrade these recalcitrant compounds. In this study, we characterized the role of the Arabidopsis thaliana AT5G05600, a putative DOX of the flavonol synthase family, in the transformation of PAHs. Phenotypic analysis of loss-of-function mutant lines showed that these plant lines were less sensitive to the toxic effects of phenanthrene, suggesting possible roles of this gene in PAH degradation in vivo. Interestingly, these mutant lines showed less accumulation of H2O2 after PAH exposure. Transgenic lines over-expressing At5g05600 showed a hypersensitive response and more oxidative stress after phenanthrene treatments. Moreover, fluorescence spectra results of biochemical assays with the recombinant His-tagged protein AT5G05600 detected chemical modifications of phenanthrene. Taken together, these results support the hypothesis that AT5G05600 is involved in the catabolism of PAHs and the accumulation of toxic intermediates during PAH biotransformation in plants. This research represents the first step in the design of transgenic plants with the potential to degrade PAHs, leading to the development of vigorous plant varieties that can reduce the levels of these pollutants in the environment. PMID:27637093

  3. The Pun1 gene for pungency in pepper encodes a putative acyltransferase.

    PubMed

    Stewart, Charles; Kang, Byoung-Cheorl; Liu, Kede; Mazourek, Michael; Moore, Shanna L; Yoo, Eun Young; Kim, Byung-Dong; Paran, Ilan; Jahn, Molly M

    2005-06-01

    Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

  4. Exogenous application of pectin-derived oligosaccharides to grape berries modifies anthocyanin accumulation, composition and gene expression.

    PubMed

    Villegas, Daniel; Handford, Michael; Alcalde, José Antonio; Perez-Donoso, Alonso

    2016-07-01

    -glucose flavonoid 3-O-glucosyltransferase (UFGT) expression, probably mediated by a rise in MYBA1 was also observed. Regarding the anthocyanin profile, despite the increase observed in MYB5A expression in PDO and Ethrel treatments, no changes in flavonoid 3'-hydroxylase (F-3'-H); flavonoid 3'5'-hydroxylase (F-3'5'-H) or O-methyltransferase (OMT) could be related with the profile modifications described. Overall, this study highlights that application of PDO is a novel means of altering specific grape berry anthocyanins, and could be a means of positively influencing wine quality without the addition of agrochemicals.

  5. Putative melatonin receptors in a human biological clock

    SciTech Connect

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  6. Noise Reduction by Signal Accumulation

    ERIC Educational Resources Information Center

    Kraftmakher, Yaakov

    2006-01-01

    The aim of this paper is to show how the noise reduction by signal accumulation can be accomplished with a data acquisition system. This topic can be used for student projects. In many cases, the noise reduction is an unavoidable part of experimentation. Several techniques are known for this purpose, and among them the signal accumulation is the…

  7. Gypsum accumulation on carbonate stone

    USGS Publications Warehouse

    McGee, E.S.; Mossotti, V.G.

    1992-01-01

    The accumulation of gypsum on carbonate stone has been investigated through exposure of fresh samples of limestone and marble at monitored sites, through examination of alteration crusts from old buildings and through laboratory experiments. Several factors contribute to gypsum accumulation on carbonate stone. Marble or limestone that is sheltered from direct washing by rain in an urban environment with elevated pollution levels is likely to accumulate a gypsum crust. Crust development may be enhanced if the stone is porous or has an irregular surface area. Gypsum crusts are a surficial alteration feature; gypsum crystals form at the pore opening-air interface, where evaporation is greatest.

  8. Strain accumulation in quasicrystalline solids

    NASA Technical Reports Server (NTRS)

    Nori, Franco; Ronchetti, Marco; Elser, Veit

    1988-01-01

    The relaxation of two-dimensional quasicrystalline elastic networks when their constituent bonds are perturbed homogeneously is studied. Whereas ideal, quasi-periodic networks are stable against such perturbations, significant accumulations of strain in a class of disordered networks generated by a growth process are found. The grown networks are characterized by root mean square phason fluctuations which grow linearly with system size. The strain accumulation observed in these networks also grows linearly with system size. Finally, dependence of strain accumulation on cooling rate is found.

  9. The "kynurenate test", a biochemical assay for putative cognition enhancers.

    PubMed

    Pittaluga, A; Vaccari, D; Raiteri, M

    1997-10-01

    Some putative cognition enhancers (oxiracetam, aniracetam and D-cycloserine) were previously shown to prevent the kynurenic acid antagonism of the N-methyl-D-aspartate (NMDA)-evoked norepinephrine (NE) release in rat hippocampal slices. This functional in vitro assay was further characterized in the present work. D-Serine, a glutamate coagonist at the NMDA receptor glycine site, concentration-dependently (EC50 approximately 0.1 microM) prevented the kynurenate (100 microM) block of the NMDA (100 microM)-evoked [3H]NE release. L-Serine was ineffective up to 10 microM. The gamma-aminobutyric acidB (GABA[B]) receptor antagonist CGP 36742, reported to improve cognitive performance, potently prevented the kynurenate antagonism. The activity of CGP 36742 (1 microM) appeared to be unaffected by 10 microM (-)-baclofen, a GABA(B) receptor agonist; furthermore, CGP 52432, a GABA(B) antagonist more potent than CGP 36742, but reportedly devoid of nootropic properties, was inactive in the "kynurenate test." The novel putative cognition enhancer CR2249, but not its enantiomer CR2361, also potently prevented the kynurenate antagonism. In contrast, linopirdine, nicotine and tacrine were inactive. In rat hippocampal synaptosomes glycine and D-cycloserine enhanced the NMDA-evoked [3H]NE release, whereas oxiracetam and CR2249 did not. These four compounds were all similarly effective in preventing kynurenate antagonism, both in slices and in synaptosomes. The NMDA potentiation caused by glycine (0.1-100 microM) was not affected by 100 microM oxiracetam, which suggested that drugs active in the "kynurenate test" may bind to sites different from the glycine site of the NMDA receptor. To conclude, the "kynurenate test" is an in vitro assay useful in the identification and characterization of putative cognition enhancers acting via NMDA receptors.

  10. An ORF from Bacillus licheniformis encodes a putative DNA repressor.

    PubMed

    Naval, J; Aguilar, D; Serra, X; Pérez-Pons, J A; Piñol, J; Lloberas, J; Querol, E

    2000-01-01

    The complete sequence of a reading frame adjacent to the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis is reported. It encodes a putative 171 amino acid residues protein with either, low significant sequence similarity in data banks or the corresponding orthologue in the recently sequenced Bacillus subtilis genome. Computer analyses predict a canonical Helix-Turn-Helix motif characteristic of bacterial repressors/DNA binding proteins. A maxicells assay shows that the encoded polypeptide is expressed. A DNA-protein binding, assay performed by gel electrophoresis shows that the expressed protein specifically binds to Bacillus licheniformis DNA.

  11. Molecular genetics: DNA analysis of a putative dog clone.

    PubMed

    Parker, Heidi G; Kruglyak, Leonid; Ostrander, Elaine A

    2006-03-09

    In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..

  12. Manganese As a Metal Accumulator

    EPA Science Inventory

    Manganese deposits in water distribution systems accumulate metals, radionuclides and oxyanions by a combination of surface complexation, adsorption and solid substitution, as well as a combination of oxidation followed by manganese reduction and sorption of the oxidized constitu...

  13. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  14. Characterization of a Putative Ancestor of Coxsackievirus B5 ▿

    PubMed Central

    Gullberg, Maria; Tolf, Conny; Jonsson, Nina; Mulders, Mick N.; Savolainen-Kopra, Carita; Hovi, Tapani; Van Ranst, Marc; Lemey, Philippe; Hafenstein, Susan; Lindberg, A. Michael

    2010-01-01

    Like other RNA viruses, coxsackievirus B5 (CVB5) exists as circulating heterogeneous populations of genetic variants. In this study, we present the reconstruction and characterization of a probable ancestral virion of CVB5. Phylogenetic analyses based on capsid protein-encoding regions (the VP1 gene of 41 clinical isolates and the entire P1 region of eight clinical isolates) of CVB5 revealed two major cocirculating lineages. Ancestral capsid sequences were inferred from sequences of these contemporary CVB5 isolates by using maximum likelihood methods. By using Bayesian phylodynamic analysis, the inferred VP1 ancestral sequence dated back to 1854 (1807 to 1898). In order to study the properties of the putative ancestral capsid, the entire ancestral P1 sequence was synthesized de novo and inserted into the replicative backbone of an infectious CVB5 cDNA clone. Characterization of the recombinant virus in cell culture showed that fully functional infectious virus particles were assembled and that these viruses displayed properties similar to those of modern isolates in terms of receptor preferences, plaque phenotypes, growth characteristics, and cell tropism. This is the first report describing the resurrection and characterization of a picornavirus with a putative ancestral capsid. Our approach, including a phylogenetics-based reconstruction of viral predecessors, could serve as a starting point for experimental studies of viral evolution and might also provide an alternative strategy for the development of vaccines. PMID:20631132

  15. Putative cryptoendolithic life in Devonian pillow basalt, Rheinisches Schiefergebirge, Germany.

    PubMed

    Peckmann, J; Bach, W; Behrens, K; Reitner, J

    2008-03-01

    Middle Devonian (Givetian) pillow basalt and inter-pillow breccia from the Rheinisches Schiefergebirge in Germany were found to contain putative biogenic filaments that indicate that life once proliferated within these volcanic rocks. Mineralized filaments are found in carbonate amygdules (vesicles filled by carbonate cement) in the volcanic rock, where they started to form on the internal surface of the once water-filled vesicles. Biogenicity of the filaments is indicated by (1) their size and shape resembling modern microorganisms including a constant diameter along the length of curved filaments, (2) their independence of crystal faces or cleavage planes, (3) branching patterns reminiscent of modern microorganisms, and (4) their spatial clustering and preferential occurrence close to the margin of pillows and in the inter-pillow breccias. A time lag between the deposition of pillow basalt and the activity of endoliths is revealed by the sequence of carbonate cements filling the amygdules. The putative filamentous microorganisms thrived after the formation of early fibrous rim cement, but before later equant calcite spar filled most of the remaining porosity. Microbial clay authigenesis analogous to the encrustation of prokaryotes in modern iron-rich environments led to the preservation of filaments. The filaments predominantly consist of the clay minerals chamosite and illite. Having dwelled in water-filled vesicles, the Devonian basalt-hosted filaments apparently represent cryptoendoliths. This finding suggests that a previously unrecognized niche for life exists within volcanic rock.

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

    SciTech Connect

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

    2009-01-01

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

  17. Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans.

    PubMed

    Srinivasa, Kavitha; Kim, Na-Rae; Kim, Jiwon; Kim, Minsun; Bae, Ju Yun; Jeong, Woojin; Kim, Wankee; Choi, Wonja

    2012-03-01

    In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H(2)O(2)) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H(2)O(2) scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H(2)O(2), menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H(2)O(2). These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H(2)O(2), but its cellular function is specified to t-BOOH.

  18. Molecular characterization of the putative transcription factor SebA involved in virulence in Aspergillus fumigatus.

    PubMed

    Dinamarco, Taísa Magnani; Almeida, Ricardo S; de Castro, Patrícia Alves; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Ramalho, Leandra Naira Zambelli; Savoldi, Marcela; Goldman, Maria Helena S; Goldman, Gustavo Henrique

    2012-04-01

    Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.

  19. Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

    PubMed Central

    Dinamarco, Taísa Magnani; Almeida, Ricardo S.; Alves de Castro, Patrícia; Brown, Neil Andrew; dos Reis, Thaila Fernanda; Zambelli Ramalho, Leandra Naira; Savoldi, Marcela; Goldman, Maria Helena S.

    2012-01-01

    Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The ΔsebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA::GFP (SebA::green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the ΔsebA mutant. The A. fumigatus ΔsebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the ΔsebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses. PMID:22345349

  20. Maximum likelihood decoding analysis of Accumulate-Repeat-Accumulate Codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    Repeat-Accumulate (RA) codes are the simplest turbo-like codes that achieve good performance. However, they cannot compete with Turbo codes or low-density parity check codes (LDPC) as far as performance is concerned. The Accumulate Repeat Accumulate (ARA) codes, as a subclass of LDPC codes, are obtained by adding a pre-coder in front of RA codes with puncturing where an accumulator is chosen as a precoder. These codes not only are very simple, but also achieve excellent performance with iterative decoding. In this paper, the performance of these codes with (ML) decoding are analyzed and compared to random codes by very tight bounds. The weight distribution of some simple ARA codes is obtained, and through existing tightest bounds we have shown the ML SNR threshold of ARA codes approaches very closely to the performance of random codes. We have shown that the use of precoder improves the SNR threshold but interleaving gain remains unchanged with respect to RA code with puncturing.

  1. Variation in Arabidopsis flooding responses identifies numerous putative "tolerance genes".

    PubMed

    Vashisht, Divya; van Veen, Hans; Akman, Melis; Sasidharan, Rashmi

    2016-11-01

    Plant survival in flooded environments requires a combinatory response to multiple stress conditions such as limited light availability, reduced gas exchange and nutrient uptake. The ability to fine-tune the molecular response at the transcriptional and/or post-transcriptional level that can eventually lead to metabolic and anatomical adjustments are the underlying requirements to confer tolerance. Previously, we compared the transcriptomic adjustment of submergence tolerant, intolerant accessions and identified a core conserved and genotype-specific response to flooding stress, identifying numerous 'putative' tolerance genes. Here, we performed genome wide association analyses on 81 natural Arabidopsis accessions that identified 30 additional SNP markers associated with flooding tolerance. We argue that, given the many genes associated with flooding tolerance in Arabidopsis, improving resistance to submergence requires numerous genetic changes.

  2. Design and synthesis of inositolphosphoglycan putative insulin mediators.

    PubMed

    López-Prados, Javier; Cuevas, Félix; Reichardt, Niels-Christian; de Paz, José-Luis; Morales, Ezequiel Q; Martín-Lomas, Manuel

    2005-03-07

    The binding modes of a series of molecules, containing the glucosamine (1-->6) myo-inositol structural motif, into the ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase using the same intermediates has been explored. The results from the biological evaluation of these molecules provide additional support to an insulin-mediated signalling system which involves the intermediacy of inositolphosphoglycans as putative insulin mediators.

  3. Akt1 as a putative regulator of Hox genes.

    PubMed

    Kong, Kyoung-Ah; Yoon, Heejei; Kim, Myoung Hee

    2013-01-25

    In mammals, precise spatiotemporal expressions of Hox genes control the main body axis during embryogenesis. However, the mechanism by which Hox genes are regulated is poorly understood. To discover the putative regulator of Hox genes, in silico analyses were performed using GEO profiles, and Akt1 emerged as a candidate regulator of Hox genes in E13.5 MEFs. The results of the RT-PCR showed that 5' Hoxc genes, including ncRNA were upregulated in Akt1 null MEF. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing showed that the CpG island of a 5' Hoxc gene was hypomethylated in Akt1 null cells. These results indicate that Hox expression could be controlled by the function of Akt1 through epigenetic modification such as DNA methylation.

  4. Motor abnormalities as a putative endophenotype for Autism Spectrum Disorders

    PubMed Central

    Esposito, Gianluca; Paşca, Sergiu P.

    2013-01-01

    Autism Spectrum Disorders (ASDs) represent a complex group of behaviorally defined conditions with core deficits in social communication and the presence of repetitive and restrictive behaviors. To date, neuropathological studies have failed to identify pathognomonic cellular features for ASDs and there remains a fundamental disconnection between the complex clinical aspects of ASDs and the underlying neurobiology. Although not listed among the core diagnostic domains of impairment in ASDs, motor abnormalities have been consistently reported across the spectrum. In this perspective article, we summarize the evidence that supports the use of motor abnormalities as a putative endophenotype for ASDs. We argue that because these motor abnormalities do not directly depend on social or linguistic development, they may serve as an early disease indicator. Furthermore, we propose that stratifying patients based on motor development could be useful not only as an outcome predictor and in identifying more specific treatments for different ASDs categories, but also in exposing neurobiological mechanisms. PMID:23781177

  5. Catalysis-based total synthesis of putative mandelalide A.

    PubMed

    Willwacher, Jens; Fürstner, Alois

    2014-04-14

    A concise synthesis of the putative structure assigned to the highly cytotoxic marine macrolide mandelalide A (1) is disclosed. Specifically, an iridium-catalyzed two-directional Krische allylation and a cobalt-catalyzed carbonylative epoxide opening served as convenient entry points for the preparation of the major building blocks. The final stages feature the first implementation of terminal-acetylene metathesis into natural product synthesis, which is remarkable as this class of substrates was beyond reach until very recently; key to success was the use of the highly selective molybdenum alkylidyne complex 42 as the catalyst. Although the constitution and stereochemistry of the synthetic samples are unambiguous, the spectra of 1 as well as of 11-epi-1 deviate from those of the natural product, which implies a subtle but deep-seated error in the original structure assignment.

  6. Quartet analysis of putative horizontal gene transfer in Crenarchaeota.

    PubMed

    Ching, Travers H; Yoza, Brandon A; Li, Qing X

    2014-02-01

    Horizontal gene transfers (HGT) between four Crenarchaeota species (Metallosphaera cuprina Ar-4T, Acidianus hospitalis W1T, Vulcanisaeta moutnovskia 768-28T, and Pyrobaculum islandicum DSM 4184T) were investigated with quartet analysis. Strong support was found for individual genes that disagree with the phylogeny of the majority, implying genomic mosaicism. One such gene, a ferredoxin-related gene, was investigated further and incorporated into a larger phylogeny, which provided evidence for HGT of this gene from the Vulcanisaeta lineage to the Acidianus lineage. This is the first application of quartet analysis of HGT for the phylum Crenarchaeota. The results have shown that quartet analysis is a powerful technique to screen homologous sequences for putative HGTs and is useful in visually describing genomic mosaicism and HGT within four taxa.

  7. Novel putative mechanisms to link circadian clocks to healthy aging.

    PubMed

    Popa-Wagner, Aurel; Catalin, Bogdan; Buga, Ana-Maria

    2015-08-01

    The circadian clock coordinates the internal physiology to increase the homeostatic capacity thereby providing both a survival advantage to the system and an optimization of energy budgeting. Multiple-oscillator circadian mechanisms are likely to play a role in regulating human health and may contribute to the aging process. Our aim is to give an overview of how the central clock in the hypothalamus and peripheral clocks relate to aging and metabolic disorders, including hyperlipidemia and hyperglycemia. In particular, we unravel novel putative mechanisms to link circadian clocks to healthy aging. This review may lead to the design of large-scale interventions to help people stay healthy as they age by adjusting daily activities, such as feeding behavior, and or adaptation to age-related changes in individual circadian rhythms.

  8. Cryptic Species in Putative Ancient Asexual Darwinulids (Crustacea, Ostracoda)

    PubMed Central

    Schön, Isa; Pinto, Ricardo L.; Halse, Stuart; Smith, Alison J.; Martens, Koen; Birky, C. William

    2012-01-01

    Background Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied. Methodology/Principal Findings We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies. Conclusions/Significance Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated. PMID:22802945

  9. Putative Regulatory Factors Associated with Intramuscular Fat Content

    PubMed Central

    Cesar, Aline S. M.; Regitano, Luciana C. A.; Koltes, James E.; Fritz-Waters, Eric R.; Lanna, Dante P. D.; Gasparin, Gustavo; Mourão, Gerson B.; Oliveira, Priscila S. N.; Reecy, James M.; Coutinho, Luiz L.

    2015-01-01

    Intramuscular fat (IMF) content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies) were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG), biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV) for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA) and co-expression network methods, such as partial correlation coefficient with information theory (PCIT), regulatory impact factor (RIF) and phenotypic impact factor (PIF) were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H) and low (L) GEBV) and 77 DEG (FDR 10%) were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1), MPC1 and UBL5 (RIF2) and a host of small RNAs, including miR-1281 (PIF). These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption. PMID:26042666

  10. Characterization of two new putative adhesins of Leptospira interrogans.

    PubMed

    Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

    2017-01-01

    We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

  11. The floral organ number4 gene encoding a putative ortholog of Arabidopsis CLAVATA3 regulates apical meristem size in rice.

    PubMed

    Chu, Huangwei; Qian, Qian; Liang, Wanqi; Yin, Changsong; Tan, Hexin; Yao, Xuan; Yuan, Zheng; Yang, Jun; Huang, Hai; Luo, Da; Ma, Hong; Zhang, Dabing

    2006-11-01

    To understand the molecular mechanism regulating meristem development in the monocot rice (Oryza sativa), we describe here the isolation and characterization of three floral organ number4 (fon4) alleles and the cloning of the FON4 gene. The fon4 mutants showed abnormal enlargement of the embryonic and vegetative shoot apical meristems (SAMs) and the inflorescence and floral meristems. Likely due to enlarged SAMs, fon4 mutants produced thick culms (stems) and increased numbers of both primary rachis branches and floral organs. We identified FON4 using a map-based cloning approach and found it encodes a small putatively secreted protein, which is the putative ortholog of the Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) gene. FON4 transcripts mainly accumulated in the small group of cells at the apex of the SAMs, whereas the rice ortholog of CLV1 (FON1) is expressed throughout the SAMs, suggesting that the putative FON4 ligand might be sequestered as a possible mechanism for rice meristem regulation. Exogenous application of the peptides FON4p and CLV3p corresponding to the CLV3/ESR-related (CLE) motifs of FON4 and CLV3, respectively, resulted in termination of SAMs in rice, and treatment with CLV3p caused consumption of both rice and Arabidopsis root meristems, suggesting that the CLV pathway in limiting meristem size is conserved in both rice and Arabidopsis. However, exogenous FON4p did not have an obvious effect on limiting both rice and Arabidopsis root meristems, suggesting that the CLE motifs of Arabidopsis CLV3 and FON4 are potentially functionally divergent.

  12. Ion Trapping in the Accumulator

    SciTech Connect

    Marriner, J.

    1985-02-18

    The beam space charge (- for {bar p}'s) will attract positive ions. In the absence of additional fields (clearing electrodes, e.g.) these ions will be trapped in the beam potential well. The depth of this potential well has been calculated for some geometries relevant for the accumulator.

  13. The two putative comS homologs of the biotechnologically important Bacillus licheniformis do not contribute to competence development.

    PubMed

    Jakobs, Mareike; Hoffmann, Kerstin; Liesegang, Heiko; Volland, Sonja; Meinhardt, Friedhelm

    2015-03-01

    In Bacillus subtilis, natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating-among others-DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis, ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis. Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis. Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis.

  14. The Putative O-Linked N-Acetylglucosamine Transferase SPINDLY Inhibits Class I TCP Proteolysis to Promote Sensitivity to Cytokinin.

    PubMed

    Steiner, Evyatar; Livne, Sivan; Kobinson-Katz, Tammy; Tal, Lior; Pri-Tal, Oded; Mosquna, Assaf; Tarkowská, Danuše; Mueller, Bruno; Tarkowski, Petr; Weiss, David

    2016-06-01

    Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.

  15. Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island.

    PubMed

    Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

    2003-10-01

    Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.

  16. Intrinsic effects of AM4113, a putative neutral CB1 receptor selective antagonist, on open-field behaviors in rats

    PubMed Central

    Järbe, T.U.C.; LeMay, B.J.; Olzewska, T.; Vemuri, V.K.; Wood, J.T.; Makriyannis, A.

    2008-01-01

    We examined open-field effects in rats of the cannabinoid 1 receptor (CB1R) agonist WIN55,212-2 (WIN; 3 mg/kg) and its interaction with the CB1R putative neutral antagonist AM4113 (0.3 to 3 mg/kg). Separate studies examined AM4113 alone (0.3 to 5.6 mg/kg). Unlike the CB1R antagonist rimonabant, in vitro (e.g., Sink et al., 2007) AM4113 produced no change in cAMP accumulation (neutral antagonism vis-a-vie inverse agonism). Recorded behaviors were: ambulation, rearing, circling, latency, scratching, grooming, defecation, urination and vocalization/squeaking. WIN reduced ambulation and rearing; AM4113 completely (ambulation) or partially (rearing) antagonized these behaviors. WIN alone resulted in circling and an increased latency to leave the start-area; effects blocked by AM4113. AM4113 increased scratching and grooming, effects attenuated but not abolished by WIN. AM4113 alone tended to reduce ambulation and rearing and had no effect on latency or circling. AM4113 alone increased scratching and grooming. Effects on defecation, urination and vocalization were non-significant. The open-field effects of AM4113 are similar to those reported for rimonabant in rats. Yet, unlike the inverse agonists rimonabant and AM251, the putative neutral CB1R antagonist AM4113 did not produce signs of nausea in ferrets and rats (Chambers et al., 2007; Sink et al., 2007). PMID:18640150

  17. Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island

    PubMed Central

    Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H.; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

    2003-01-01

    Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria. PMID:13129949

  18. Copper and cobalt accumulation in plants: a critical assessment of the current state of knowledge.

    PubMed

    Lange, Bastien; van der Ent, Antony; Baker, Alan John Martin; Echevarria, Guillaume; Mahy, Grégory; Malaisse, François; Meerts, Pierre; Pourret, Olivier; Verbruggen, Nathalie; Faucon, Michel-Pierre

    2017-01-01

    This review synthesizes contemporary understanding of copper-cobalt (Cu-Co) tolerance and accumulation in plants. Accumulation of foliar Cu and Co to > 300 μg g(-1) is exceptionally rare globally, and known principally from the Copperbelt of Central Africa. Cobalt accumulation is also observed in a limited number of nickel (Ni) hyperaccumulator plants occurring on ultramafic soils around the world. None of the putative Cu or Co hyperaccumulator plants appears to comply with the fundamental principle of hyperaccumulation, as foliar Cu-Co accumulation is strongly dose-dependent. Abnormally high plant tissue Cu concentrations occur only when plants are exposed to high soil Cu with a low root to shoot translocation factor. Most Cu-tolerant plants are Excluders sensu Baker and therefore setting nominal threshold values for Cu hyperaccumulation is not informative. Abnormal accumulation of Co occurs under similar circumstances in the Copperbelt of Central Africa as well as sporadically in Ni hyperaccumulator plants on ultramafic soils; however, Co-tolerant plants behave physiologically as Indicators sensu Baker. Practical application of Cu-Co accumulator plants in phytomining is limited due to their dose-dependent accumulation characteristics, although for Co field trials may be warranted on highly Co-contaminated mineral wastes because of its relatively high metal value.

  19. Serial analysis of gene expression in sugarcane (Saccharum spp.) leaves revealed alternative C4 metabolism and putative antisense transcripts.

    PubMed

    Calsa, Tercilio; Figueira, Antonio

    2007-04-01

    Sugarcane (Saccharum spp.) is a highly efficient biomass and sugar producing crop. Leaf reactions have been considered as potential rate-limiting step for sucrose accumulation in sugarcane stalks. To characterize the sugarcane leaf transcriptome, field-grown mature leaves from cultivar "SP80-3280" were analyzed using Serial Analysis of Gene Expression (SAGE). From 480 sequenced clones, 9,482 valid tags were extracted, with 5,227 unique sequences, from which 3,659 (70%) matched at least a sugarcane assembled sequence (SAS) with putative function; while 872 tags (16.7%) matched SAS with unknown function; 523 (10%) matched SAS without a putative annotation; and only 173 (3.3%) did not match any sugarcane ESTs. Based on gene ontology (GO), photosystem (PS) I reaction center was identified as the most frequent gene product location, followed by the remaining sites of PS I, PS II and thylakoid complexes. For metabolic processes, photosynthesis light harvesting complexes; carbon fixation; and chlorophyll biosynthesis were the most enriched GO-terms. Considering the alternative photosynthetic C(4) cycles, tag frequencies related to phosphoenolpyruvate carboxykinase (PEPCK) and aspartate aminotransferase compared to those for NADP(+)-malic enzyme (NADP-ME) and NADP-malate dehydrogenase, suggested that PEPCK-type decarboxylation appeared to predominate over NADP-ME in mature leaves, although both may occur, opposite to currently assumed in sugarcane. From the unique tag set, 894 tags (17.1%) were assigned as potentially derived from antisense transcripts, while 73 tags (1.4%) were assigned to more than one SAS, suggesting the occurrence of alternative processing. The occurrence of antisense was validated by quantitative reverse transcription amplification. Sugarcane leaf transcriptome provided new insights for functional studies associated with sucrose synthesis and accumulation.

  20. A Putative Multiple-Demand System in the Macaque Brain

    PubMed Central

    Bell, Andrew H.; Buckley, Mark J.; Mitchell, Anna S.; Sallet, Jerome; Duncan, John

    2016-01-01

    In humans, cognitively demanding tasks of many types recruit common frontoparietal brain areas. Pervasive activation of this “multiple-demand” (MD) network suggests a core function in supporting goal-oriented behavior. A similar network might therefore be predicted in nonhuman primates that readily perform similar tasks after training. However, an MD network in nonhuman primates has not been described. Single-cell recordings from macaque frontal and parietal cortex show some similar properties to human MD fMRI responses (e.g., adaptive coding of task-relevant information). Invasive recordings, however, come from limited prespecified locations, so they do not delineate a macaque homolog of the MD system and their positioning could benefit from knowledge of where MD foci lie. Challenges of scanning behaving animals mean that few macaque fMRI studies specifically contrast levels of cognitive demand, so we sought to identify a macaque counterpart to the human MD system using fMRI connectivity in 35 rhesus macaques. Putative macaque MD regions, mapped from frontoparietal MD regions defined in humans, were found to be functionally connected under anesthesia. To further refine these regions, an iterative process was used to maximize their connectivity cross-validated across animals. Finally, whole-brain connectivity analyses identified voxels that were robustly connected to MD regions, revealing seven clusters across frontoparietal and insular cortex comparable to human MD regions and one unexpected cluster in the lateral fissure. The proposed macaque MD regions can be used to guide future electrophysiological investigation of MD neural coding and in task-based fMRI to test predictions of similar functional properties to human MD cortex. SIGNIFICANCE STATEMENT In humans, a frontoparietal “multiple-demand” (MD) brain network is recruited during a wide range of cognitively demanding tasks. Because this suggests a fundamental function, one might expect a similar

  1. The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals

    PubMed Central

    Pan, Zezheng; Sun, Mengli; Liang, Xia; Li, Jia; Zhou, Fangyue; Zhong, Zhisheng; Zheng, Yuehui

    2016-01-01

    The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years. PMID:26788065

  2. The Controversy, Challenges, and Potential Benefits of Putative Female Germline Stem Cells Research in Mammals.

    PubMed

    Pan, Zezheng; Sun, Mengli; Liang, Xia; Li, Jia; Zhou, Fangyue; Zhong, Zhisheng; Zheng, Yuehui

    2016-01-01

    The conventional view is that female mammals lose their ability to generate new germ cells after birth. However, in recent years, researchers have successfully isolated and cultured a type of germ cell from postnatal ovaries in a variety of mammalian species that have the abilities of self-proliferation and differentiation into oocytes, and this finding indicates that putative germline stem cells maybe exist in the postnatal mammalian ovaries. Herein, we review the research history and discovery of putative female germline stem cells, the concept that putative germline stem cells exist in the postnatal mammalian ovary, and the research progress, challenge, and application of putative germline stem cells in recent years.

  3. Characterization and Expression of DNA Sequences Encoding Putative Type-II Metallothioneins in the Seagrass Posidonia oceanica1

    PubMed Central

    Giordani, Tommaso; Natali, Lucia; Maserti, Bianca Elena; Taddei, Sonia; Cavallini, Andrea

    2000-01-01

    Posidonia oceanica is a marine phanerogam, largely widespread in the Mediterranean sea, representing an important food substrate for many marine organisms. A progressive reduction of P. oceanica meadows has been reported, due to anthropogenic coastal activity. Studying mechanisms by which this species responds to environmental stresses, three DNA sequences putatively encoding metallothioneins (MTs) have been isolated, by PCR. Two sequences, Pomt2a (accession no. AJ249603) and Pomt2b (accession no. AJ249602), show high similarities with genes encoding type-II MTs and are interrupted by two and one intron, respectively. The third sequence, Pomt2c (accession no. AJ249604), is supposed to be a pseudogene, originated by retrotranscription of the Pomt2b mRNA. These sequences belong to a multigene family with at least five members. Northern hybridizations indicated that MT transcripts accumulation is constitutive and seasonally regulated. MT encoding RNAs increase after rhyzome harvesting and (at a lesser extent) after 15 d of cultivation in an aquarium. As for animal MTs, transcripts accumulation is observed also after exposure to trace metals such as copper and cadmium. In the case of copper, the effect depends on concentration. Finally, taking into consideration the great interest in studying the biogeochemical cycle of mercury in the Mediterranean basin and since P. oceanica is commonly considered a bioindicator of this metal, the effect of mercury treatments on the accumulation of MT transcripts has been analyzed: in only a few experiments a small increase in the level of transcripts was recorded, suggesting that MTs are not key elements in the mercury accumulation by this species. PMID:10938373

  4. Bacterial accumulation in viscosity gradients

    NASA Astrophysics Data System (ADS)

    Waisbord, Nicolas; Guasto, Jeffrey

    2016-11-01

    Cell motility is greatly modified by fluid rheology. In particular, the physical environments in which cells function, are often characterized by gradients of viscous biopolymers, such as mucus and extracellular matrix, which impact processes ranging from reproduction to digestion to biofilm formation. To understand how spatial heterogeneity of fluid rheology affects the motility and transport of swimming cells, we use hydrogel microfluidic devices to generate viscosity gradients in a simple, polymeric, Newtonian fluid. Using video microscopy, we characterize the random walk motility patterns of model bacteria (Bacillus subtilis), showing that both wild-type ('run-and-tumble') cells and smooth-swimming mutants accumulate in the viscous region of the fluid. Through statistical analysis of individual cell trajectories and body kinematics in both homogeneous and heterogeneous viscous environments, we discriminate passive, physical effects from active sensing processes to explain the observed cell accumulation at the ensemble level.

  5. Metal accumulating plants: Medium's role

    NASA Astrophysics Data System (ADS)

    Rabier, J.; Prudent, P.; Szymanska, B.; Mevy, J.-P.

    2003-05-01

    To evaluate phytoremediation potentialities by metal accumulation in tolerant plants, trials are carried out using in vitro cultures. Organie compounds influence on metal accumulation is studied with metals supplemented media. The tested compounds on zinc and lead absorption by Brassica juncea, are chelating agents (EDTA, citric acid) and soluble organic fractions of compost. EDTA seems to enhance the transfer of lead in plant but it is the opposite in the case of zinc. Citric acid stimulates root absorption for both zinc and lead. For the aqueous extracts of compost, variable effects are obtained according to the origin of compost (green wastes and food wastes). In'all tested conditions of cultures, zinc is mainly exported towards shoot while lead is stored in root.

  6. Mechanisms of intrahepatic triglyceride accumulation

    PubMed Central

    Ress, Claudia; Kaser, Susanne

    2016-01-01

    Hepatic steatosis defined as lipid accumulation in hepatocytes is very frequently found in adults and obese adolescents in the Western World. Etiologically, obesity and associated insulin resistance or excess alcohol intake are the most frequent causes of hepatic steatosis. However, steatosis also often occurs with chronic hepatitis C virus (HCV) infection and is also found in rare but potentially life-threatening liver diseases of pregnancy. Clinical significance and outcome of hepatic triglyceride accumulation are highly dependent on etiology and histological pattern of steatosis. This review summarizes current concepts of pathophysiology of common causes of hepatic steatosis, including non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, chronic HCV infections, drug-induced forms of hepatic steatosis, and acute fatty liver of pregnancy. Regarding the pathophysiology of NAFLD, this work focuses on the close correlation between insulin resistance and hepatic triglyceride accumulation, highlighting the potential harmful effects of systemic insulin resistance on hepatic metabolism of fatty acids on the one side and the role of lipid intermediates on insulin signalling on the other side. Current studies on lipid droplet morphogenesis have identified novel candidate proteins and enzymes in NAFLD. PMID:26819531

  7. Rapid Discrimination Among Putative Mechanistic Models of Biochemical Systems

    PubMed Central

    Lomnitz, Jason G.; Savageau, Michael A.

    2016-01-01

    An overarching goal in molecular biology is to gain an understanding of the mechanistic basis underlying biochemical systems. Success is critical if we are to predict effectively the outcome of drug treatments and the development of abnormal phenotypes. However, data from most experimental studies is typically noisy and sparse. This allows multiple potential mechanisms to account for experimental observations, and often devising experiments to test each is not feasible. Here, we introduce a novel strategy that discriminates among putative models based on their repertoire of qualitatively distinct phenotypes, without relying on knowledge of specific values for rate constants and binding constants. As an illustration, we apply this strategy to two synthetic gene circuits exhibiting anomalous behaviors. Our results show that the conventional models, based on their well-characterized components, cannot account for the experimental observations. We examine a total of 40 alternative hypotheses and show that only 5 have the potential to reproduce the experimental data, and one can do so with biologically relevant parameter values. PMID:27578053

  8. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

    PubMed Central

    Kierny, Michael R.; Cunningham, Thomas D.; Bouhenni, Rachida A.; Edward, Deepak P.; Kay, Brian K.

    2015-01-01

    Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots. PMID:25902199

  9. A putative corticosteroid hormone in Pacific lamprey, Entosphenus tridentatus.

    PubMed

    Rai, Satbir; Szeitz, András; Roberts, Brent W; Christie, Quill; Didier, Wesley; Eom, Junho; Yun, Sang-Seon; Close, David A

    2015-02-01

    Great efforts have been put forth to elucidate the mechanisms of the stress response in vertebrates and demonstrate the conserved response across different vertebrate groups, ranging from similarities in the activation of the hypothalamic-pituitary-adrenal axis to the release and role of corticosteroids. There is however, still very little known about stress physiology in the Pacific lamprey (Entosphenus tridentatus), descendants of the earliest vertebrate lineage, the agnathans. In this paper we demonstrate that 11-deoxycortisol, a steroid precursor to cortisol in the steroidogenic pathway, may be a functional corticosteroid in Pacific lamprey. We identified the putative hormone in Pacific lamprey plasma by employing an array of methods such as RIA, HPLC and mass spectrometry analysis. We demonstrated that plasma levels of 11-deoxycortisol significantly increased in Pacific lamprey 0.5 and 1 h after stress exposure and that lamprey corticotropin releasing hormone injections increased circulating levels of 11-deoxycortisol, suggesting that the stress response is under the control of the HPA/I axis as it is in higher vertebrates. A comprehensive understanding of vertebrate stress physiology may help shed light on the evolution of the corticosteroid signaling system within the vertebrate lineage.

  10. Putative transmembrane transporter modulates higher-level aggression in Drosophila.

    PubMed

    Chowdhury, Budhaditya; Chan, Yick-Bun; Kravitz, Edward A

    2017-02-28

    By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

  11. Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury.

    PubMed

    Kierny, Michael R; Cunningham, Thomas D; Bouhenni, Rachida A; Edward, Deepak P; Kay, Brian K

    2015-01-01

    Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.

  12. Phytophthora infestans specific phosphorylation patterns and new putative control targets.

    PubMed

    Frades, Itziar; Andreasson, Erik

    2016-04-01

    In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation.

  13. Flamingo cadherin: a putative host receptor for Streptococcus pneumoniae.

    PubMed

    Blau, Karin; Portnoi, Maxim; Shagan, Marilou; Kaganovich, Antonina; Rom, Slava; Kafka, Daniel; Chalifa Caspi, Vered; Porgador, Angel; Givon-Lavi, Noga; Gershoni, Jonathan M; Dagan, Ron; Mizrachi Nebenzahl, Yaffa

    2007-06-15

    Streptococcus pneumoniae fructose bisphosphate aldolase (FBA) is a cell wall-localized lectin. We demonstrate that recombinant (r) FBA and anti-rFBA antibodies inhibit encapsulated and unencapsulated S. pneumoniae serotype 3 adherence to A549 type II lung carcinoma epithelial cells. A random combinatorial peptide library expressed by filamentous phage was screened with rFBA. Eleven of 30 rFBA-binding phages inhibited 90% of S. pneumoniae adhesion to A549 cells. The insert peptide sequence of 9 of these phages matched the Flamingo cadherin receptor (FCR) when aligned against the human genome. A peptide comprising a putative FBA-binding region of FCR (FCRP) inhibited 2 genetically and capsularly unrelated pairs of encapsulated and unencapsulated S. pneumoniae strains from binding to A549 cells. Moreover, FCRP inhibited S. pneumoniae nasopharyngeal and lung colonization and, possibly, pneumonia development in the mouse intranasal inoculation model system. These data indicate that FBA is an S. pneumoniae adhesin and that FCR is its host receptor.

  14. Putative impact of RNA editing on drug discovery.

    PubMed

    Decher, Niels; Netter, Michael F; Streit, Anne K

    2013-01-01

    Virtually all organisms use RNA editing as a powerful post-transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre-mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the 'one gene encodes one protein' hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the 'wrong' protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high-throughput screens, rational drug design or choosing the right model organism for target validation.

  15. General pharmacology of the putative cognition enhancer linopirdine.

    PubMed

    Flagmeyer, I; Gebert, I; van der Staay, F J

    1995-04-01

    The putative cognition enhancer linopirdine (3,3-bis(4-pyrindinylmethyl)-1-phenylindolin-2-one, CAS 105431-72-9) is supposed to act by enhancing the release of neurotransmitters, especially acetylcholine. The present study assessed the effects of a single administration of this compound on the central nervous system in eight different rat and mouse models (CNS general pharmacology). In each test performed, linopirdine was administered subcutaneously in doses of 3, 10, and 30 mg/kg. The compound did not affect traction ability and nociceptive responsiveness nor did it induce catalepsy. Linopirdine impaired motor coordination in the balance rod test. The compound showed a distinct proconvulsive action in the pentylenetetrazole threshold dose test and induced in the highest dose tested (30 mg/kg) lethal seizures in some mice. It increased the duration of hexobarbital-induced anaesthesia in mice. Rats treated with linopirdine showed ptosis, salivation, slight sedation, paw beating and slight hypothermia. These results support the hypothesis that linopirdine acts by elevating the release of different neurotransmitters such as acetylcholine and dopamine. The compound has a low potential to produce side effects at pharmacodynamic active doses.

  16. Inhalation of two putative Gulf War toxins by mice.

    PubMed

    Repine, John E; Wilson, Paul; Elkins, Nancy; Klawitter, Jelena; Christians, Uwe; Peters, Ben; Smith, Dwight M

    2016-01-01

    We employed our inhalation methodology to examine whether biomarkers of inflammation and oxidative stress would be produced in mice following inhalation of aerosols containing carbonaceous particles or the vapor of pesticides prevalent during the first Gulf War. Exposure to two putative Gulf War Illness toxins, fine airborne particles and the pesticide malathion, increased biomarkers of inflammation and oxidative stress in Friend virus B (FVB) female mice. Mice inhaling particles 24 h before had increased lung lavage and plasma Leukotriene B4 (LTB4) (a biomarker of inflammation) and PGF2α (a biomarker of oxidative stress) levels, lung lavage protein and lung lavage lactic dehydrogenase (LDH) levels. These changes were a function of particle density and exposure time. Compared to particle inhalation, mice inhaling malathion 24 h before had small increase in plasma LTB4 and PGF2α levels but no increase in lung lavage LTB4, lung lavage protein, lung lavage LDH, and lung lavage alveolar macrophage (AM) levels compared to unexposed control mice. AM from particle-exposed mice contained phagocytosed particles, while AM from malathion-exposed mice showed no abnormalities. Our results indicate that inhaling particles or malathion can alter inflammatory and oxidative biomarkers in mice and raise the possibility that these toxins may have altered inflammation and oxidative stress biomarkers in Gulf War-exposed individuals.

  17. Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration.

    PubMed

    Hansen, H H; Schmid, P C; Bittigau, P; Lastres-Becker, I; Berrendero, F; Manzanares, J; Ikonomidou, C; Schmid, H H; Fernández-Ruiz, J J; Hansen, H S

    2001-09-01

    Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.

  18. A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana*

    PubMed Central

    Qi, Yafei; Liu, Xiayan; Liang, Shuang; Wang, Rui; Li, Yuanfeng; Zhao, Jun; Shao, Jingxia; An, Lijun; Yu, Fei

    2016-01-01

    The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis. PMID:26702056

  19. The aerosols' fate in a putative ammonia ocean on Titan

    NASA Astrophysics Data System (ADS)

    Ramírez, S. I.; Coll, P.; Buch, A.; Brassé, C.; Poch, O.; Raulin, F.

    2010-04-01

    A laboratory study on the chemical transformation of Titan's aerosol analogues placed under putative surface conditions of the satellite was performed. The surface of Titan was one of the targets of the Cassini-Huygens mission and of several of the Cassini orbiter instruments, especially ISS, VIMS and Radar. The first images revealed an interesting solid surface with features that suggest aeolian, tectonic, fluvial processes and even an impact structure[1]. Since then, more detailed descriptions of dunes, channels, lakes, impact craters and cryovolcanic structures have been documented[2]. The existence of an internal liquid water ocean, containing a few percent ammonia has been proposed[2, 3]. It has also been proposed that ammonia-water mixtures can erupt from the putative subsurface ocean leading to cryovolcanism[4]. The Cassini Titan Radar Mapper obtained Synthetic Aperture Radar (SAR) images during 2004 and 2005 that revealed a highly complex geology occurring at Titan's surface[5], among which cryovolcanic features play a central role. The composition of the cryomagma is mainly proposed to be a mixture of water ice and ammonia[6, 7, 8], although ammonia has not been directly detected on Titan, but suggested by recent Cassini-VIMS observations[9]. In order to understand the role that ammonia may play on the chemical transformation of atmospheric aerosols once they reach the surface, we designed the following protocol: laboratory analogues of Titan's aerosols were synthesized from a N2:CH4 (98:2) mixture irradiated under a continuous flow regime of 845 sccm inside which, a cold plasma of 180 W was established. The synthesized analogues were recovered and partitioned in several 10.0 mg samples that were placed in 4.0 mL-volume of aqueous ammonia solutions (25.00, 12.50, 6.25 and 3.125%) at different temperatures (298, 277, 253 and 93 K) for 10 weeks. After a derivatization process performed to the aerosols' refractory phase with N

  20. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    ERIC Educational Resources Information Center

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  1. Memory accumulation mechanisms in human cortex are independent of motor intentions.

    PubMed

    Sestieri, Carlo; Tosoni, Annalisa; Mignogna, Valeria; McAvoy, Mark P; Shulman, Gordon L; Corbetta, Maurizio; Romani, Gian Luca

    2014-05-14

    Previous studies on perceptual decision-making have often emphasized a tight link between decisions and motor intentions. Human decisions, however, also depend on memories or experiences that are not closely tied to specific motor responses. Recent neuroimaging findings have suggested that, during episodic retrieval, parietal activity reflects the accumulation of evidence for memory decisions. It is currently unknown, however, whether these evidence accumulation signals are functionally linked to signals for motor intentions coded in frontoparietal regions and whether activity in the putative memory accumulator tracks the amount of evidence for only previous experience, as reflected in "old" reports, or for both old and new decisions, as reflected in the accuracy of memory judgments. Here, human participants used saccadic-eye and hand-pointing movements to report recognition judgments on pictures defined by different degrees of evidence for old or new decisions. A set of cortical regions, including the middle intraparietal sulcus, showed a monotonic variation of the fMRI BOLD signal that scaled with perceived memory strength (older > newer), compatible with an asymmetrical memory accumulator. Another set, including the hippocampus and the angular gyrus, showed a nonmonotonic response profile tracking memory accuracy (higher > lower evidence), compatible with a symmetrical accumulator. In contrast, eye and hand effector-specific regions in frontoparietal cortex tracked motor intentions but were not modulated by the amount of evidence for the effector outcome. We conclude that item recognition decisions are supported by a combination of symmetrical and asymmetrical accumulation signals largely segregated from motor intentions.

  2. Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper.

    PubMed

    Zhang, Zi-Xin; Zhao, Shu-Niu; Liu, Gao-Feng; Huang, Zu-Mei; Cao, Zhen-Mu; Cheng, Shan-Han; Lin, Shi-Sen

    2016-10-19

    The Indian pepper 'Guijiangwang' (Capsicum frutescens L.), one of the world's hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper's expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000. More than 19 million clean reads were obtained from each library, and greater than 50% of the reads were assignable to reference genes. Digital gene expression (DGE) profile analysis using Solexa sequencing was performed at five fruit developmental stages and resulted in the identification of 135 genes of known function; their expression patterns were compared to the capsaicin accumulation pattern. Ten genes of known function were identified as most likely to be involved in regulating capsaicin synthesis. Additionally, 20 new candidate genes were identified related to capsaicin synthesis. We use a combination of RNA-Seq and DGE analyses to contribute to the understanding of the biosynthetic regulatory mechanism(s) of secondary metabolites in a nonmodel plant and to identify candidate enzyme-encoding genes.

  3. Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments.

    PubMed

    Lanfranconi, Mariana P; Alvarez, Adrián F; Alvarez, Héctor M

    2015-12-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest.

  4. Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper

    PubMed Central

    Zhang, Zi-Xin; Zhao, Shu-Niu; Liu, Gao-Feng; Huang, Zu-Mei; Cao, Zhen-Mu; Cheng, Shan-Han; Lin, Shi-Sen

    2016-01-01

    The Indian pepper ‘Guijiangwang’ (Capsicum frutescens L.), one of the world’s hottest chili peppers, is rich in capsaicinoids. The accumulation of the alkaloid capsaicin and its analogs in the epidermal cells of the placenta contribute to the pungency of Capsicum fruits. To identify putative genes involved in capsaicin biosynthesis, RNA-Seq was used to analyze the pepper’s expression profiles over five developmental stages. Five cDNA libraries were constructed from the total RNA of placental tissue and sequenced using an Illumina HiSeq 2000. More than 19 million clean reads were obtained from each library, and greater than 50% of the reads were assignable to reference genes. Digital gene expression (DGE) profile analysis using Solexa sequencing was performed at five fruit developmental stages and resulted in the identification of 135 genes of known function; their expression patterns were compared to the capsaicin accumulation pattern. Ten genes of known function were identified as most likely to be involved in regulating capsaicin synthesis. Additionally, 20 new candidate genes were identified related to capsaicin synthesis. We use a combination of RNA-Seq and DGE analyses to contribute to the understanding of the biosynthetic regulatory mechanism(s) of secondary metabolites in a nonmodel plant and to identify candidate enzyme-encoding genes. PMID:27756914

  5. Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.

    PubMed Central

    Knauer, M F; Soreghan, B; Burdick, D; Kosmoski, J; Glabe, C G

    1992-01-01

    The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively

  6. Suppression subtractive hybridization identifies genes induced in response to UV-B irradiation in apple skin: isolation of a putative UDP-glucose 4-epimerase.

    PubMed

    Ban, Yusuke; Honda, Chikako; Bessho, Hideo; Pang, Xiao-Ming; Moriguchi, Takaya

    2007-01-01

    Suppression subtractive hybridization (SSH) successfully identified 11 cDNAs in apple skin with highly induced expression as a result of ultraviolet (UV)-B irradiation. Apart from three putative flavonoid biosynthetic genes, chalcone synthase (CHS; A5C), flavanone-3-hydroxylase (F3H; B5F), and flavonol synthase (FLS; D1F), five clones (A1H, A10E, B11G, D5F, and D11H) were induced by low temperature (17 degrees C) as well, which is also known to induce anthocyanin accumulation in apple skin. Moreover, four clones (A1H, A10E, B11G, and D11H), showing higher expression levels in the skin, accumulated higher anthocyanin concentrations than their counterparts. Of the four clones, only A10E, a putative UDP-glucose 4-epimerase (UGE), was deemed to play an important role in anthocyanin accumulation in apple skin based on the facts that: (i) its transcription level was higher in the deep red cultivar, 'Jonathan', than in the pale red cultivar, 'Tsugaru'; and (ii) it could reversibly catalyse UDP-glucose to UDP-galactose, and the latter molecule is a major sugar donor for cyanidin-glycoside in apple. Therefore, the full-length cDNA of A10E was isolated by rapid amplification of cDNA ends (RACE) and designated as MdUGE1. Further analysis demonstrated that UGE enzymatic activity was positively correlated with anthocyanin accumulation in apple skin. Thus, MdUGE1 isolated by SSH could play an important role in anthocyanin biosynthesis in apple skin in concert with other flavonoid biosynthetic genes.

  7. Neurodegeneration with brain iron accumulation (NBIA)

    MedlinePlus

    ... gov/ency/article/001225.htm Neurodegeneration with brain iron accumulation (NBIA) To use the sharing features on this page, please enable JavaScript. Neurodegeneration with brain iron accumulation (NBIA) are a group of rare nervous ...

  8. Racial Differences in Patterns of Wealth Accumulation

    ERIC Educational Resources Information Center

    Gittleman, Maury; Wolff, Edward N.

    2004-01-01

    The race differences in patterns of asset accumulations were examined using PSD data for 1984, 1989 and 1994. The results indicate that inheritances led to wealth accumulations among whites as compared to the African Americans.

  9. Mind Operational Semantics and Brain Operational Architectonics: A Putative Correspondence

    PubMed Central

    Benedetti, Giulio; Marchetti, Giorgio; Fingelkurts, Alexander A; Fingelkurts, Andrew A

    2010-01-01

    ) of different complexity within OA’s theory: EOMC could correspond to simple OMs, correlators to complex OMs and the correlational network to a set of simple and complex OMs. Finally, a set of experiments is proposed to verify the putative correspondence between OS and OA and prove the existence of an integrated continuum between brain and mind. PMID:21113277

  10. 47 CFR 32.3100 - Accumulated depreciation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 2 2010-10-01 2010-10-01 false Accumulated depreciation. 32.3100 Section 32... Accumulated depreciation. (a) This account shall include the accumulated depreciation associated with the... with depreciation amounts concurrently charged to Account 6561, Depreciation...

  11. Dbp7p, a putative ATP-dependent RNA helicase from Saccharomyces cerevisiae, is required for 60S ribosomal subunit assembly.

    PubMed Central

    Daugeron, M C; Linder, P

    1998-01-01

    Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit. PMID:9582098

  12. Ectoine accumulation in Brevibacterium epidermis.

    PubMed

    Onraedt, Annelies; De Muynck, Cassandra; Walcarius, Bart; Soetaert, Wim; Vandamme, Erick

    2004-10-01

    As a halotolerant bacterial species, Brevibacterium epidermis DSM 20659 can grow at relatively high salinity, tolerating up to 2 M NaCl. It synthesizes ectoine and the intracellular content increases with the medium salinity, with a maximum of 0.14 g ectoine/g CDW at 1 M NaCl. Sugar-stressed cells do not synthesize ectoine. Ectoine synthesis is also affected by the presence of external osmolytes. Added betaine is taken up and completely replaced ectoine, while L-proline is only temporarily accumulated after which ectoine is synthesized. The strain can metabolize ectoine; L-glutamate is a better carbon source for ectoine synthesis than L-aspartate.

  13. Characterization of a putative grapevine Zn transporter, VvZIP3, suggests its involvement in early reproductive development in Vitis vinifera L

    PubMed Central

    2012-01-01

    Background Zinc (Zn) deficiency is one of the most widespread mineral nutritional problems that affect normal development in plants. Because Zn cannot passively diffuse across cell membranes, it must be transported into intracellular compartments for all biological processes where Zn is required. Several members of the Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) gene family have been characterized in plants, and have shown to be involved in metal uptake and transport. This study describes the first putative Zn transporter in grapevine. Unravelling its function may explain an important symptom of Zn deficiency in grapevines, which is the production of clusters with fewer and usually smaller berries than normal. Results We identified and characterized a putative Zn transporter from berries of Vitis vinifera L., named VvZIP3. Compared to other members of the ZIP family identified in the Vitis vinifera L. genome, VvZIP3 is mainly expressed in reproductive tissue - specifically in developing flowers - which correlates with the high Zn accumulation in these organs. Contrary to this, the low expression of VvZIP3 in parthenocarpic berries shows a relationship with the lower Zn accumulation in this tissue than in normal seeded berries where its expression is induced by Zn. The predicted protein sequence indicates strong similarity with several members of the ZIP family from Arabidopsis thaliana and other species. Moreover, VvZIP3 complemented the growth defect of a yeast Zn-uptake mutant, ZHY3, and is localized in the plasma membrane of plant cells, suggesting that VvZIP3 has the function of a Zn uptake transporter. Conclusions Our results suggest that VvZIP3 encodes a putative plasma membrane Zn transporter protein member of the ZIP gene family that might play a role in Zn uptake and distribution during the early reproductive development in Vitis vinifera L., indicating that the availability of this micronutrient may be relevant for

  14. A novel variant of the putative demethylase gene, s-JMJD1C, is a coactivator of the AR.

    PubMed

    Wolf, Siegmund S; Patchev, Vladimir K; Obendorf, Maik

    2007-04-01

    Evidence is accumulating in support of the view that tissue-specific effects of steroid hormones depend on the recruitment of nuclear receptor comodulator proteins. The latter interact directly with the hormone receptors and modify their transcriptional effects on specific target genes. The mechanisms of comodulator influence on nuclear receptor-controlled gene transcription is only partially understood. Here, we describe the discovery of a new AR coactivator which belongs to the JmjC containing enzyme family as a novel variant of JMJD1C (jumonji domain-containing 1C). By using a fragment of the human AR (aa 325-919) as bait in a yeast two-hybrid screen, a region of the human JMJD1C gene was identified as interacting with AR. A novel splice variant s-JMJD1C was amplified by RACE, and the binding to AR was analysed by GST-pull-down and mammalian one-hybrid experiments. As a nuclear-localized protein, the s-JMJD1C gene is expressed in a variety of human tissues. In the brain, this protein is present in several, but not confined to, AR-expressing neuronal populations and its abundance varies with the hormonal status in a region-specific fashion. Interestingly, the expression of s-JMJD1C is reduced in breast cancer tumors and significantly higher in normal breast tissues indicating a putative role in tumor suppression. As s-JMJD1C has putative demethylase activity, removal of methylation seems to be important for nuclear receptor-based gene regulation.

  15. Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus

    PubMed Central

    Houldcroft, Charlotte J.; Bryant, Josephine M.; Depledge, Daniel P.; Margetts, Ben K.; Simmonds, Jacob; Nicolaou, Stephanos; Tutill, Helena J.; Williams, Rachel; Worth, Austen J. J.; Marks, Stephen D.; Veys, Paul; Whittaker, Elizabeth; Breuer, Judith

    2016-01-01

    Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1–27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome. PMID:27667983

  16. Guidelines for Waste Accumulation Areas (WAAs)

    SciTech Connect

    Not Available

    1991-07-01

    The purpose of this document is to set conditions for establishing and maintaining areas for the accumulation of hazardous waste at LBL. Areas designed for accumulation of these wastes in quantities greater than 100 kg (220 lb) per month of solid waste or 55 gallons per month of liquid waste are called Waste Accumulation Areas (WAAs). Areas designed for accumulation of wastes in smaller amounts are called Satellite Accumulation Areas (SAAs). This document provides guidelines for employee and organizational responsibilities for WAAs; constructing a WAA; storing waste in a WAA; operating and maintaining a WAA, and responding to spills in a WAA. 4 figs.

  17. Sequential biases in accumulating evidence

    PubMed Central

    Huggins, Richard; Dogo, Samson Henry

    2015-01-01

    Whilst it is common in clinical trials to use the results of tests at one phase to decide whether to continue to the next phase and to subsequently design the next phase, we show that this can lead to biased results in evidence synthesis. Two new kinds of bias associated with accumulating evidence, termed ‘sequential decision bias’ and ‘sequential design bias’, are identified. Both kinds of bias are the result of making decisions on the usefulness of a new study, or its design, based on the previous studies. Sequential decision bias is determined by the correlation between the value of the current estimated effect and the probability of conducting an additional study. Sequential design bias arises from using the estimated value instead of the clinically relevant value of an effect in sample size calculations. We considered both the fixed‐effect and the random‐effects models of meta‐analysis and demonstrated analytically and by simulations that in both settings the problems due to sequential biases are apparent. According to our simulations, the sequential biases increase with increased heterogeneity. Minimisation of sequential biases arises as a new and important research area necessary for successful evidence‐based approaches to the development of science. © 2015 The Authors. Research Synthesis Methods Published by John Wiley & Sons Ltd. PMID:26626562

  18. Natural radionuclide accumulation by raindrops

    NASA Astrophysics Data System (ADS)

    Gusev, Anatoly; Martin, Inacio; Shkevov, Rumen; Alves, Mauro

    2016-07-01

    The laboratory of environmental radiation of ITA (São José dos Campos, 23°11'11″S, 45°52'43″W, 650 MAMSL) performs simultaneous monitoring of a natural radiation background and meteorological parameters. A time resolution of up to 1 minute allows a detailed comparison of changes in meteorological parameters with those of a concentration of ambient radon progenies in the atmosphere. Results of a study of variation of a fallout of radon progenies ^{214}Pb and ^{214}Bi concomitanting rainfalls are present. The radionuclide fallout rate is reconstructed from the observed gamma rate through a simulation of the first kind Volterra integral equation with difference kernel, determined by ratio of precipitating rates of 214Pb and 214Bi and their decay half times. An original straightforward step-by-step procedure was used for the numerical solution of the equation. The radionuclide concentration in the rainwater is calculated as a ratio of the reconstructed fallout to the measured rainfall. It was observed that the radionuclide fallout rate increases as the rainfall one in approximately power 0.6, i.e. the same as the mean raindrop volume. The concentration thereafter decreases as the rainfall rate in power 0.4. A numerical simulation of the process of accumulation of the radionuclides during diffusion and coalescence drop growth and aerosol scavenging during a passage from a cloud to the ground was performed. The results of the simulations agree with the experimental data.

  19. Response times from ensembles of accumulators

    PubMed Central

    Zandbelt, Bram; Purcell, Braden A.; Palmeri, Thomas J.; Logan, Gordon D.; Schall, Jeffrey D.

    2014-01-01

    Decision-making is explained by psychologists through stochastic accumulator models and by neurophysiologists through the activity of neurons believed to instantiate these models. We investigated an overlooked scaling problem: How does a response time (RT) that can be explained by a single model accumulator arise from numerous, redundant accumulator neurons, each of which individually appears to explain the variability of RT? We explored this scaling problem by developing a unique ensemble model of RT, called e pluribus unum, which embodies the well-known dictum “out of many, one.” We used the e pluribus unum model to analyze the RTs produced by ensembles of redundant, idiosyncratic stochastic accumulators under various termination mechanisms and accumulation rate correlations in computer simulations of ensembles of varying size. We found that predicted RT distributions are largely invariant to ensemble size if the accumulators share at least modestly correlated accumulation rates and RT is not governed by the most extreme accumulators. Under these regimes the termination times of individual accumulators was predictive of ensemble RT. We also found that the threshold measured on individual accumulators, corresponding to the firing rate of neurons measured at RT, can be invariant with RT but is equivalent to the specified model threshold only when the rate correlation is very high. PMID:24550315

  20. Accumulation boundaries: codimension-two accumulation of accumulations in phase diagrams of semiconductor lasers, electric circuits, atmospheric and chemical oscillators.

    PubMed

    Bonatto, Cristian; Gallas, Jason Alfredo Carlson

    2008-02-28

    We report high-resolution phase diagrams for several familiar dynamical systems described by sets of ordinary differential equations: semiconductor lasers; electric circuits; Lorenz-84 low-order atmospheric circulation model; and Rössler and chemical oscillators. All these systems contain chaotic phases with highly complicated and interesting accumulation boundaries, curves where networks of stable islands of regular oscillations with ever-increasing periodicities accumulate systematically. The experimental exploration of such codimension-two boundaries characterized by the presence of infinite accumulation of accumulations is feasible with existing technology for some of these systems.

  1. Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    PubMed Central

    Young, John D.; Young, Lena; Wu, Cheng-Yeu; Young, Andrew

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures

  2. Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis.

    PubMed

    Young, John D; Martel, Jan; Young, Lena; Wu, Cheng-Yeu; Young, Andrew; Young, David

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50-500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described

  3. Structural connectivity patterns associated with the putative visual word form area and children's reading ability.

    PubMed

    Fan, Qiuyun; Anderson, Adam W; Davis, Nicole; Cutting, Laurie E

    2014-10-24

    With the advent of neuroimaging techniques, especially functional MRI (fMRI), studies have mapped brain regions that are associated with good and poor reading, most centrally a region within the left occipito-temporal/fusiform region (L-OT/F) often referred to as the visual word form area (VWFA). Despite an abundance of fMRI studies of the putative VWFA, research about its structural connectivity has just started. Provided that the putative VWFA may be connected to distributed regions in the brain, it remains unclear how this network is engaged in constituting a well-tuned reading circuitry in the brain. Here we used diffusion MRI to study the structural connectivity patterns of the putative VWFA and surrounding areas within the L-OT/F in children with typically developing (TD) reading ability and with word recognition deficits (WRD; sometimes referred to as dyslexia). We found that L-OT/F connectivity varied along a posterior-anterior gradient, with specific structural connectivity patterns related to reading ability in the ROIs centered upon the putative VWFA. Findings suggest that the architecture of the putative VWFA connectivity is fundamentally different between TD and WRD, with TD showing greater connectivity to linguistic regions than WRD, and WRD showing greater connectivity to visual and parahippocampal regions than TD. Findings thus reveal clear structural abnormalities underlying the functional abnormalities in the putative VWFA in WRD.

  4. Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum

    PubMed Central

    2012-01-01

    Background Phaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination. Results The specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases). Conclusions The results indicate that P. tricornutum continued

  5. Putative crater-floor pingos, paleolakes and periglacial landscapes in north Utopia Planitia, Mars.

    NASA Astrophysics Data System (ADS)

    Soare, R. J.; Conway, S. J.; Dohm, J. M.

    2012-04-01

    are formed in these craters by the accumulation of ice precipitated from regional polar-winds laden with water vapour. Under slightly different obliquity solution these polar winds might have precipitated ice further to the south in those craters where the putative pingos are observed. Subsequently, were temperatures to have migrated close to 0°C the accumulated crater-ice could have thawed, forming endogenic paleolakes. El Maarry et al. (2010) suggest that the fracture geometry of some polygons on the floors of impact craters in northern Utopia Planitia points to an origin by desiccation, the end-stage of paleo-lake evolution. Although we explore two non-periglacial mound-formation hypotheses, the periglacial hypothesis is shown to be more robust, encompassing geomorphological, stratrigraphical and climatological observations, and is less subject to inconsistencies than the alternatives. Conway S.J., et al. 2011. Climate-driven deposition of water ice and the formation of mounds in craters in Mars' North Polar Region. Icarus (in review) El Maarry, M.R., et al. 2010. Crater Floor Polygons: Desiccation Patterns of Ancient Lakes on Mars? Journal of Geophysical Research 115, doi:10.1029/2010JE003609. Soare R.J., et al. 2005. Possible pingos and a periglacial landscape in northwest Utopia Planitia Icarus 174, 373-382.

  6. Exposure to chlorpyrifos increases neutral lipid accumulation with accompanying increased de novo lipogenesis and decreased triglyceride secretion in McArdle-RH7777 hepatoma cells.

    PubMed

    Howell, George Eli; Mulligan, Charlee; Young, Darian; Kondakala, Sandeep

    2016-04-01

    Hepatic steatosis is associated with hepatic insulin resistance as well as hypertriglyceridemia. Recent studies have determined exposure to organophosphate (OP) pesticides can cause dyslipidemia and hepatic steatosis. However, the mechanisms through which OPs induced hepatic steatosis are not completely understood. Therefore, the current study was designed to determine if direct exposure to an OP insecticide, chlorpyrifos (CPS), could promote hepatic steatosis and identify putative mechanisms of CPS-induced steatosis. To determine if CPS exposure increased intracellular lipid accumulation, McA-RH7777 cells were incubated with CPS for 48 h then lipid accumulation was determined by Oil Red O staining. Exposure to CPS significantly increased neutral lipid accumulation in a concentration-dependent manner. This increase in Oil Red O staining appears to be due to increased intracellular triglyceride accumulation. In addition to increasing neutral lipid accumulation under normal growth conditions, exposure to CPS increased free fatty acid-induced intracellular neutral lipid accumulation. CPS induced increases in intracellular neutral lipid/triglyceride accumulation appear to be due to increased extracellular free fatty acid accumulation, increased de novo lipogenesis, and decreased fatty acidinduced triglyceride secretion. In summary, the present studies indicate exposure to CPS can have a direct effect on the hepatocyte to promote hepatic steatosis by increasing intracellular lipid/triglyceride accumulation through increased extracellular free fatty acid accumulation, increased hepatic de novo lipogenesis, and decreased triglyceride efflux.

  7. The putative guanine nucleotide exchange factor RicA mediates upstream signaling for growth and development in Aspergillus.

    PubMed

    Kwon, Nak-Jung; Park, Hee-Soo; Jung, Seunho; Kim, Sun Chang; Yu, Jae-Hyuk

    2012-11-01

    Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.

  8. 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.

  9. 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

  10. Isolation, expression and functional analysis of a putative RNA-dependent RNA polymerase gene from maize (Zea mays L.).

    PubMed

    He, Junguang; Dong, Zhigang; Jia, Zhiwei; Wang, Jianhua; Wang, Guoying

    2010-02-01

    RNA-dependent RNA polymerases (RdRPs) in plants have been reported to be involved in post-transcriptional gene silencing (PTGS) and antiviral defense. In this report, an RdRP gene from maize (ZmRdRP1) was obtained by rapid amplification of cDNA ends (RACE) and RT-PCR. The mRNA of ZmRdRP1 was composed of 3785 nucleotides, including a 167 nt 5' untranslated region (UTR), a 291 nt 3'UTR and a 3327 nt open reading frame (ORF), which encodes a putative protein of 1108 amino acids with an estimated molecular mass of 126.9 kDa and a predicated isoelectric point (pI) of 8.37. Real-time quantitative RT-PCR analysis showed that ZmRdRP1 was elicited by salicylic acid (SA) treatment, methyl jasmonate (MeJA) treatment and sugarcane mosaic virus (SCMV) infection. We silenced ZmRdRP1 by constitutively expressing an inverted-repeat fragment of ZmRdRP1 (ir-RdRP1) in transgenic maize plants. Further studies revealed that the ir-RdRP1 transgenic plants were more susceptible to SCMV infection than wild type plants. Virus-infected transgenic maize plants developed more serious disease symptoms and accumulated more virus than wild type plants. These findings suggested that ZmRdRP1 was involved in antiviral defense in maize.

  11. The choC gene encoding a putative phospholipid methyltransferase is essential for growth and development in Aspergillus nidulans.

    PubMed

    Tao, Li; Gao, Na; Chen, Sanfeng; Yu, Jae-Hyuk

    2010-06-01

    Phosphatidylcholines (PCs) are a class of major cell membrane phospholipids that participate in many physiological processes. Three genes, choA, choB and choC, have been proposed to function in the endogenous biosynthesis of PC in Aspergillus nidulans. In this study, we characterize the choC gene encoding a putative highly conserved phospholipid methyltransferase. The previously reported choC3 mutant allele results from a mutation leading to the E177K amino acid substitution. The transcript of choC accumulates at high levels during vegetative growth and early asexual developmental phases. The deletion of choC causes severe impairment of vegetative growth, swelling of hyphal tips and the lack of both asexual and sexual development, suggesting the requirement of ChoC and PC in growth and development. Noticeably, supplementation of the mutant with the penultimate precursor of PC N, N-dimethylaminoethanol leads to full recovery of vegetative growth, but incomplete progression of asexual and sexual development, implying differential roles of PC and its intermediates in fungal growth and development. Importantly, while the choC deletion mutant shows reduced vegetative growth and precocious cell death until day 4, it regains hyphal proliferation and cell viability from day 5, indicating the presence of an alternative route for cellular membrane function in A. nidulans.

  12. Rice-Infecting Pseudomonas Genomes Are Highly Accessorized and Harbor Multiple Putative Virulence Mechanisms to Cause Sheath Brown Rot

    PubMed Central

    Quibod, Ian Lorenzo; Grande, Genelou; Oreiro, Eula Gems; Borja, Frances Nikki; Dossa, Gerbert Sylvestre; Mauleon, Ramil; Cruz, Casiana Vera; Oliva, Ricardo

    2015-01-01

    Sheath rot complex and seed discoloration in rice involve a number of pathogenic bacteria that cannot be associated with distinctive symptoms. These pathogens can easily travel on asymptomatic seeds and therefore represent a threat to rice cropping systems. Among the rice-infecting Pseudomonas, P. fuscovaginae has been associated with sheath brown rot disease in several rice growing areas around the world. The appearance of a similar Pseudomonas population, which here we named P. fuscovaginae-like, represents a perfect opportunity to understand common genomic features that can explain the infection mechanism in rice. We showed that the novel population is indeed closely related to P. fuscovaginae. A comparative genomics approach on eight rice-infecting Pseudomonas revealed heterogeneous genomes and a high number of strain-specific genes. The genomes of P. fuscovaginae-like harbor four secretion systems (Type I, II, III, and VI) and other important pathogenicity machinery that could probably facilitate rice colonization. We identified 123 core secreted proteins, most of which have strong signatures of positive selection suggesting functional adaptation. Transcript accumulation of putative pathogenicity-related genes during rice colonization revealed a concerted virulence mechanism. The study suggests that rice-infecting Pseudomonas causing sheath brown rot are intrinsically diverse and maintain a variable set of metabolic capabilities as a potential strategy to occupy a range of environments. PMID:26422147

  13. Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

    PubMed Central

    Longstaff, M; Brigneti, G; Boccard, F; Chapman, S; Baulcombe, D

    1993-01-01

    Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells. Images PMID:8440232

  14. Rice-Infecting Pseudomonas Genomes Are Highly Accessorized and Harbor Multiple Putative Virulence Mechanisms to Cause Sheath Brown Rot.

    PubMed

    Quibod, Ian Lorenzo; Grande, Genelou; Oreiro, Eula Gems; Borja, Frances Nikki; Dossa, Gerbert Sylvestre; Mauleon, Ramil; Cruz, Casiana Vera; Oliva, Ricardo

    2015-01-01

    Sheath rot complex and seed discoloration in rice involve a number of pathogenic bacteria that cannot be associated with distinctive symptoms. These pathogens can easily travel on asymptomatic seeds and therefore represent a threat to rice cropping systems. Among the rice-infecting Pseudomonas, P. fuscovaginae has been associated with sheath brown rot disease in several rice growing areas around the world. The appearance of a similar Pseudomonas population, which here we named P. fuscovaginae-like, represents a perfect opportunity to understand common genomic features that can explain the infection mechanism in rice. We showed that the novel population is indeed closely related to P. fuscovaginae. A comparative genomics approach on eight rice-infecting Pseudomonas revealed heterogeneous genomes and a high number of strain-specific genes. The genomes of P. fuscovaginae-like harbor four secretion systems (Type I, II, III, and VI) and other important pathogenicity machinery that could probably facilitate rice colonization. We identified 123 core secreted proteins, most of which have strong signatures of positive selection suggesting functional adaptation. Transcript accumulation of putative pathogenicity-related genes during rice colonization revealed a concerted virulence mechanism. The study suggests that rice-infecting Pseudomonas causing sheath brown rot are intrinsically diverse and maintain a variable set of metabolic capabilities as a potential strategy to occupy a range of environments.

  15. Analysis of the expression of putative heat-stress related genes in relation to thermotolerance of cork oak.

    PubMed

    Correia, Barbara; Rodriguez, José Luis; Valledor, Luis; Almeida, Tânia; Santos, Conceição; Cañal, Maria Jesús; Pinto, Glória

    2014-03-15

    Cork oak (Quercus suber L.) is a research priority in the Mediterranean area and because of cork oaks' distribution these stands are experiencing daily stress. Based on projections of intensifying climate change and considering the key role of exploring the recovery abilities, cork oak seedlings were subjected to a cumulative temperature increase from 25°C to 55°C and subsequent recovery. CO2 assimilation rate, chlorophyll fluorescence, anthocyanins, proline and lipid peroxidation were used to evaluate plant performance, while the relative abundance of seven genes encoding for proteins of cork oak with a putative role in thermal/stress regulation (POX1, POX2, HSP10.4, HSP17a.22, CHS, MTL and RBC) was analyzed by qPCR (quantitative Polymerase Chain Reaction). A temperature change to 35°C showed abundance alterations in the tested genes; at 45°C, the molecular changes were associated with an antioxidant response, possibly modulated by anthocyanins. At 55°C, HSP17a.22, MTL and proline accumulation were evident. After recovery, physiological balance was restored, whereas POX1, HSP10.4 and MTL abundances were suggested to be involved in increased thermotolerance. The data presented here are expected to pinpoint some pathways changes occurring during such stress and further recovery in this particular Mediterranean species.

  16. 46 CFR 58.30-25 - Accumulators.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... RELATED SYSTEMS Fluid Power and Control Systems § 58.30-25 Accumulators. (a) An accumulator is an unfired pressure vessel in which energy is stored under high pressure in the form of a gas or a gas and hydraulic... result in contamination of the hydraulic fluid and loss of gas through absorption. (c) Each...

  17. 46 CFR 58.30-25 - Accumulators.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... RELATED SYSTEMS Fluid Power and Control Systems § 58.30-25 Accumulators. (a) An accumulator is an unfired pressure vessel in which energy is stored under high pressure in the form of a gas or a gas and hydraulic... result in contamination of the hydraulic fluid and loss of gas through absorption. (c) Each...

  18. Proline accumulation in plants: a review.

    PubMed

    Verbruggen, Nathalie; Hermans, Christian

    2008-11-01

    Proline (Pro) accumulation is a common physiological response in many plants in response to a wide range of biotic and abiotic stresses. Controversy has surrounded the possible role(s) of proline accumulation. In this review, knowledge on the regulation of Pro metabolism during development and stress, results of genetic manipulation of Pro metabolism and current debate on Pro toxicity in plants are presented.

  19. 40 CFR 262.34 - Accumulation time.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.34 Accumulation time. (a) Except as provided in paragraphs (d), (e), and (f) of this section, a generator may accumulate...: (1) The waste is placed: (i) In containers and the generator complies with the...

  20. 40 CFR 262.34 - Accumulation time.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.34 Accumulation time. (a) Except as provided in paragraphs (d), (e), and (f) of this section, a generator may accumulate...: (1) The waste is placed: (i) In containers and the generator complies with the...

  1. 40 CFR 262.34 - Accumulation time.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.34 Accumulation time. (a) Except as provided in paragraphs (d), (e), and (f) of this section, a generator may accumulate...: (1) The waste is placed: (i) In containers and the generator complies with the...

  2. 40 CFR 262.34 - Accumulation time.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.34 Accumulation time. (a) Except as provided in paragraphs (d), (e), and (f) of this section, a generator may accumulate...: (1) The waste is placed: (i) In containers and the generator complies with the...

  3. 40 CFR 262.34 - Accumulation time.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) STANDARDS APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.34 Accumulation time. (a) Except as provided in paragraphs (d), (e), and (f) of this section, a generator may accumulate...: (1) The waste is placed: (i) In containers and the generator complies with the...

  4. 19 CFR 10.2017 - Accumulation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 19 Customs Duties 1 2014-04-01 2014-04-01 false Accumulation. 10.2017 Section 10.2017 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION, DEPARTMENT OF HOMELAND SECURITY; DEPARTMENT OF THE TREASURY... Rules of Origin § 10.2017 Accumulation. (a) Originating materials from the territory of a Party that...

  5. Dissociation between iron accumulation and ferritin upregulation in the aged substantia nigra: attenuation by dietary restriction

    PubMed Central

    Walker, Thomas; Michaelides, Christos; Ekonomou, Antigoni; Geraki, Kalotina; Parkes, Harold G; Suessmilch, Maria; Herlihy, Amy H; Crum, William R; So, Po-Wah

    2016-01-01

    Despite regulation, brain iron increases with aging and may enhance aging processes including neuroinflammation. Increases in magnetic resonance imaging transverse relaxation rates, R2 and R2*, in the brain have been observed during aging. We show R2 and R2* correlate well with iron content via direct correlation to semi-quantitative synchrotron-based X-ray fluorescence iron mapping, with age-associated R2 and R2* increases reflecting iron accumulation. Iron accumulation was concomitant with increased ferritin immunoreactivity in basal ganglia regions except in the substantia nigra (SN). The unexpected dissociation of iron accumulation from ferritin-upregulation in the SN suggests iron dyshomeostasis in the SN. Occurring alongside microgliosis and astrogliosis, iron dyshomeotasis may contribute to the particular vulnerability of the SN. Dietary restriction (DR) has long been touted to ameliorate brain aging and we show DR attenuated agerelated in vivo R2 increases in the SN over ages 7 – 19 months, concomitant with normal iron-induction of ferritin expression and decreased microgliosis. Iron is known to induce microgliosis and conversely, microgliosis can induce iron accumulation, which of these may be the initial pathological aging event warrants further investigation. We suggest iron chelation therapies and anti-inflammatory treatments may be putative ‘antibrain aging’ therapies and combining these strategies may be synergistic. PMID:27743512

  6. Maps of context-dependent putative regulatory regions and genomic signal interactions.

    PubMed

    Diamanti, Klev; Umer, Husen M; Kruczyk, Marcin; Dąbrowski, Michał J; Cavalli, Marco; Wadelius, Claes; Komorowski, Jan

    2016-11-02

    Gene transcription is regulated mainly by transcription factors (TFs). ENCODE and Roadmap Epigenomics provide global binding profiles of TFs, which can be used to identify regulatory regions. To this end we implemented a method to systematically construct cell-type and species-specific maps of regulatory regions and TF-TF interactions. We illustrated the approach by developing maps for five human cell-lines and two other species. We detected ∼144k putative regulatory regions among the human cell-lines, with the majority of them being ∼300 bp. We found ∼20k putative regulatory elements in the ENCODE heterochromatic domains suggesting a large regulatory potential in the regions presumed transcriptionally silent. Among the most significant TF interactions identified in the heterochromatic regions were CTCF and the cohesin complex, which is in agreement with previous reports. Finally, we investigated the enrichment of the obtained putative regulatory regions in the 3D chromatin domains. More than 90% of the regions were discovered in the 3D contacting domains. We found a significant enrichment of GWAS SNPs in the putative regulatory regions. These significant enrichments provide evidence that the regulatory regions play a crucial role in the genomic structural stability. Additionally, we generated maps of putative regulatory regions for prostate and colorectal cancer human cell-lines.

  7. Genetic Control and Evolution of Anthocyanin Methylation1[W

    PubMed Central

    Provenzano, Sofia; Spelt, Cornelis; Hosokawa, Satoko; Nakamura, Noriko; Brugliera, Filippa; Demelis, Linda; Geerke, Daan P.; Schubert, Andrea; Tanaka, Yoshikazu; Quattrocchio, Francesca; Koes, Ronald

    2014-01-01

    Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins’ structural diversity. PMID:24830298

  8. Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

    PubMed Central

    Sahin-Tóth, M.; Kaback, H. R.

    1993-01-01

    Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+. PMID

  9. Eliciting maltreated and nonmaltreated children's transgression disclosures: narrative practice rapport building and a putative confession.

    PubMed

    Lyon, Thomas D; Wandrey, Lindsay; Ahern, Elizabeth; Licht, Robyn; Sim, Megan P Y; Quas, Jodi A

    2014-01-01

    This study tested the effects of narrative practice rapport building (asking open-ended questions about a neutral event) and a putative confession (telling the child an adult "told me everything that happened and he wants you to tell the truth") on 4- to 9-year-old maltreated and nonmaltreated children's reports of an interaction with a stranger who asked them to keep toy breakage a secret (n = 264). Only one third of children who received no interview manipulations disclosed breakage; in response to a putative confession, one half disclosed. Narrative practice rapport building did not affect the likelihood of disclosure. Maltreated children and nonmaltreated children responded similarly to the manipulations. Neither narrative practice rapport building nor a putative confession increased false reports.

  10. Molecular diagnosis of putative Stargardt disease by capture next generation sequencing.

    PubMed

    Zhang, Xiao; Ge, Xianglian; Shi, Wei; Huang, Ping; Min, Qingjie; Li, Minghan; Yu, Xinping; Wu, Yaming; Zhao, Guangyu; Tong, Yi; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-01-01

    Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.

  11. Complete genome sequences of a putative new alphapartitivirus detected in Rosa spp.

    PubMed

    Phelan, James; James, Delano

    2016-09-01

    A putative new alphapartitivirus was detected by next-generation sequencing (NGS) in Rosa spp. and identified as rose partitivirus isolate Phyllis Bide (RoPV-PB). The virus is bipartite with a dsRNA1 fragment (1937 bp) encoding a putative RdRp and a dsRNA2 fragment (1811 bp) encoding the putative CP subunit of the virus. dsRNA1 of RoPV-BP is closely related to Vicia faba partitivirus 1, with identities of 67 % and 72 % for the nucleotide (nt) and deduced amino acid (aa) sequences, respectively. In NGS analysis of RoPV-BP, coverage was uneven across both dsRNA fragments, with GC/AT content appearing to be a major determinant of depth of coverage.

  12. A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis

    PubMed Central

    Matsumura, Yoko; Ohbayashi, Iwai; Takahashi, Hiro; Kojima, Shoko; Ishibashi, Nanako; Keta, Sumie; Nakagawa, Ayami; Hayashi, Rika; Saéz-Vásquez, Julio; Echeverria, Manuel; Sugiyama, Munetaka; Nakamura, Kenzo; Machida, Chiyoko

    2016-01-01

    ABSTRACT Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1) and AS2 (AS1-AS2) is critical to repress abaxial (ventral) genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal) development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1) synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP) that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development. PMID:27334696

  13. Geochemistry Model Validation Report: External Accumulation Model

    SciTech Connect

    K. Zarrabi

    2001-09-27

    The purpose of this Analysis and Modeling Report (AMR) is to validate the External Accumulation Model that predicts accumulation of fissile materials in fractures and lithophysae in the rock beneath a degrading waste package (WP) in the potential monitored geologic repository at Yucca Mountain. (Lithophysae are voids in the rock having concentric shells of finely crystalline alkali feldspar, quartz, and other materials that were formed due to entrapped gas that later escaped, DOE 1998, p. A-25.) The intended use of this model is to estimate the quantities of external accumulation of fissile material for use in external criticality risk assessments for different types of degrading WPs: U.S. Department of Energy (DOE) Spent Nuclear Fuel (SNF) codisposed with High Level Waste (HLW) glass, commercial SNF, and Immobilized Plutonium Ceramic (Pu-ceramic) codisposed with HLW glass. The scope of the model validation is to (1) describe the model and the parameters used to develop the model, (2) provide rationale for selection of the parameters by comparisons with measured values, and (3) demonstrate that the parameters chosen are the most conservative selection for external criticality risk calculations. To demonstrate the applicability of the model, a Pu-ceramic WP is used as an example. The model begins with a source term from separately documented EQ6 calculations; where the source term is defined as the composition versus time of the water flowing out of a breached waste package (WP). Next, PHREEQC, is used to simulate the transport and interaction of the source term with the resident water and fractured tuff below the repository. In these simulations the primary mechanism for accumulation is mixing of the high pH, actinide-laden source term with resident water; thus lowering the pH values sufficiently for fissile minerals to become insoluble and precipitate. In the final section of the model, the outputs from PHREEQC, are processed to produce mass of accumulation

  14. Microbial accumulation of uranium, radium, and cesium

    SciTech Connect

    Strandberg, G.W.; Shumate, S.E. II; Parrott, J.R. Jr.; North, S.E.

    1981-05-01

    Diverse microbial species varied considerably in their ability to accumulate uranium, cesium, and radium. Mechanistic differences in uranium uptake by Saccharomyces cerevisiae and Pseudomonas aeruginosa were indicated. S. serevisiae exhibited a slow (hours) surface accumulation of uranium which was subject to environmental factors, while P. aeruginosa accumulated uranium rapidly (minutes) as dense intracellular deposits and did not appear to be affected by environmental parameters. Metabolism was not required for uranium uptake by either organism. Cesium and radium were concentrated to a considerably lesser extent than uranium by the several species tested.

  15. Coexistence of Several Putative Neurotransmitters in Single Identified Neurosn of Aplysia

    PubMed Central

    Brownstein, Michael J.; Saavedra, Juan M.; Axelrod, Julius; Zeman, Gary H.; Carpenter, David O.

    1974-01-01

    By sensitive enzymatic micromethods several putative neurotransmitters were measured in four identifiable neurons of Aplysia californica (R-2, R-14, L-11, and C-1). Serotonin was found in all of these neurons, and octopamine in all but C-1. Acetylcholine has been previously reported to be present in R-2 and L-11. The catecholamines, norepinephrine and dopamine, were not detected in the four cells examined. The possible biological consequence of the presence of several putative transmitters in single identifiable neurons is discussed. PMID:4373726

  16. The atf2 gene is involved in triacylglycerol biosynthesis and accumulation in the oleaginous Rhodococcus opacus PD630.

    PubMed

    Hernández, Martín A; Arabolaza, Ana; Rodríguez, Eduardo; Gramajo, Hugo; Alvarez, Héctor M

    2013-03-01

    Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf1 and atf2 exhibited a significant DGAT activity when expressed in E. coli, as revealed in a previous study. The contribution of atf1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327-2335, 2008). In this study, we investigated the in vivo role of atf2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25-30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf1 and atf2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf1 and atf2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf1 gene, atf2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.

  17. 40 CFR 94.220 - Service accumulation.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... accumulation. (a) Each test emission data engine in the test fleet may be operated with all emission control systems operating properly for a period, up to 125 hours of operation, that is sufficient to...

  18. The accumulation and structure of comets

    NASA Technical Reports Server (NTRS)

    Donn, Bertram

    1991-01-01

    The paper reviews evidence for the accumulation of the terrestrial planets and comets from solid grains, with emphasis on the various proposals for the formation of cometary nuclei. With three exceptions, all hypotheses conclude or imply that a single compact object forms. Several hypotheses start with Goldreich-Ward-type gravitational instabilities. The collapse for this case also occurs at low velocities in the cm/s to m/s range. Experiment and theory show that under these conditions, low-density, filamentary clusters form that are fractal aggregates with a fractal dimension approximately equal to 2. In order to form cometary nuclei, the initial temperature must be about 50 K and not undergo a significant temperature rise during the accumulation process. The calculations show that accumulation will occur at low temperatures. Models of cometary nuclei are reviewed, and a simple model of the structure that results fom the accumulation of fluffy aggregates is described.

  19. Waste tank ventilation system waste material accumulations

    SciTech Connect

    Van Vleet, R.J., Westinghouse Hanford

    1996-08-06

    This paper calculates the amount of material that accumulates in the ventilation systems of various Tank Waste Remediation System facilities and estimates the amount of material that could be released due to a rapid pressurization.

  20. Accumulation of nickel in transgenic tobacco

    NASA Astrophysics Data System (ADS)

    Sidik, Nik Marzuki; Othman, Noor Farhan

    2013-11-01

    The accumulation of heavy metal Ni in the roots and leaves of four T1 transgenic lines of tobacco (T(1)20E, T(1)24C, T(1)18B1 and T(1)20B) expressing eiMT1 from E.indica was assessed. The aim of the study was to investigate the level of Ni accumulation in the leaves and roots of each transgenic lines and to evaluate the eligibility of the plants to be classified as a phytoremediation agent. All of the transgenic lines showed different ability in accumulating different metals and has translocation factor (TF) less than 1 (TF<1) at all levels of metal treatment. Among the 4 transgenic lines, transgenic line T(1)24C showed the highest accumulation of Ni (251.9 ± 0.014 mg/kg) and the lowest TF value (TFT(1)24C=0.0875) at 60 ppm Ni.

  1. 19 CFR 10.534 - Accumulation.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Singapore Free Trade Agreement Rules of Origin § 10.534 Accumulation. (a) Originating materials of Singapore or the United States...

  2. 19 CFR 10.534 - Accumulation.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Singapore Free Trade Agreement Rules of Origin § 10.534 Accumulation. (a) Originating materials of Singapore or the United States...

  3. 19 CFR 10.534 - Accumulation.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Singapore Free Trade Agreement Rules of Origin § 10.534 Accumulation. (a) Originating materials of Singapore or the United States...

  4. 19 CFR 10.534 - Accumulation.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Singapore Free Trade Agreement Rules of Origin § 10.534 Accumulation. (a) Originating materials of Singapore or the United States...

  5. 19 CFR 10.534 - Accumulation.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... ARTICLES CONDITIONALLY FREE, SUBJECT TO A REDUCED RATE, ETC. United States-Singapore Free Trade Agreement Rules of Origin § 10.534 Accumulation. (a) Originating materials of Singapore or the United States...

  6. Deletion of a MFS transporter-like gene in Cercospora nicotianae reduces cercosporin toxin accumulation and fungal virulence.

    PubMed

    Choquer, Mathias; Lee, Miin-Huey; Bau, Huey-Jiunn; Chung, Kuang-Ren

    2007-02-06

    Many phytopathogenic Cercospora species produce a host-nonselective polyketide toxin, called cercosporin, whose toxicity exclusively relies on the generation of reactive oxygen species. Here, we describe a Cercospora nicotianae CTB4 gene that encodes a putative membrane transporter and provide genetic evidence to support its role in cercosporin accumulation. The predicted CTB4 polypeptide has 12 transmembrane segments with four conserved motifs and has considerable similarity to a wide range of transporters belonging to the major facilitator superfamily (MFS). Disruption of the CTB4 gene resulted in a mutant that displayed a drastic reduction of cercosporin production and accumulation of an unknown brown pigment. Cercosporin was detected largely from fungal hyphae of ctb4 disruptants, but not from the surrounding medium, suggesting that the mutants were defective in both cercosporin biosynthesis and secretion. Cercosporin purified from the ctb4 disruptants exhibited toxicity to tobacco suspension cells, insignificantly different from wild-type, whereas the disruptants formed fewer lesions on tobacco leaves. The ctb4 null mutants retained normal resistance to cercosporin and other singlet oxygen-generating photosensitizers, indistinguishable from the parental strain. Transformation of a functional CTB4 clone into a ctb4 null mutant fully revived cercosporin production. Thus, we propose that the CTB4 gene encodes a putative MFS transporter responsible for secretion and accumulation of cercosporin.

  7. Sodium accumulation in Atriplex. Final report

    SciTech Connect

    Norton, J.A.; Caldwell, M.M.; Richardson, S.G.

    1984-09-01

    This study was undertaken to determine the ecological significance and the significance to arid land reclamation of sodium accumulation and nonaccumulation in Atriplex. There was a continuum in the genetic tendency of Atriplex canescens to accumulate sodium, from populations which accumulated almost no sodium to populations which accumulated up to 7% in the leaves. There were also substantial differences in sodium uptake between populations of A. tridentata, A. falcata and A. gardneri, with some populations having less than 0.1% leaf sodium and other populations having up to 5 or 6%. In three experiments (a field study, a greenhouse pot study and a hydroponics study) there were no significant differences in salinity tolerance between sodium accumulating and nonaccumulating A. canescens: both genotypes were highly salt tolerant. There was a significant buildup of sodium in the soil beneath sodium accumulating Atriplex plants, both in natural populations and on revegetated oil shale study plots. The sodium buildup was not sufficient to be detrimental to the growth or establishment of most herbaceous species, but with older Atriplex plants or with more saline soil, the buildup could potentially be detrimental. 14 references, 42 figures, 3 tables.

  8. Blueberry mosaic associated virus – A putative, new member of Ophioviridae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Blueberry mosaic initially was reported more than 50 years ago and is now known from different parts of the world. A new virus, closely associated with the disease has been identified recently. The virus tentatively named as Blueberry mosaic associated virus (BlMaV), is a putative member of the gen...

  9. Identification of putative TSWV resistance gene and development of gene-specific marker in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato spotted wilt virus (TSWV) is one of the most destructive viral diseases threatening peanut production in the Southeastern U.S. Among different strategies of controlling this disease, the use of resistant cultivars is more efficient. The objective of this study is to develop putative TSWV res...

  10. Identification of putative TSWV resistance genes and development of gene-specific marker in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato spotted wilt virus (TSWV) is one of the most destructive viral diseases threatening peanut production in the Southeastern U.S. Among different strategies of controlling this disease, the use of resistant cultivars is more efficient. The objective of this study is to develop putative TSWV res...

  11. Purification and characterization pecan (Carya Illinoinensis) vicilin, a putative food allergen (abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The pecan seed storage protein vicilin, a putative food allergen, was recombinantly expressed for and purified by a combination of metal affinity and gel filtration chromatography. The protein was crystallized and studied by crystallography. The obtained crystals belonged to space group P212121 with...

  12. Characterization of a putative endoxylanase in the migratory plant-parasitic nematode Radopholus similis.

    PubMed

    Haegeman, Annelies; Vanholme, Bartel; Gheysen, Godelieve

    2009-05-01

    Plant-parasitic nematodes have developed an arsenal of enzymes to degrade the rigid plant cell wall. In this article, we report the presence of a putative endoxylanase in the migratory endoparasitic nematode Radopholus similis. This enzyme is thought to facilitate the migration of the nematode, as it breaks down xylan, the major component of hemicellulose. The corresponding gene (Rs-xyl1) was cloned and the sequence revealed three small introns. Interestingly, the position of all three introns was conserved in a putative endoxylanase from Meloidogyne hapla, and the position of one intron was conserved in two endoxylanases from Meloidogyne incognita, which suggests a common ancestral gene. The spatial and temporal expression of the Rs-xyl1 gene was examined by in situ hybridization and semi-quantitative reverse transcriptase-polymerase chain reaction. The putative protein consists of a signal peptide, a catalytic domain and a carbohydrate-binding module (CBM). The catalytic domain showed similarity to both glycosyl hydrolase family 5 (GHF5) and GHF30 enzymes. Using Hidden Markov Model profiles and phylogenetic analysis, we were able to show that Rs-XYL1 and its closest homologues are not members of GHF5, as suggested previously, but rather form a subclass within GHF30. Silencing the putative endoxylanase by double-stranded RNA targeting of the CBM region resulted in an average decrease in infection of 60%, indicating that the gene is important for the nematode to complete its life cycle.

  13. A rapid approach to evaluate putative nursery sites for penaeid prawns

    NASA Astrophysics Data System (ADS)

    Taylor, Matthew D.; Smith, James A.; Boys, Craig A.; Whitney, Hannah

    2016-08-01

    Identifying nursery habitats for an aquatic species generally requires tracing adult individuals back through time and space to the area or habitat in which they developed as juveniles. We develop and trial a study design and analytical approach to evaluate the suitability of using stable isotopes to trace emigrating prawns to putative nursery sites, and evaluate assumptions inherent in the application of the approach using two penaeid species with Type-II life cycles: Penaeus (Melicertus) plebejus and Metapenaeus macleayi. Prawns were collected in putative nursery sites within the Hunter River, Australia, and analysed as composite samples of 6 individuals to provide habitat-specific isotopic signatures. Prawns emigrating from the mouth of the river were used as a proxy for individuals recruiting to the adult population, and assigned to putative nursery sites using a probabilistic mixing model and a simple, distance-based approach. Bivariate (δ15N and δ13C) isotopic data was sufficient to distinguish prawns from different putative nursery sites, and isotopic composition correlated closely with salinity. Approximately 90% of emigrating prawns collected could be assigned to these sites using bivariate isotopic data, and both analytical approaches gave similar results. The design developed here is broadly applicable to a suite of penaeid species, but its application will be most powerful when sampling is also aimed at understanding nursery function by simultaneous monitoring of size structure/growth, density, and trophic relationships within nursery habitats.

  14. A simplified sequence-based identification scheme for Bordetella reveals several putative novel species.

    PubMed

    Spilker, Theodore; Leber, Amy L; Marcon, Mario J; Newton, Duane W; Darrah, Rebecca; Vandamme, Peter; Lipuma, John J

    2014-02-01

    The differentiation of Bordetella species, particularly those causing human infection, is problematic. We found that sequence analysis of an internal fragment of nrdA allowed differentiation of the currently named Bordetella species. Analysis of 107 "Bordetella" isolates recovered almost exclusively from human respiratory tract specimens identified several putative novel species.

  15. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    PubMed

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir, we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C (V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  16. Occurrence of putative pathogenicity islands in enterococci from distinct species and of differing origins.

    PubMed

    Semedo-Lemsaddek, Teresa; Barreto-Crespo, Maria Teresa; Tenreiro, Rogério

    2009-11-01

    Enterococci isolated from ewe's milk and cheese, clinical isolates of human and veterinary origins, and reference strains obtained from culture collections were screened for the occurrence of putative pathogenicity island (PAIs). Results obtained after PCR amplification and hybridization point toward PAI dissemination among enterococci of diverse origins (food/clinical) and species (Enterococcus faecalis/non-E. faecalis).

  17. Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15- 02/2011. The genomic characterization of the isolate s...

  18. Isolation and characterization of 17 different genes encoding putative endopolygalacturonase genes from Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase enzymes are a valuable aid in the retting of flax for production of linens and, more recently, production of biofuels from citrus wastes. In a search of the recently sequenced Rhizopus oryzae strain 99-880 genome database, 18 putative endopolygalacturonase genes were identified, w...

  19. Preinspiratory calcium rise in putative pre-Bötzinger complex astrocytes

    PubMed Central

    Okada, Yasumasa; Sasaki, Takuya; Oku, Yoshitaka; Takahashi, Naoya; Seki, Megumi; Ujita, Sakiko; Tanaka, Kenji F; Matsuki, Norio; Ikegaya, Yuji

    2012-01-01

    The neural inspiratory activity originates from a ventrolateral medullary region called the pre-Bötzinger complex (preBötC), yet the mechanism underlying respiratory rhythmogenesis is not completely understood. Recently, the role of not only neurons but astrocytes in the central respiratory control has attracted considerable attention. Here we report our discovery that an intracellular calcium rise in a subset of putative astrocytes precedes inspiratory neuronal firing in rhythmically active slices. Functional calcium imaging from hundreds of preBötC cells revealed that a subset of putative astrocytes exhibited rhythmic calcium elevations preceding inspiratory neuronal activity with a time lag of approximately 2 s. These preinspiratory putative astrocytes maintained their rhythmic activities even during the blockade of neuronal activity with tetrodotoxin, whereas the rhythm frequency was lowered and the intercellular phases of these rhythms were decoupled. In addition, optogenetic stimulation of preBötC putative astrocytes induced firing of inspiratory neurons. These findings raise the possibility that astrocytes in the preBötC are actively involved in respiratory rhythm generation in rhythmically active slices. PMID:22777672

  20. Diversity of putative archaeal RNA viruses in metagenomic datasets of a yellowstone acidic hot spring.

    PubMed

    Wang, Hongming; Yu, Yongxin; Liu, Taigang; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

    2015-01-01

    Two genomic fragments (5,662 and 1,269 nt in size, GenBank accession no. JQ756122 and JQ756123, respectively) of novel, positive-strand RNA viruses that infect archaea were first discovered in an acidic hot spring in Yellowstone National Park (Bolduc et al., 2012). To investigate the diversity of these newly identified putative archaeal RNA viruses, global metagenomic datasets were searched for sequences that were significantly similar to those of the viruses. A total of 3,757 associated reads were retrieved solely from the Yellowstone datasets and were used to assemble the genomes of the putative archaeal RNA viruses. Nine contigs with lengths ranging from 417 to 5,866 nt were obtained, 4 of which were longer than 2,200 nt; one contig was 204 nt longer than JQ756122, representing the longest genomic sequence of the putative archaeal RNA viruses. These contigs revealed more than 50% sequence similarity to JQ756122 or JQ756123 and may be partial or nearly complete genomes of novel genogroups or genotypes of the putative archaeal RNA viruses. Sequence and phylogenetic analyses indicated that the archaeal RNA viruses are genetically diverse, with at least 3 related viral lineages in the Yellowstone acidic hot spring environment.

  1. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    PubMed

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis.

  2. Effect of Curare on Responses to Different Putative Neurotransmitters in Aplysia.

    DTIC Science & Technology

    1976-06-01

    The effects of curare on responses resulting from ionophoretic application of several putative neurotransmitters onto Aplysia neurons were studied...In Aplysia nervous tissue, curare appears to be a specfic blocking agent for a class of receptor-activated Na and Cl responses.

  3. Identification, recombinant expression, and biochemical analysis of putative secondary product glucosyltransferases from Citrus paradisi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavonoid and limonoid glycosides influence taste properties as well as marketability of citrus fruit and products, particularly in grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semi-conser...

  4. Diet Does Not Affect Putative Mammary Epithelial Stem Cells in Pre-weaned Holstein Heifers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Overfeeding prepubertal heifers can impair mammary epithelial growth and development, processes that depend on stem cells. In this study we evaluated effects of diet composition on putative bovine mammary epithelial stem cell populations using a 5-bromo-2-deoxyrudine (BrdU; a thymidine analog) label...

  5. A new putative alphapartitivirus recovered from the powdery mildew fungus Erysiphe palczewskii.

    PubMed

    Xiong, Guihong; Qiu, Ping; Li, Cong; Chen, Zhuo; Islam, Saif Ul; Fang, Shouguo; Wu, Zujian; Zhang, Songbai; Du, Zhenguo

    2017-02-27

    Two double-stranded RNAs (dsRNA) likely representing the genome of a novel alphapartitivirus which we provisionally named Erysiphe palczewskii alphapartitivirus 1 (EpV1) were recovered from the powdery mildew fungus E. palczewskii infecting Sophora japonica in Jingzhou, Hubei province of China. The two dsRNAs, 1955 (dsRNA1) and 1917 (dsRNA2) bp in size, respectively, each contains a single open reading frame (ORF) encoding a 585- and 528-aa protein, respectively. The 585-aa protein contains a conserved RNA-dependent RNA polymerase (RdRp) domain and shows significant homology to RdRps of approved or putative partitiviruses, particularly those belonging to the genus Alphapartitivirus. However, it shares an aa sequence identity lower than 80% with its closest relative, the RdRp of the putative alphapartitivirus Grapevine partitivirus, and lower than 60% with the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRp aa sequences of selected partitiviruses, the putative virus EpV1 clustered with Grapevine partitivirus and formed a well-supported monophyletic clade with known or putative alphapartitiviruses.

  6. Cymbidium ringspot virus harnesses RNA silencing to control the accumulation of virus parasite satellite RNA.

    PubMed

    Pantaleo, Vitantonio; Burgyán, József

    2008-12-01

    Cymbidium ringspot virus (CymRSV) satellite RNA (satRNA) is a parasitic subviral RNA replicon that replicates and accumulates at the cost of its helper virus. This 621-nucleotide (nt) satRNA species has no sequence similarity to the helper virus, except for a 51-nt-long region termed the helper-satellite homology (HSH) region, which is essential for satRNA replication. We show that the accumulation of satRNA strongly depends on temperature and on the presence of the helper virus p19 silencing suppressor protein, suggesting that RNA silencing plays a crucial role in satRNA accumulation. We also demonstrate that another member of the Tombusvirus genus, Carnation Italian ringspot virus (CIRV), supports satRNA accumulation at a higher level than CymRSV. Our results suggest that short interfering RNA (siRNA) derived from CymRSV targets satRNA more efficiently than siRNA from CIRV, possibly because of the higher sequence similarity between the HSH regions of the helper and CIRV satRNAs. RNA silencing sensor RNA carrying the putative satRNA target site in the HSH region was efficiently cleaved when transiently expressed in CymRSV-infected plants but not in CIRV-infected plants. Strikingly, replacing the CymRSV HSH box2 sequence with that of CIRV restores satRNA accumulation both at 24 degrees C and in the absence of the p19 suppressor protein. These findings demonstrate the extraordinary adaptation of this virus to its host in terms of harnessing the antiviral silencing response of the plant to control the virus parasite satRNA.

  7. Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy.

    PubMed

    Dang, Thu-Thuy T; Facchini, Peter J

    2014-01-03

    Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (Km=4.63±0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.

  8. Salt stress encourages proline accumulation by regulating proline biosynthesis and degradation in Jerusalem artichoke plantlets.

    PubMed

    Huang, Zengrong; Zhao, Long; Chen, Dandan; Liang, Mingxiang; Liu, Zhaopu; Shao, Hongbo; Long, Xiaohua

    2013-01-01

    Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation.

  9. Geomorphic control of landscape carbon accumulation

    USGS Publications Warehouse

    Rosenbloom, N.A.; Harden, J.W.; Neff, J.C.; Schimel, D.S.

    2006-01-01

    We use the CREEP process-response model to simulate soil organic carbon accumulation in an undisturbed prairie site in Iowa. Our primary objectives are to identify spatial patterns of carbon accumulation, and explore the effect of erosion on basin-scale C accumulation. Our results point to two general findings. First, redistribution of soil carbon by erosion results in a net increase in basin-wide carbon storage relative to a noneroding environment. Landscape-average mean residence times are increased in an eroding landscape owing to the burial/preservation of otherwise labile C. Second, field observations taken along a slope transect may overlook significant intraslope variations in carbon accumulation. Spatial patterns of modeled deep C accumulation are complex. While surface carbon with its relatively short equilibration time is predictable from surface properties, deep carbon is strongly influenced by the landscape's geomorphic and climatic history, resulting in wide spatial variability. Convergence and divergence associated with upland swales and interfluves result in bimodal carbon distributions in upper and mid slopes; variability in carbon storage within modeled mid slopes was as high as simulated differences between erosional shoulders and depositional valley bottoms. The bimodality of mid-slope C variability in the model suggests that a three-dimensional sampling strategy is preferable over the traditional two-dimensional analog or "catena" approach. Copyright 2006 by the American Geophysical Union.

  10. Impact of Insulin Degrading Enzyme and Neprilysin in Alzheimer's Disease Biology: Characterization of Putative Cognates for Therapeutic Applications.

    PubMed

    Jha, Niraj Kumar; Jha, Saurabh Kumar; Kumar, Dhiraj; Kejriwal, Noopur; Sharma, Renu; Ambasta, Rashmi K; Kumar, Pravir

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative process primarily characterized by amyloid-β (Aβ) agglomeration, neuroinflammation, and cognitive dysfunction. The prominent cause for dementia is the deposition of Aβ plaques and tau-neurofibrillary tangles that hamper the neuronal organization and function. Aβ pathology further affects numerous signaling cascades that disturb the neuronal homeostasis. For instance, Aβ deposition is responsible for altered expression of insulin encoding genes that lead to insulin resistance, and thereby affecting insulin signaling pathway and glucose metabolism in the brain. As a result, the common pathology of insulin resistance between Type-2 diabetes mellitus and AD has led AD to be proposed as a form of diabetes and termed 'Type-3 diabetes'. Since accumulation of Aβ is the prominent cause of neuronal toxicity in AD, its clearance is the prime requisite for therapeutic prospects. This purpose is expertly fulfilled by the potential role of Aβ degrading enzymes such as insulin degrading enzyme (IDE) and Neprilysin (NEP). Therefore, their molecular study is important to uncover the proteolytic and regulatory mechanism of Aβ degradation. Herein, (i) In silico sequential and structural analysis of IDE and NEP has been performed to identify the molecular entities for proteolytic degradation of Aβ in the AD brain, (ii) to analyze their catalytic site to demonstrate the enzymatic action played by IDE and NEP, (iii) to identify their structural homologues that could behave as putative partners of IDE and NEP with similar catalytic action and (iv) to illustrate various IDE- and NEP-mediated therapeutic approaches and factors for clearing Aβ in AD.

  11. Identification of Putative Stage-Specific Grapevine Berry Biomarkers and Omics Data Integration into Networks1[C][W][OA

    PubMed Central

    Zamboni, Anita; Di Carli, Mariasole; Guzzo, Flavia; Stocchero, Matteo; Zenoni, Sara; Ferrarini, Alberto; Tononi, Paola; Toffali, Ketti; Desiderio, Angiola; Lilley, Kathryn S.; Pè, M. Enrico; Benvenuto, Eugenio; Delledonne, Massimo; Pezzotti, Mario

    2010-01-01

    The analysis of grapevine (Vitis vinifera) berries at the transcriptomic, proteomic, and metabolomic levels can provide great insight into the molecular events underlying berry development and postharvest drying (withering). However, the large and very different data sets produced by such investigations are difficult to integrate. Here, we report the identification of putative stage-specific biomarkers for berry development and withering and, to our knowledge, the first integrated systems-level study of these processes. Transcriptomic, proteomic, and metabolomic data were integrated using two different strategies, one hypothesis free and the other hypothesis driven. A multistep hypothesis-free approach was applied to data from four developmental stages and three withering intervals, with integration achieved using a hierarchical clustering strategy based on the multivariate bidirectional orthogonal projections to latent structures technique. This identified stage-specific functional networks of linked transcripts, proteins, and metabolites, providing important insights into the key molecular processes that determine the quality characteristics of wine. The hypothesis-driven approach was used to integrate data from three withering intervals, starting with subdata sets of transcripts, proteins, and metabolites. We identified transcripts and proteins that were modulated during withering as well as specific classes of metabolites that accumulated at the same time and used these to select subdata sets of variables. The multivariate bidirectional orthogonal projections to latent structures technique was then used to integrate the subdata sets, identifying variables representing selected molecular processes that take place specifically during berry withering. The impact of this holistic approach on our knowledge of grapevine berry development and withering is discussed. PMID:20826702

  12. Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species ( Lupinus angustifolius L. and L. mutabilis L.).

    PubMed

    Francki, Michael G; Whitaker, Peta; Smith, Penelope M; Atkins, Craig A

    2002-11-01

    Seed triacylglycerols (TAGs) are stored as energy reserves and extracted for various end-product uses. In lupins, seed oil content varies from 16% in Lupinus mutabilisto 8% in L. angustifolius. We have shown that TAGs rapidly accumulate during mid-stages of seed development in L. mutabilis compared to the lower seed oil species, L. angustifolius. In this study, we have targeted the key enzymes of the lipid biosynthetic pathway, acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), to determine factors regulating TAG accumulation between two lupin species. A twofold increase in ACCase activity was observed in L. mutabilis relative to L. angustifolius and correlated with rapid TAG accumulation. No difference in DAGAT activity was detected. We have identified, cloned and partially characterised a novel gene differentially expressed during TAG accumulation between L. angustifolius and L. mutabilis. The gene has some identity to the glucose dehydrogenase family previously described in barley and bacteria and the significance of its expression levels during seed development in relation to TAG accumulation is discussed. DNA sequence analysis of the promoter in both L. angustifolius and L. mutabilis identified putative matrix attachment regions and recognition sequences for transcription binding sites similar to those found in the Adh1 gene from Arabidopsis. The identical promoter regions between species indicate that differential gene expression is controlled by alternative transcription factors, accessibility to binding sites or a combination of both.

  13. Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

  14. Cohabitation history, marriage, and wealth accumulation.

    PubMed

    Vespa, Jonathan; Painter, Matthew A

    2011-08-01

    This study extends research on the relationship between wealth accumulation and union experiences, such as marriage and cohabitation. Using data from the National Longitudinal Survey of Youth 1979, we explore the wealth trajectories of married individuals in light of their premarital cohabitation histories. Over time, marriage positively correlates with wealth accumulation. Most married persons with a premarital cohabitation history have wealth trajectories that are indistinguishable from those without cohabitation experience, with one exception: individuals who marry their one and only cohabiting partner experience a wealth premium that is twice as large as that for married individuals who never cohabited prior to marrying. Results remain robust over time despite cohabiters' selection out of marriage, yet vary by race/ethnicity. We conclude that relationship history may shape long-term wealth accumulation, and contrary to existing literature, individuals who marry their only cohabiting partners experience a beneficial marital outcome. It is therefore important to understand the diversity of cohabitation experiences among the married.

  15. Ordinary differential equation for local accumulation time.

    PubMed

    Berezhkovskii, Alexander M

    2011-08-21

    Cell differentiation in a developing tissue is controlled by the concentration fields of signaling molecules called morphogens. Formation of these concentration fields can be described by the reaction-diffusion mechanism in which locally produced molecules diffuse through the patterned tissue and are degraded. The formation kinetics at a given point of the patterned tissue can be characterized by the local accumulation time, defined in terms of the local relaxation function. Here, we show that this time satisfies an ordinary differential equation. Using this equation one can straightforwardly determine the local accumulation</